matador and the regulation of cyclin e1 in … placental development and placental pathology ......
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MATADOR AND THE REGULATION OF CYCLIN E1 IN
NORMAL HUMAN PLACENTAL DEVELOPMENT AND
PLACENTAL PATHOLOGY
BY
JOCELYN ELAINE RAY
A THESIS SUBMITTED IN CONFORMITY WITH THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
GRADUATE DEPARTMENT OF PHYSIOLOGY
UNIVERSITY OF TORONTO
© COPYRIGHT BY JOCELYN ELAINE RAY, 2010
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Matador and the Regulation of cyclin E1 in Normal
Human Placental Development and Placental Pathology
Jocelyn Ray, Doctor of Philosophy, 2010
Graduate Department of Physiology, University of Toronto
Toronto, Ontario, Canada
ABSTRACT
Preeclampsia and molar pregnancy are two devastating placental pathologies characterized by an
immature proliferative trophoblast phenotype accompanied by excessive cell death. It is
therefore of paramount importance to study the regulation of cell fate in the placenta, to gain a
further understanding of the mechanisms that contribute to these diseases.
In this dissertation we report that during normal placental development and in preeclampsia,
Matador (Mtd), a pro-apoptotic member of the Bcl-2 family, has a dual function in regulating
trophoblast cell proliferation and death. Importantly, we reveal a novel role of Mtd-L in
promoting cyclin E1 expression and cell cycle progression.
Of clinical importance, we also identify that both cyclin E1 and the CDK inhibitor p27, are
increased in severe early onset preeclampsia. However, the inhibitory function of p27 in this
pathology may be hampered due to its increased phosphorylation at Ser10, resulting in its nuclear
export. Of equal importance, data presented demonstrate that placentae from severe early onset
preeclampsia display a molecular profile distinct from late onset preeclampsia or intrauterine
growth restricted pregnancies.
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In the final data chapter we demonstrate that Mtd is highly expressed in molar tissue, where it
localizes to both apoptotic and proliferative cells. Our data suggests that an abundance of Mtd
and cyclin E1 in conjunction with the low level of p27 may contribute to the hyperproliferative
nature of the disorder.
The body of work in this dissertation uncovers novel insights into the regulation of trophoblast
cell fate. Importantly, the impact of Mtd on cyclin E1 to promote G1-S transition is a novel
mechanism found to regulate trophoblast cell proliferation in normal and pathological
placentation. Equally important is our identification of molecular differences between placental
pathologies that may help to differentiate early and late onset preeclampsia, IUGR and molar
pregnancy.
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ACKNOWLEDGEMENTS
I would like to express my appreciation to my supervisors, Dr. Isabella Caniggia and Dr. Andrea
Jurisicova for their guidance throughout my PhD. Through this experience I have learned how to
be a better scientist and mentor. Thank you for the scientific discussions and for the lessons on
life. I have learned a great deal that I will keep with me forever.
I would also like to extend my gratitude to the members of my PhD supervisory committee, Drs.
Stephen Matthews, Mingyao Liu, and Lowelle Langille, who gave me valuable advice
throughout my PhD. I am forever grateful.
I would also like to thank Drs. Lye, Brown, Casper, Rogers, and Kingdom who encouraged me
through the last six years, listened to my ideas and helped me to become a better scientist. Thank
you to Bev Bessey and Cindy Todoroff for all the administrative help, support, and kind words.
I would also like to extend my thanks to the scientists with whom I previously worked; Drs. Jay
Wunder, Ben Alman, Carl Ware, and Peter Greer. They deserve the utmost credit for teaching
me the basics, helping to expand my ability to think logically and creatively, and for stimulating
and cultivating my interest in science.
I would especially like to show my appreciation to all the members of the Caniggia, Jurisicova,
and Kingdom labs for their stimulating scientific discussions, technical support, and
encouragement. I would like to thank Dr. Yuan Xu for taking me under her wing in my initial
years, Julia Garcia, and my Caniggia lab sisters, Livia Deda, Antonella Racano, Tara
Sivasubramaniyam and Manpreet Kalkat, who made the six years in the Caniggia lab feel like a
home. I would like to especially thank Livia, my first student and now a great friend and
confidante. She is one of the most compassionate, understanding, and gracious women I know.
Thank you, Antonella, a breath of fresh air, always quick witted and entertaining, and Julia, Tara
and Manpreet for lighting up the lab. I would like to thank Dr. Jacquie Detmar for being
someone I could always turn to for help, for teaching me technical skills and for working with
me to troubleshoot many experiments. I would like to thank Dr. Alicia Tone, for her great
scientific mind and for her amazing friendship. I would like to send a special thanks Dr. Sascha
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Drewlo for his constant support both scientifically and personally, he was my rock through the
hard times and will remain a best friend and colleague.
Most importantly I would like to thank my family. To my parents who let me fend for myself; I
know you were always there if I needed you. The two of you have been my true motivation.
Lastly I would like to thank my sister who has been an inspiration as a woman of strength. She is
a reminder of what is truly important in life, and has kept me balanced.
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CONTRIBUTIONS
The following people have contributed to the generation of data reported in the present thesis.
Introduction: Portions of the introduction have been published in the following form Ray,J.,
Jurisicova,A., and Caniggia,I. (2008). IFPA Trophoblast Research Award Lecture: The
Dynamic Role of Bcl-2 Family Members in Trophoblast Cell Fate. Placenta.
Chapter 3: Yuan Wu contributed to the data presented in Figure 3.5 by performing the JEG-3
cell fractionation experiment. Dr. Julia Garcia created the GFP-hMtd-L doxycycline inducible
cell line used to generate data in Figure 3.8. Dr. Caniggia aided in the explant culture work
performed in chapter 3 and chapter 4. Placental tissue was collected by the research nursing staff
at Mount Sinai Hospital. Data in chapter three have been published in the following form
Ray,J.E., Garcia,J., Jurisicova,A., and Caniggia,I. (2009). Mtd/Bok takes a swing:
proapoptotic Mtd/Bok regulates trophoblast cell proliferation during human placental
development and in preeclampsia. Cell Death and Differentiation.
Chapter 4: Dr. Barbara Cifra contributed to the data presented in Figure 4.10 by performing
western blot and immunofluorescence analysis of phospho-p27 Ser 10 in developmental samples.
Dr. Tullia Todros kindly provided PE and IUGR placentae used in this chapter.
Chapter 5: Dr. Ori Nevo aided in the collection of the molar placentae.
Future directions: Caspase-3 defient mice were obtained from Dr. Tak Mak‟s laboratory, OCI
UHN. Dr. Jurisicova collected the blastocysts and helped to establish the TS cell lines.
Work presented in this thesis was supported by the Genesis Foundation and CIHR.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ........................................................................................................ iv
CONTRIBUTIONS ...................................................................................................................... vi
TABLE OF CONTENTS ........................................................................................................... vii
List of Tables ............................................................................................................................... xii
List of Figures ............................................................................................................................. xiii
Abbreviations .............................................................................................................................. xv
1 Introduction .............................................................................................................................. 1
1.1 Human Placental Development ....................................................................................... 1
1.1.1 Early placental development ................................................................................... 1
1.1.2 Trophoblast Differentiation and Placental Establishment ..................................... 7
1.1.3 The Mature Placenta ............................................................................................. 10
1.1.4 Oxygen and Human Placentation ......................................................................... 12
1.2 Cell Death and the Regulation of Apoptosis in the Placenta ...................................... 13
1.2.1 Classical Function: Apoptosis and the Intrinsic Pathway .................................... 14
1.2.2 Apoptosis and the Bcl-2 Family in Placentation .................................................. 18
1.2.3 Mtd in Placental Apoptosis ................................................................................... 21
1.3 Regulation of the Cell Cycle in the Placenta ................................................................ 23
1.3.1 The Cell Cycle ....................................................................................................... 23
1.3.2 Cell Cycle Inhibitors ............................................................................................. 26
1.3.3 Regulation of Proliferation and the Cell Cycle in the Placenta ........................... 31
1.3.4 Role of Bcl-2 Family Members in Cell Fate ......................................................... 34
1.4 Placental Pathology ......................................................................................................... 35
viii
1.4.1 Preeclampsia ......................................................................................................... 37
1.4.2 IUGR ..................................................................................................................... 41
1.4.3 Mtd and the Bcl-2 family in preeclampsia ............................................................ 43
1.5 Complete Molar Pregnancy ........................................................................................... 44
1.5.1 Clinical Detection and Classification of Molar Pregnancy ................................. 45
1.5.2 Trophoblast Biology of the Complete Molar Placenta: Morphological
characteristics and Histopathology ...................................................................... 46
1.5.3 Trophoblast Biology of the Complete Molar Placenta: Molecular
Characteristics ...................................................................................................... 47
1.6 Thesis Hypothesis and Objectives ................................................................................. 48
2 Materials and Methods .......................................................................................................... 51
2.1 Placental Tissue Collection ............................................................................................... 51
2.1.1 Placental samples for studies on preeclamptic pathology .................................... 51
2.1.2 Samples for studies on molar twin pathology ....................................................... 53
2.1.3 Samples for laser capture microdissection ........................................................... 53
2.2 First trimester Villous Explant Culture ............................................................................. 55
2.2.1 Mtd antisense knockdown ..................................................................................... 55
2.2.2 TGF treatment .................................................................................................... 55
2.3 Laser Capture Microdissection ......................................................................................... 56
2.4 RNA Analysis ................................................................................................................... 56
2.5 Antibodies ......................................................................................................................... 57
2.6 Western Blot Analysis ...................................................................................................... 58
2.7 Immuno-precipitation ....................................................................................................... 58
2.8 Peroxidase Based Immunohistochemistry ........................................................................ 58
2.9 Immunofluorescence (IF) Staining ................................................................................... 59
2.10 TUNEL (Terminal Deoxynucleotidyl Transferase-dUTP-Nick End Labeling) ............... 60
2.11 Cell Line Culture and Analysis ......................................................................................... 61
ix
2.11.1 SNP (Sodium nitroprusside) treatment ................................................................. 61
2.11.2 Trypan Blue Exclusion Assay ................................................................................ 61
2.11.3 Cell Viability (MTT) .............................................................................................. 61
2.11.4 Cell Fractionation ................................................................................................. 62
2.11.5 Localization of Mtd to Mitochondria .................................................................... 62
2.11.6 siRNA Treatment ................................................................................................... 62
2.11.7 BrdU Incorporation .............................................................................................. 63
2.11.8 TGF treatment .................................................................................................... 63
2.12 Construction of Stable Cell Line Expressing GFP-hMtdL ............................................... 63
2.13 Statistical analysis ............................................................................................................. 63
3 Pro-apoptotic Mtd/Bok Regulates Trophoblast Cell Proliferation during Human
Placental Development and in Preeclampsia ....................................................................... 65
3.1 Abstract ............................................................................................................................ 65
3.2 Introduction ..................................................................................................................... 66
3.3 Results .............................................................................................................................. 67
3.3.1 Mtd expression in proliferating trophoblast cells ................................................. 67
3.3.2 Mtd localizes to villous trophoblast cells in the G1-phase of the cell cycle .......... 69
3.3.3 Mtd expression can occur independently of cell death during early
placentation ........................................................................................................... 72
3.3.4 Mtd-L is the predominant isoform expressed in proliferative trophoblast cells ... 72
3.3.5 Mtd isoforms are differentially localized within proliferative JEG-3 cells .......... 75
3.3.6 SNP-induced apoptosis promotes mitochondrial localization of Mtd in JEG-3
cells ....................................................................................................................... 75
3.3.7 Inhibition of Mtd-L suppresses cyclin E1 expression ........................................... 78
3.4 Discussion ......................................................................................................................... 81
4 Altered trophoblast proliferation in preeclampsia is associated with increased cyclin
E1expression and abnormal regulation of the cell cycle inhibitor p27 ............................. 86
4.1 ABSTRACT ..................................................................................................................... 86
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4.2 Introduction ..................................................................................................................... 87
4.3 Results .............................................................................................................................. 89
4.3.1 Mtd expression in proliferating trophoblast cells in preeclampsia ...................... 89
4.3.2 Cyclin E1 and the CDK inhibitor p27 show opposing expression during
normal placentation .............................................................................................. 89
4.3.3 Expression of cyclin E1 and p27 is altered in severe early onset preeclamptic
placentae compared to age matched and term controls ....................................... 95
4.3.4 Post translation regulation of p27 is altered in preeclampsia ............................. 98
4.3.5 Regulation of cyclin E1 and p27 are altered in several placental pathologies .... 98
4.3.6 Phosphorylation of p27 is increased in the early stages of normal placental
development ........................................................................................................ 102
4.3.7 TGF influences cyclin E1 and p27 expression in villous explants cultured
under varying oxygen conditions ........................................................................ 102
4.3.8 TGF influences cyclin E1 and p27 expression in JEG-3 choriocarcinoma cell
line cultured under varying oxygen conditions ................................................... 104
4.4 Discussion ....................................................................................................................... 107
5 Dual role for Mtd in trophoblast proliferation and apoptosis in molar pathology ........ 113
5.1 Abstract .......................................................................................................................... 113
5.2 Introduction ................................................................................................................... 114
5.3 Results ............................................................................................................................ 116
5.3.1 Two cases of a twin pregnancy with a complete hydatidiform mole and
coexistent twin fetus ............................................................................................ 116
5.3.2 Second trimester complete molar placentae display increased trophoblast
proliferation and apoptosis ................................................................................. 118
5.3.3 Pro-apoptotic Mtd is elevated in the molar placenta compared to its co-
existing twin and it is associated with apoptotic cells in the trophoblastic and
stromal areas ...................................................................................................... 121
5.3.4 Mtd localizes to the nuclei of proliferative trophoblast cells in molar
pathology ............................................................................................................. 123
5.3.5 Mtd is associated with increased cyclin E1 in villous trophoblast cells of
molar placentae .................................................................................................. 123
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5.3.6 Molar placentae exhibit decreased levels of cell cycle inhibitor p27 ................. 127
5.3.7 Molar placentae exhibit altered expression of molecules involved in
regulating the G1 phase of the cell cycle ............................................................ 127
5.4 Discussion ....................................................................................................................... 127
6 Summary and Future Directions ........................................................................................ 136
7 Future Directions – Experimental Design and Preliminary Data ................................... 147
7.1 Determine if caspase-3 is the connecting link between Mtd and cyclin E1 through the
cleavage of p21 and p27. ................................................................................................ 147
7.2 Determine the upstream pathway in preeclampsia leading to the phosphorylation of
p27at Ser10 and its translocation to the cytoplasm ......................................................... 149
7.3 Determine the mechanism leading to low p27 expression in molar tissue and
determine if it contributes to increased Mtd and cyclin E1 expression in the pathology 151
xii
List of Tables
Table 2-1: Clinical data for preeclamptic, intra uterine growth restricted, and control cases ...... 52
Table 2-2: Clinical data for molar twin pregnancies and age matched control twin cases ........... 54
Table 3-1 Expression of Ki67 and Mtd in trophoblast cells ......................................................... 70
xiii
List of Figures
Figure 1-1: Implantation of the blastocyst ...................................................................................... 2
Figure 1-2: Primitive placenta ........................................................................................................ 4
Figure 1-3: Development of placental villi ..................................................................................... 5
Figure 1-4: Placental floating and anchoring villi .......................................................................... 6
Figure 1-5: The placental membrane .............................................................................................. 8
Figure 1-6: The mature placenta ................................................................................................... 11
Figure 1-7: The extrinsic and intrinsic cell death pathway ........................................................... 15
Figure 1-8: BCL-2 family members ............................................................................................. 17
Figure 1-9: Mtd isoforms ............................................................................................................. 22
Figure 1-10: The cell cycle ........................................................................................................... 24
Figure 1-11: Function of cyclin E1 ............................................................................................... 27
Figure 1-12: Regulation and function of p27 ................................................................................ 29
Figure 1-13: Dual role of Bcl-2 family members in cell death and proliferation ......................... 36
Figure 3-1: Mtd expression in proliferating trophoblast cells. ..................................................... 68
Figure 3-2: Association of Mtd with cyclin E1. ........................................................................... 71
Figure 3-3: Apoptosis in early first trimester placental sections. ................................................. 73
Figure 3-4: Mtd isoform mRNA expression in trophoblast subpopulations................................. 74
Figure 3-5: Subcellular localization of Mtd isoforms in JEG-3 cells. .......................................... 76
Figure 5-1 Morphologic characteristics of placentae from the mole and its co-existing twin ... 117
xiv
Figure 5-2 Proliferative assessment of placentae from the mole and its co-existing twin ......... 119
Figure 5-3 Apoptotic assessment of placentae from the mole and its co-existing twin by TUNEL
staining ........................................................................................................................................ 120
Figure 5-4 Expression of apoptotic molecules in molar twins and control twins ....................... 122
Figure 5-5 Co-localization of Mtd with Ki67 expression in mole and twin placentae ............... 124
Figure 5-6 Mtd is expressed in proliferative trophoblast cells associated with various molar
characteristics .............................................................................................................................. 125
Figure 5-7 Cyclin E1 is overexpressed in molar placentae compared to twin controls .............. 126
Figure 5-8 p27 expression in molar placentae and twin controls ............................................... 128
Figure 5-9 Expression of G1 phase cell cycle regulators in the molar and control twins .......... 129
Figure 6-1 Putative model of the mechanism linking Mtd to cyclin E1 expression ................... 139
Figure 7-1 Caspase-3 cleavage of CDK inhibitors in the placenta ............................................. 148
Figure 7-2 Caspase-3 null TS cell derivation ............................................................................. 150
xv
Abbreviations
ABC avidin biotin complex
AMC age matched control
AS antisence
Bcl-2 B cell lymphoma-2
Bok Bcl-2 related ovarian killer
BSA bovine serum albumin
oC degree Celsius
Casp-3 caspase-3
CDK cyclin dependent kinase
CT cytotrophoblast cells
Ct threshold cycle
CTB cytotrophoblast
Cy D/E cyclin D, cyclin E
DAB diaminobenzidine tetraaminobiphenyl
DAPI 4‟.6-diamidino-2-phenylindole
DEPC diethyl pyrocarbonate
dH2O distilled water
DMEM dulbecco‟s modified essential medium
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
ECM extracellular matrix
EGF epidermal growth factor
EVT extravillous trophoblast
FBS fetal bovine serum
GTD gestation trophoblastic disease
H2O2 hydrogen peroxide
hCG human chorionic gonadotropin
HIF-1 hypoxia-inducible factor-1
HLA-G histocompatibility-linked antigen-G
HRE hypoxia response element
IF immunofluorescence
INK inhibitor of CDK4
IUGR intra uterine growth restriction
xvi
ug microgram
ul microlitre
Mcl-1 myeloid cell leukemia sequence 1
mg milligram
mL millilitre
mM millimolar
mmHg millimeters of mercury
MMP matrix metalloproteinase
mRNA messenger ribonucleic acid
Mtd matador
MTT 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide
n number of samples
O2 molecular oxygen
OCT optimal cutting temperature
PBS phosphate-buffered saline
PCNA proliferating cell nuclear antigen
PCR polymerase chain reaction
PE preeclampsia
PFA paraformaldehyde
pO2 partial pressure of oxygen
qRT-PCR quantitative real-time PCR
S sense
SS scramble sequence
SEM standard error of the mean
SK syncytial knot
SNP sodium nitroprusside
ST syncytium/syncytiotrophoblast
STBM syncytiotrophoblast microfragments
TBS tris buffered saline
TdT terminal deoxynucleotide transferase
TGF-B transforming growth factor beta
TS trophoblast stem cells (murine)
TUNEL Terminal Deoxynucleotidyl Transferase-dUTP-Nick End Labeling
Ub ubiquitin
VEGF vascular endothelial growth factor
Vol volume
Wks weeks
Wt weight
1
1 Introduction
The placenta is an extremely unique organ that, although transient, has the capacity to sustain
life. During pregnancy it functions as a life line, mediating the physiological exchange between
mother and fetus. Proper formation of the placenta is therefore essential for fetal development
and a successful healthy pregnancy. In humans, cell proliferation, differentiation and death are
the driving forces behind the process of placentation, determining the fate of trophoblast cells.
Abnormality at any stage of this development, due to altered proliferation, differentiation or cell
death may lead to improper placental function and subsequent pregnancy related complications.
It is therefore imperative that mechanisms regulating these cell fate events be elucidated to aid in
prevention, diagnosis, and treatment of placental related disorders.
1.1 Human Placental Development
1.1.1 Early placental development
Human placental development begins with fertilization, a process where the male and female
gametes unite to create a zygote containing a unique set of 46 chromosomes of equal maternal
and paternal contribution. This process, is followed directly by the cortical reaction of the cells
surrounding the ovum to prevent the chance of polyspermy (Moore and Persaud, 1998). In rare
cases gametes of unequal genetic contribution are formed, leading to abnormal pregnancy. This
topic will be discussed further under the context of placental pathologies.
Following fertilization, the resulting zygote travels down the fallopian tube reaching the uterine
cavity within four to five days. Meanwhile the zygote undergoes a number of mitotic cell
divisions in a process referred to as cleavage. Around the fourth or fifth day, fluid penetrates this
mass of cells to form a hollow ball of around 100 cells referred to as a blastocyst. The blastocyst
consists of an inner mass of cells (known as the inner cell mass) that will give rise to the embryo
proper and extraembryonic tissue, and an outer ring of cells that develop to form the trophoblast
of the placenta (Moore and Persaud, 1998) (Figure 1.1).
By the end of the first week the blastocyst adheres to the endometrial epithelium of the uterine
wall, and the process of implantation begins (Figure 1.1). The trophoblast differentiates into two
distinct layers, an inner layer of proliferative multi-potent mononuclear cytotrophoblast cells and
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Figure 1-1: Implantation of the blastocyst
Approximately one week after fertilization the blastocyst attaches to the uterine wall at its embryonic pole. Syncytial
projections begin to penetrate and invade the maternal endometrium thereby permitting the process of implantation.
Diagram modified from (Moore and Persaud, 1998) with permission.
3
an outer multinucleated layer with no discernable cell boundaries, called the syncytiotrophoblast.
Implantation is initiated by syncytial projections that arise at the embryonic pole of the conceptus
(Figure 1.1). These projections penetrate the endometrium in a process mediated by factors
secreted from the trophoblast, including integrins, matrix metalloproteinases, laminin, and
fibronectin that allow for trophoblast attachment and invasion. In addition, the
syncytiotrophoblast layer also functions to secrete human chorionic gonadotropin (hCG), a
hormone required for the maintenance of pregnancy (Moore and Persaud, 1998). HCG can be
detected as early as the second week after fertilization and is the most commonly used indicator
of pregnancy.
As the blastocyst continues to invade deeper into the endometrium, vacuoles appear in the
syncytium and fuse to form lacunar spaces (Figure 1.2). These lacunae partially contact the
maternal arteries, veins and glands, effectively filling with maternal blood and glandular
secretions from ruptured endometrial capillaries and eroded uterine glands (Figure 1.2). This
produces a nutritional mix (termed embryotroph) that reaches the embryo proper by diffusion
(Moore and Persaud, 1998).
By the end of the second week the underlying cytotrophoblast begins to proliferate and extend
into the syncytiotrophoblast, establishing the formation of the primitive chorionic villi. As the
primitive chorionic villi continue to develop, they begin to branch, expanding into the
intervillous spaces created from the fusion of the syncytial lacunae (Figure 1.3 top panels).
Mesenchyme begins to enter the villous core and soon after capillaries arise, which eventually
fuse to form the arteriocapillary network (Figure 1.3 bottom panel). Meanwhile,
cytotrophoblast cells continue to proliferate and expand out through the syncytium, creating
trophoblast cell columns that form a cytotrophoblastic shell surrounding the chorionic sac, and
effectively anchor the conceptus to the endometrium (Figure 1.3 bottom panel). By the end of
the third week two villous structures have evolved; the anchoring villi that contact the maternal
endometrium, and the floating villi produced from villous branches that remain bathed in the
fluid of the intervillous space (Figure 1.3, 1.4).
4
Figure 1-2: Primitive placenta
The blastocyst invades deeper in to the uterine wall. Cytotrophoblast cells extend into the overlying syncytium to
form the primitive chorionic villi. Lacunar spaces form in the syncytium and accumulate nutritional fluids from
erosion of uterine glands and vasculature that will provide nutrients to the growing conceptus. Diagram modified
from (Moore and Persaud, 1998) with permission.
5
Figure 1-3: Development of placental villi
The chorionic villi begin to branch into the intervillous spaces created from fusion of the syncytial lacunae. In
addition cytotrophoblast cells proliferate and invade past the syncytium to form the trophoblastic shell and establish
the primary extravillous columns and villous tree. Meanwhile mesenchyme and eventually a capillary network
develop in the villous stroma. Diagram modified from (Moore and Persaud, 1998) with permission.
6
Figure 1-4: Placental floating and anchoring villi
The floating villi float in maternal blood within the intervillous space. They are comprised of two trophoblast layers;
an inner layer of proliferative cytotrophoblast cells (green) and an outer layer of differentiated multinucleated
syncytium (white). The core of the villi consists of mesenchymal cells as well as villous vasculature. Anchoring villi
physically attach the placenta to the uterine endothelium (decidua). Anchoring villi are composed of extra villous
trophoblast cells (EVT). The cells at the proximal portion of the column are proliferative (green) and the EVT cells
at the distal end of the column become invasive and migrate into to the deciduas (blue and purple). EVT cells
infiltrate the arterial walls of the maternal spiral arteries and replace cells of the maternal endothelium and smooth
muscle producing uterine arteriole walls consisting of fetal and maternal cells. Green – proliferative trophoblast
cells; blue – differentiating extra villous trophoblast cells; purple – invasive extra villous trophoblast cells.
7
1.1.2 Trophoblast Differentiation and Placental Establishment
1.1.2.1 Formation of the floating villi
The floating villi are comprised of two trophoblast layers, an inner layer of progenitor stem cells,
termed cytotrophoblast, and an outer layer of differentiated syncytium (Figure 1.4). The core of
the villi consists of allantoic mesoderm and the endothelium of the feto-placental circulation. As
the name suggests, these villi float in the maternal fluid, where the branched architecture allow
for maximal surface area for gas and nutrient transport. In early gestation gas and nutrients must
pass through the four layer barrier consisting of syncytium, cytotrophoblast, connective tissue
and endothelium (Figure 1.5).
The layer of syncytium is a ciliated, multinucleated sheath of cellular material with no lateral cell
borders. It is in direct contact with the maternal circulation and acts as a physical barrier between
mother and fetus. The syncytium is also a major source of endocrine activity in the placenta. It
produces and secretes hormones including cortico gonadotropin, placental lactogen, placental
growth hormone and human chorionic gonadotropin (hCG). These secretions are necessary for
placental growth and maternal adaptation to pregnancy.
The syncytial layer is highly differentiated and incapable of self-renewal; a continuous influx of
cellular material is therefore required to keep it replenished so that it can maintain placental
function. This is accomplished through fusion of underlying post-mitotic cytotrophoblast cells
into the syncytial layer, (Huppertz et al., 1998;Potgens et al., 2002;Huppertz et al., 2006). The
addition of cellular material into the syncytiotrophoblast layer is balanced by the deportation of
aged material from the syncytium into the maternal circulation, in the form of membrane
enclosed vesicles termed syncytial knots (Figure 1.4, 1.5). These knots are comprised mostly of
aged nuclei with little cytoplasm. Syncytial knots are carried away through the maternal
circulation and filtered in the maternal lung where they are eventually engulfed by macrophages.
Studies have shown that the process of differentiation from cytotrophoblast to syncytium occurs
in a period of 2-3 days. The material then resides in the syncytium for 3-4 weeks before being
extruded (Huppertz and Kingdom, 2004). The complete process from proliferation through
cytotrophoblast-syncytial fusion, to syncytial shedding is referred to as trophoblast turnover.
Proliferation of cytotrophoblast cells is maximal early in the first trimester in order to support the
initial stages of placental growth, but proliferation is also maintained throughout gestation to
allow for continual trophoblast turnover. Thus trophoblast turnover is highly regulated as
8
Figure 1-5: The placental membrane
The image depicts cross sections through floating placental villi from early gestation and at term. The placental
membrane in the first trimester consists of four layers; the syncytium, the cytotrophoblast cells, the mesenchyme and
the endothelium of the fetal capillaries. At term the membrane is thinned and in some areas consists of only
syncytium and endothelium. Note that at term cytotrophoblast cells persist and are important in replenishing the
syncytial layer that is naturally sloughed off as syncytial knots. Diagram modified from (Moore and Persaud, 1998)
with permission.
9
demonstrated by the time dependent coordination of events. (Specific regulation of trophoblast
cell proliferation and cell death will be covered in more detail in later sections). After the
twentieth week of gestation the placental membrane adapts to the increasing nutritional demands
of the growing fetus. At this point the membrane consists of few cytotrophoblast cells and the
syncytium can become markedly thinned. In addition the capillary endothelium will directly
contact the syncytium in areas, to allow for maximal gas and nutrient transfer (Figure 1.5).
1.1.2.2 Formation of the anchoring villi
The same cytotrophoblast cells that give rise to the syncytial trophoblast also give rise to the
cells that form the anchoring columns and maintain the attachment of the placenta to the uterine
wall. In this case, the majority of the daughter cells produced by the cytotrophoblast, remain as
single non-polarized cells and detach from the basement membrane. As they accumulate, they
form a multilayer of cells that push against the overlying syncytium and break through into the
extravillous space. These cells are referred to as extravillous trophoblast (EVT), since they have
left the confines of the villi (Figure 1.4). The emergence of the EVT constitutes the beginning of
anchoring column formation. Importantly, the cells at the proximal end of the columns maintain
their proliferative potential and function by continuing to feed cells into the column (Figure 1.4).
The EVT cells at the distal portion of the column begin to differentiate, losing their proliferative
capacity, and begin to acquire a migratory phenotype. As the column develops, the EVT cells at
the distal end of the column become invasive and migrate further into to the decidua, up to the
first third of the myometrium (interstitial invasion) (Graham and Lala, 1992) (Figure 1.4).
Differentiation of the extra villous trophoblast cells involves a switch in the expression of a
variety of proteins. As cells differentiate along the invasion pathway they lose cell-cell contact,
and they change their expression pattern of adhesion molecules (Damsky et al., 1994;Caniggia et
al., 1997). Spatially and temporally regulated changes are also seen with respect to the synthesis
and degradation of extracellular matrix proteins and their receptors (Fisher et al., 1989;Caniggia
et al., 1997). The switch in the protein expression accompanying EVT differentiation is governed
in part by a change in the growth factor and cytokine milieu (Morrish et al., 1998). In addition
this process can be opposed by members of the TGF pathway. Specifically, TGF1 and TGF3
have been found to inhibit trophoblast differentiation along the invasive pathway (Graham and
10
Lala, 1991;Caniggia et al., 1999), through a process that is likely mediated by endoglin, a TGF
receptor expressed by the invading EVT (St Jacques et al., 1994;Caniggia et al., 1997).
Trophoblast cells migrate along the capillary walls and essentially plug the maternal spiral
arteries. These endovascular plugs of EVT regress around the 12th week, to allow perfusion of
the inter-villous space by maternal blood. Meanwhile, EVT continue to invade the uterine wall,
and remodel the spiral arteries, to maximize uteroplacental blood flow (Ashton et al., 2005).
Cytotrophoblast cells infiltrate the arterial walls of the maternal spiral arteries and replace cells
of the maternal endothelium and smooth muscle, producing uterine arteriole walls consisting of
fetal and maternal cells (Figure 1.4). This phenomenon has been replicated in vitro using a first
trimester placental-decidual explant model, that showed endothelial cell and smooth muscle
disruption around the maternal vessels, concomitant with trophoblast invasion (Dunk et al.,
2003). This results in the transformation from muscular, narrow, high-resistance uterine
arterioles to non-muscular, distended, low-resistance vessels that are unresponsive to endocrine
stimuli (Kurman, 1991b). This process is critical to the establishment of a successful pregnancy,
as indicated by the various placental pathologies associated with insufficient spiral artery
remodeling (Kurman, 1991a). This is a topic of high importance and will be elaborated upon in
more detail in Section 1.4 of the Introduction.
1.1.3 The Mature Placenta
The mature placenta is thus composed of a fetal chorionic portion and a maternal endometrial
(decidual) component that make up the fetomaternal junction (Figure 1.6). The fetal chorionic
villi anchor the chorionic sac, containing the fetus, to the decidua through the cytotrophoblastic
shell (Moore and Persaud, 1998). Erosion of the decidua, by the extra villous trophoblast,
generates wedge shaped sections of decidua, left behind to form placental septa. These septa
effectively divide the placenta into segmented areas, referred to as cotyledons. Each cotyledon
contains two or more branched stem villi that float in the maternal blood filled intervillous
spaces, created by fusion of the syncytial lacunae (Figure 1.6). Though the intervillous space is
divided by placental septa, maternal blood in the intervillous space can travel freely between
compartments, as the septa do not reach as far down as the chorionic plate. Maternal blood enters
and exits the intervillous spaces through maternal endometrial arteries and veins that pass
through gaps in the trophoblastic shell (Figure 1.6). The maternal blood, rich in oxygen and
11
Figure 1-6: The mature placenta
The mature placenta is a fully functioning organ that mediates gas and nutrient exchange between maternal and fetal
capillary system. Invasive extra villous trophoblast infiltrate the maternal spiral arteries and remodel their
endothelial lining and muscular wall, transforming them into dilated vessels that promote the unencumbered flow of
maternal blood into the maternal space. Floating villi bathe in the maternal blood where their outer covering of
syncytial material performs the exchange of gas and nutrients between fetal and maternal blood. Diagram modified
from (Moore and Persaud, 1998) with permission.
12
nutrients, enters the intervillous space in pulsatile spurts, generated from the maternal blood
pressure. As the blood enters the intervillous space, the pressure lessens and the blood flows
more gently around the branches of placental villi. Gas and nutrients from the maternal blood
diffuse across the placental membrane of the floating villi towards the placental vasculature,
where they are transported to the growing fetus via the fetal blood stream. The fetal capillaries
therefore come in close contact to the maternal blood, but remain separated by the thin placental
membranes.
An intricate artery-capillary-venous return system is established within each chorionic floating
villous. These arteries and veins merge forming larger vessels until they become the two
umbilical arteries and the umbilical vein that connect the placenta to the embryonic heart of the
fetus. Fetal blood begins to flow through this capillary system by the fourth week, allowing
transfer of oxygen and nutrients from the villous space, across the placental villi to the fetal
capillaries, and carbon dioxide and fetal waste are eliminated from the fetus to the maternal
circulation (Moore and Persaud, 1998).
The main function of the placenta is therefore to ensure sufficient nutrient and gas exchange
between the mother and fetus. Additionally the placenta, mediates proper structural attachment
of the conceptus to the uterine wall, is involved in endocrine secretion, provides immunological
protection to the growing fetus, and remodels the maternal spiral arteries to establish a constant,
unhindered blood flow for nutritional purposes.
1.1.4 Oxygen and Human Placentation
As previously mentioned, one of the most important functions of the placenta is to remodel the
maternal vasculature, to allow a sufficient flow of maternal blood into the placental space, so that
oxygen and nutrients can be extracted for embryonic development. However, prior to
establishment of the utero-placental circulation, early embryonic development takes place in a
relatively low oxygenated environment (Burton et al., 1999). It was originally postulated that
oxygenation of the intervillous space occurs in the fourth week of development, following the
initial vascular remodeling events. However, it is now recognized that the placental environment
remains poorly oxygenated well into the late stages of the first trimester, due to obstruction of
the maternal vessels by the formation of the trophoblastic plugs. This environment of low
oxygenation has been well documented as a critical factor leading to proper early placental
13
development, where oxygen itself acts as a key regulator of a variety of cellular events associated
with trophoblast differentiation (Jaffe et al., 1997). The Caniggia laboratory and others have
reported that low oxygen tension can maintain trophoblast cells in a proliferative, non-invasive,
intermediate phenotype, characteristic of early placental development (Genbacev et al.,
1996;Genbacev et al., 1997;Caniggia et al., 2000;MacPhee et al., 2001), and that markers
associated with an immature/intermediate trophoblast phenotype are elevated in a low oxygen
environment (Caniggia et al., 2000;Caniggia and Winter, 2002). In addition, the expression of
hypoxia-inducible factor-1 (HIF-1), a transcription factor that regulates genes in response to low
oxygen, is elevated during this early gestational time period, where it plays a critical role in
mediating the biological effects of oxygen on early trophoblast differentiation (Caniggia et al.,
2000;Rajakumar and Conrad, 2000;MacPhee et al., 2001;Caniggia et al., 2002;Nevo et al.,
2006;Ietta et al., 2007;Yinon et al., 2008). Sufficient remodeling of the spiral arteries and
dislodging of the trophoblastic plugs occurs late in the first trimester, around the 10th
to 12th
week of gestation. This allows for maternal blood to enter the villous space leading to an
increase in oxygenation, which initiates a switch in the expression of a wide variety of genes and
proteins (Rodesch et al., 1992;Burton et al., 1999). Thus, the placenta undergoes a significant
environmental change in the late first trimester. Oxygen electrode studies have shown that prior
to the opening of the intervillous space (around 5-8 weeks of gestation) oxygen tension is around
20 mmHg, (equivalent to 3% O2), whereas at 14-16 wks, after the intervillous space has opened
to maternal blood, oxygen levels rise to ~55-60 mmHg (8-10% O2) (Rodesch et al., 1992;Burton
et al., 1999). In the late stages of pregnancy, the oxygen tension drops slightly closer to ~40
mmHg as a result of increased oxygen extraction from the growing fetus (Soothill et al., 1986).
The rapid increase of oxygen tension in the first trimester has been associated with increased
expression of genes associated with oxidative stress (Jauniaux et al., 2000) and is believed to
drive trophoblast differentiation and death as well as placental maturation (Boyd and Hamilton,
1970;Genbacev et al., 1996;Genbacev et al., 1997;Caniggia et al., 2000). Thus varying degrees
of oxygenation greatly impact upon the behaviour and differentiation of the trophoblast, however
this phenomenon is often overlooked in studies of placental development.
1.2 Cell Death and the Regulation of Apoptosis in the Placenta
Cell death plays a critical role in multiple aspects of placentation, from the attachment of the
blastocyst to the endometrium and its subsequent implantation (Galan et al., 2000), to maternal-
14
placental tolerance (Abrahams et al., 2004), and remodeling of the spiral arteries (Cartwright et
al., 2002;Ashton et al., 2005). Moreover, the development of the placental tissue relies on the
process of cell proliferation, differentiation and death, as they are the driving forces that
determine the fate of each trophoblast cell. Importantly, during placental development cell death
is thought to occur predominantly through the apoptotic pathway. Apoptosis, otherwise referred
to as programmed cell death, is a highly regulated process of cellular self-destruction, employed
to eliminate unnecessary, dysfunctional, or aged cells, so that normal tissue function can be
maintained. As opposed to cell death by way of necrosis, which is controlled by a defined
molecular pathway, however energy independent and often detrimental to the surrounding tissue;
apoptosis is controlled and uses energy dependent processes to facilitate efficient breakdown of
the cell, while avoiding an acute inflammatory response. Cell death by apoptosis is important in
decidualization and remodelling of the spiral arteries. In addition, aspects of the apoptosis
process are essential to the fusion of the trophoblast in the floating villi, and in the deportation of
the syncytial knots. Huppertz et al. have proposed the concept that, during trophoblast turnover,
the apoptotic cascade is initiated in the villous cytotrophoblast, facilitating cell fusion. Apoptosis
is then repressed within the renewed syncytium to maintain cellular function, and then re-
established during the extrusion of apoptotic nuclei in the form of “syncytial knots” (Huppertz et
al., 1998). Therefore, various aspects of the apoptotic pathway contribute to various
differentiation events throughout placentation. A number of studies have assessed late stages of
cell death by TUNEL staining (a marker of the end stages of apoptosis), and found that while the
incidence of trophoblast cell death in chorionic villi is quite low and primarily restricted to the
syncytial layer during the first trimester, the rate of trophoblast cell death significantly increases
with advancing gestation (Smith et al., 1997;Smith et al., 2000). Following a brief introduction to
apoptosis, and the regulation of the intrinsic pathway, the importance and regulation of apoptosis
in placental development will be discussed.
1.2.1 Classical Function: Apoptosis and the Intrinsic Pathway
Two distinct but converging pathways lead to apoptosis; the extrinsic pathway initiated by
activation of cell death receptors located at the plasma membrane, and the intrinsic pathway
mediated by internal cues reflecting cellular homeostasis that target the integrity of the
mitochondrial membrane (Figure 1.7). Both pathways, governed either by external or internal
15
Figure 1-7: The extrinsic and intrinsic cell death pathway
Signalling of apoptosis through the extrinsic and intrinsic pathways is illustrated. The extrinsic pathway is initiated
by cell death receptors located at the plasma membrane which activate initiator caspases (caspase 2,8,9,10). This
results in the cleavage and activation of downstream effector caspases (caspase 3,6,7) leading to apoptosis. The
intrinsic pathway is governed by pro-apoptotic and anti-apoptotic members of the Bcl-2 family. Internal cues
reflecting cellular homeostasis induce or suppress their expression. Elevated levels of pro-apoptotic molecules (Mtd,
Bax, Bak, tBid are depicted) associate and form pores in the mitochondrial membrane leading to the release of
apoptogenic factors including cytochrome c into the cytoplasm. This event leads to the formation of the apoptosome
and activation of effector caspases. The extrinsic and intrinsic pathway converge following activation of effector
caspases or through caspase 8 activation of tBid. The effect of pro-apoptotic Bcl-2 family members can be
suppressed by their interaction with anti-apoptotic Bcl-2 family members (Mcl-1 is depicted).
16
signals, converge to complete a series of events that define apoptosis. Hallmarks of apoptosis
include nuclear condensation, DNA fragmentation, blebbing of the plasma membrane, and cell
shrinkage into dense apoptotic bodies (Straszewski-Chavez et al., 2005). These cellular changes
result largely from the activation of “executioner” or “effector” caspases, a family of cysteine-
aspartate proteases that cleave specific consensus sites on selected target proteins, to facilitate the
characteristic events of apoptosis.
The caspase family can be divided into initiator (caspases -2,8,9,10) and effector (caspases -
3,6,7) members (Degterev et al., 2003), each of which is synthesized as an inactive proenzyme
that is activated upon its cleavage. Initiator caspases are activated by an allosteric mechanism,
facilitated by dimerization or oligomerization. Once activated these members function primarily
in the cleavage and activation of downstream effector caspases, thereby initiating what is known
as the caspase cascade (Degterev et al., 2003;Fuentes-Prior and Salvesen, 2004) (Figure 1.7).
Effector caspases then cleave further procaspases, creating a feed forward cycle, as well as
targeting a variety of vital cellular proteins (Timmer and Salvesen, 2007), resulting in cell
demise.
In addition to caspases, the most widely studied molecules involved in the intrinsic pathway of
apoptosis are members of the bcl-2 family (Figure 1.8). Members of the Bcl-2 family function
predominantly to regulate the integrity of the mitochondrial membrane. This family includes
both pro-apoptotic and anti-apoptotic members that function to either promote the formation of
pores in the mitochondrial membrane, or to prevent pore-formation, respectively. These family
members are identified by the presence of a Bcl-2 homology (BH) domain, of which there are
four (BH1-4). More than 20 members of this family have now been recognized (Gross et al.,
1999), each member containing at least one BH domain (Figure 1.8). These family members are
subdivided in to three groups based on their function and number of BH domains. The anti-
apoptotic or „cell death suppressors‟ (Bcl-2, Bcl-xL, Mcl-1, A1) contain four BH domains
whereas the cell death inducers contain either multiple BH domains (Bax, Bak, and Mtd/Bok) or
a single (BH3-only) domain (Hrk, Bim, Bad, Bik). Importantly, it is the BH3 domain that allows
these family members to interact with one another. Members of this gene family act through a
complex network of promiscuous homo- and hetero-dimers with the exception of Mtd, which
interacts almost exclusively with Mcl-1 (Hsu et al., 1997).
17
Figure 1-8: BCL-2 family members
The three categories of Bcl-2 family members represented by the more well known family members are depicted.
The anti-apoptotic or „cell death suppressors‟ (Bcl-2, Bcl-xL, Bcl-W, Mcl-1, A1) contain four Bcl-2 homology
regions (BH1, purple; BH2, dark blue; BH3, green; BH4, light blue) whereas the cell death inducers contain either
multiple BH domains (Bax, Bak, and Mtd/Bok) or a single (BH3-only) domain (Bid, Bad, Bik, Bim, BNip3, Nix).
Importantly, it is the BH3 domain that allows these family members to interact with one another. In addition many
of the family members possess a hydrophobic carboxy-terminal transmembrane domain (TM, pink) that allows them
to bind to intracellular membranes.
18
The anti-apoptotic members localize to the mitochondrial outer membrane where they protect
against pore formation and leakage of apoptotic inducing factors. Activation of the pro-apoptotic
members, which contain a pore-forming region, are believed to target the mitochondria, causing
release of apoptotic factors which, in turn, results in the activation of the caspase cascade
(Figure 1.7). In contrast, the BH3-only proteins confer their pro-apoptotic function by activating
pro-apoptotic Bax and Bak directly or via neutralizing anti-apoptotic family members. The
balance between pro- and anti-apoptotic molecules thus regulates cell death by controlling the
permeability of the mitochondrial membrane (Figure 1.7).
Depolarization of the mitochondrial membrane results in the release of apoptogenic factors
including cytochrome c (Yang et al., 1997;Brunelle and Chandel, 2002), Smac/Diablo (Du et al.,
2000), and apoptosis inducing factor (AIF) (Susin et al., 1996). Together, with ATP and
apoptosis inducing factor-1 (Apaf-1), these molecules aid in the recruitment of initiator caspase 9
and the formation of the “apoptosome”. Dimerization and allosteric activation of caspase-9
occurs at the apoptosome and consequently leads to caspase-9 mediated activation of the effector
caspases 3, 6 and 7. Protease function of the effector caspases results in cleavage of nuclear
lamins, DNA repair enzymes, and cytoskeletal proteins leading to the events culminating in
cellular apoptosis (Figure 1.7).
1.2.2 Apoptosis and the Bcl-2 Family in Placentation
As mentioned earlier, a variety of processes necessary for proper placentation involve aspects of
apoptosis. Implantation, maternal immune tolerance, and spiral artery remodeling require
trophoblast mediated apoptosis of surrounding cells of the endometrium and decidua (Cartwright
et al., 2002;Ashton et al., 2005). These events are thought to result from trophoblast cell
expression of Fas ligand (FasL), a peptide that interacts with the Fas receptor to stimulate
apoptosis through the extrinsic pathway. The Fas receptor is expressed by endothelial cells of the
endometrium and the villous vessels, as well as maternal T lymphocytes, resulting in trophoblast
mediated apoptosis of these cell types when the two come into contact (Ashton et al., 2005).
Studies conducted in vitro have shown through use of a co-culture system that unmodified (not
placental bed) spiral arteries obtained from cesarean sections co-cultured with extra villous
trophoblast cells exhibited a loss of the endothelial layer. This was associated with signs of
caspase cleaved PARP (poly ADP-ribose polymerase), an apoptosis marker, and could be
19
prevented by pretreatment with inhibitors of caspase or FasL (Ashton et al., 2005;Cartwright and
Wareing, 2006). Destruction of these cells allows for trophoblast migration into the uterine wall
and spiral arteries and inhibits an immune response from maternal leukocytes so that pregnancy
can continue unscathed.
In contrast, apoptotic events that take place within the placenta are thought to occur through a
process that involves regulators of the intrinsic cell death machinery (Huppertz et al.,
1998;Potgens et al., 2002). As previously mentioned, the process of trophoblast turnover is
believed to involve a multi-step cascade of apoptotic events that begin as the cytotrophoblast
prepares for fusion with the overlying syncytium, and is temporarily repressed within the
syncytial layer before its completion during syncytial knot formation and deportation (Huppertz
et al., 1998). Furthermore, it has been determined that members of the Bcl-2 and caspase family
are key mediators of these events (Huppertz et al., 1998;Ray et al., 2008;Heazell and Crocker,
2008a).
Trophoblast cell fate in the floating villi appears to be regulated at different stages by various
members of the Bcl-2 or caspase family. The initial stages of apoptosis occur in the
cytotrophoblast layer, as demonstrated by the expression of the initiator caspase 8 and cleavage
of a-fodrin (a cytoskeleton protein), as well as by the externalization of phosphatidylserine (an
aminophospholipid) from the inner to the outer leaflet of the plasma membrane (Huppertz et al.,
1998;Huppertz et al., 1999). Although these events are markers of early apoptosis, their
expression at this point may play more of a role in trophoblast differentiation then in cell death.
Studies indicate that caspase 8 activity and phosphatidylserine externalization are important for
the fusion of cytotrophoblast cells to the syncytium, as inhibition of either phosphatidylserine or
caspase-8 activation (Black et al., 2004) reduces syncytial fusion in vitro. Other apoptotic
molecules have also been found to be expressed in the cytotrophoblast layer including p53, Bax
and Mtd which may contribute to the process of trophoblast differentiation (Ratts et al.,
2000;Soleymanlou et al., 2005b).
Once the cellular material enters the syncytium further progression of the caspase cascade is
prevented until the extrusion period. Cytoplasmic expression of Mcl-1 in the cytotrophoblast and
syncytium (Huppertz et al., 1998), and Bcl-2 expression primarily in the syncytiotrophoblast
(Ratts et al., 2000;Axt-Fliedner et al., 2001;Danihel et al., 2002), have been postulated to protect
20
against final execution stages of apoptosis (Huppertz et al., 1998;Danihel et al., 2002).
Consistent with this hypothesis is the finding that the expression of Bcl-2 and Mcl-1 is absent
from syncytial knots displaying TUNEL positivity (Huppertz et al., 1998), and the observation
that both Mcl-1 and Bcl-2 levels are elevated during early placentation when trophoblast
apoptosis is minimal (Huppertz et al., 1998).
As the syncytial material ages, portions prepare for the final stages of apoptosis and deportation
of the apoptotic material in the form of membrane sealed fragments termed syncytial knots,
marking the final stages of trophoblast apoptosis (Yasuda et al., 1995;Huppertz et al., 1998).
Cytoplasmic expression of pro-apoptotic Bax and Bak has been found in discrete areas of the
syncytium primarily around regions associated with syncytial knot formation and fibrin
deposition (Ratts et al., 2000). Levels of Bax and Bak, however, have been found to be
consistently low throughout gestation suggesting that they may not be the principal molecules
driving apoptosis in the human placenta (Ratts et al., 2000;De Falco et al., 2001). As pregnancy
progresses the incidence of trophoblast apoptosis increases and by the third trimester as much as
3 grams of syncytial material is shed into the maternal circulation a day (Huppertz et al., 1998).
Efficient clearing of the foreign material, by the maternal immune system, is a natural part of
pregnancy that prevents systemic endothelial cell activation and maintains maternal health
(Redman and Sargent, 2003). However, in some patients the maternal immune system is not
capable of coping with the syncytial debris produced. This can lead to the placental pathology of
preeclampsia (discussed in Introduction 1.4). Furthermore, it has been proposed that in severe
placental pathologies the mode of cell death may change from one of apoptosis towards that of
necrosis, a process referred to as aponecrosis (Huppertz et al., 2003).
Oxygen status has also been shown to influence cell fate of the trophoblast. A low oxygen
environment, such as that experienced in the early first trimester, is associated with increased
trophoblast proliferation and retention of trophoblast cells in the cytotrophoblast layer due to a
lack of trophoblast fusion (Huppertz et al., 2003). In cases of severe hypoxia, or oxidative stress,
poor ATP production may push the regulated process of apoptosis to that of aponecrosis
(Huppertz et al., 2003). Hypoxia has also been shown to increase Bax expression and lower Bcl-
2 expression in cultured first trimester cytotrophoblast cells in some studies, whereas other
studies have found increased p53 and mdm2 under hypoxic conditions, but no alteration in the
expression of either Bax or Bcl-2 (Hu et al., 2006;Heazell et al., 2008b). The Caniggia laboratory
21
has shown that Mcl-1 increases in physiological low oxygen conditions (3% O2) and that this
molecule can protect against Mtd-mediated apoptosis (Soleymanlou et al., 2007). Under
conditions of oxidative stress however, Mcl-1 is cleaved to a pro-apoptotic molecule
(Soleymanlou et al., 2007), underscoring the importance of the oxygen status in the human
placenta. Furthermore, the Caniggia lab has identified a novel Mtd splice variant, abundantly
expressed by the human placenta (Soleymanlou et al., 2005b;Soleymanlou et al., 2007), and
shown that the balance between the anti-apoptotic Mcl-1 and the pro-apoptotic Mtd, is dependent
upon oxygen, and is pivotal in determining placental homeostasis (Soleymanlou et al., 2007).
1.2.3 Mtd in Placental Apoptosis
Mtd/Bok (Mtd: Matador/Bok: Bcl-2-related ovarian killer) is a pro-apoptotic multi-domain pore-
forming member of the Bcl-2 family (Hsu et al., 1997). Mtd mRNA is alternatively spliced and
encodes for three protein isoforms Mtd-L, Mtd-S, and a third placental specific isoform Mtd-P
(Figure 1.9), which has been characterized in normal placental development, as well as in
placental tissue from pregnancies complicated by preeclampsia (Hsu et al., 1997;Soleymanlou et
al., 2005b). Unlike Bax and Bak, Mtd-L and Mtd-P are expressed at very high levels in the
placenta and in tissues of reproductive origin (Hsu et al., 1997;Soleymanlou et al., 2005b)
suggesting that Mtd may be the primary pro-apoptotic Bcl-2 family member regulating
trophoblast apoptosis. Soleymanlou et al previously reported that Mtd is localized to the
syncytial knots in first trimester of human placentae where it is associated with trophoblast
apoptosis and localized to the cytotrophoblast layer and extra villous trophoblast cells where it
may have a regulatory role in trophoblast differentiation (Soleymanlou et al., 2005b).
Similarly to Bax, all isoforms of Mtd contain three BH domains and a transmembrane domain
which are believed to facilitate pro-apoptotic activity via mitochondrial depolarization
(Soleymanlou et al., 2005b) (Figure 1.9). This has been further supported by the finding that
overexpression of Mtd-L or Mtd-P in CHO cells leads to mitochondrial depolarization and
subsequent cleavage and activation of the caspase cascade (Soleymanlou et al., 2005b). It is
suggested, that due to disruption in the BH3 domain of Mtd-S and Mtd-P that occurs as a
consequence of splicing, neither isoform can interact with other Bcl-2 family members. It is
therefore likely that Mtd-S and Mtd-P exert their apoptotic function primarily by forming pores
directly in the mitochondrial membrane, facilitating release of various apoptogenic proteins,
22
Figure 1-9: Mtd isoforms
The three isoforms of Matador (Mtd) are illustrated. Top panel depicts the human chromosomal structure and
transcript maps of the Mtd isoforms. The full length isoform (Mtd long, Mtd-L) consists of five exons. Mtd-S is a
short isoform resulting from exon 3 skipping, whereas the placental specific isoform (Mtd-P) arises from exon 2
skipping. Figure adapted by permission from Macmillan Publishers Ltd: Cell Death and Differentiation,
Soleymanlou et al, copyright 2005. The protein domains are depicted in the lower panel. Of note, all three isoforms
maintain the pore forming region encoded by exon 4. Only Mtd-L maintains an intact BH3 domain required for
member to member interaction.
23
whereas Mtd-L likely mediates its apoptotic activity by antagonizing the function of the anti-
apoptotic Mcl-1, in addition to targeting the mitochondria (Hsu et al., 1997;Soleymanlou et al.,
2005b). Similarly to Mcl-1, low oxygen tension stimulates the expression of Mtd-L and Mtd-P,
which may explain their increased expression in the early stages of placentation and their
increased expression in placental pathologies. As previously mentioned, Mtd expression is high
during early placental development (Soleymanlou et al., 2005b) and is localized to the dynamic
cytotrophoblast and extra villous trophoblast layers. As this gestational period and site of
expression are characterized by intense trophoblast cell proliferation and little trophoblast cell
death, it is likely that Mtd, in addition to its classical role in apoptosis, may have a function in
regulating other areas of trophoblast cell fate, such as trophoblast proliferation.
1.3 Regulation of the Cell Cycle in the Placenta
1.3.1 The Cell Cycle
Adequate proliferation and differentiation of the trophoblast cells is necessary to establish
successful growth of the placenta. Proliferation includes a specific set of events, coordinated
precisely, to progress the cell cycle forward through G1, S, G2 and M phases (Figure 1.10).
During the G1 phase the cell senses and responds to environmental cues prompting the cell to
begin cycling, it then grows duplicating its organelles and preparing for DNA synthesis. During
this initial phase the cell passes a restriction point, after which mitogen stimulation is no longer
required for the cell to continue cycling. This is effectively a point of no return whereby the cell,
from this point onward, is committed to completion of cell division, or will undergo cell death
(Sherr, 1994). At the end of the G1 phase the cell passes the G1/S checkpoint and enters the S
phase where the cell undergoes DNA synthesis. This is followed by progression to the G2 phase
where the integrity of the replicated DNA is assessed before crossing the G2/M checkpoint.
Once in M phase the chromosomes segregate and the cell physically divides. The two most
important decision points therefore occur during the G1 phase: 1) the decision to begin the
cycling process and 2) the decision to commit to cell division (Sherr, 1994). Not surprisingly,
control of the cell cycle occurs largely during the G1 phase, and has subsequently become a
classic target in malignant transformation.
As shown in Figure 1.10, the transition through each stage of a typical mammalian cell cycle is
governed by cyclin dependent kinases (CDKs) that are regulated by specific cyclins (activators)
24
Figure 1-10: The cell cycle
A schematic of the cell cycle is depicted. Mitogenic cues stimulate cyclin D expression to initiate entry into the G1
phase. The D type cyclins then activate cyclin dependent kinases 4 and 6 (CDK4/6) which phosphorylate Rb. Rb is
further phosphorylated by cyclin E activated CDK2. Phosphorylation of Rb prevents its interaction with the E2F
transcription factor leading to E2F dependent gene transcription and entry in to the S phase. Cyclin A in complex
with CDK2 drives cells through the S phase in conjunction with PCNA. Entry into the G2 phase is associated with
cyclin A-CDK1 interaction which is followed by a switch to cyclin B- CDK1 interaction as the cell transitions from
the G2 to M phase. Interaction between cyclin B and CDK1 is then maintained and drives cells through the M phase.
The cell cycle is inhibited by INK4 (p15, p16, p18, and p19) and the Cip/Kip (p21, p27, and p57) family. INK4
(inhibitor of CDK4) family members bind CDK4 and CDK6 to inhibit the G1-S transition by preventing their
association with D type cyclins, while Cip/Kip proteins bind a variety of cyclins, CDKs or cyclin bound CDKs, and
can inhibit the cell cycle at various points.
25
and CDK inhibitors (CDKIs: repressors). Expression of the CDKs remains relatively constant
over the cell cycle period, whereas cyclin expression is phase specific (Sherr, 1994). The phase
sensitivity of the cyclins, in combination with their short half life (approximately 10-25 minutes),
assures that progression of the cell cycle is tightly controlled (Sherr, 1994). Entry into the G1
phase is accompanied by the expression of D type cyclins (D1, D2, D3) known as growth factor
sensing cyclins (Figure 1.10). Their expression is initiated in response to mitogen stimuli,
effectively linking the activation of the cell cycle with environmental cues (Sherr, 1995).
Independently, each D type cyclin can bind to and allosterically regulate one of two CDK
subunits, CDK4 or CDK6 (Morgan, 1997). Assembly of D cyclins into cyclin D-CDK(4/6)
complexes occurs in the cytoplasm through a process mediated by the interaction with a Cip/Kip
protein (Labaer et al., 1997). This interaction is believed to stabilize the complex and aid in its
translocation from the cytoplasm to the nucleus, as neither cyclin D nor CDK4/6 contains its own
nuclear translocation sequence (Labaer et al., 1997;Cheng et al., 1999). Once nuclear the kinase
function of the CDK is activated, and phosphorylation of its target proteins begins. Shortly
thereafter, cyclin E (E1 or E2) is expressed and forms complexes with CDK2, to promote the G1
to S transition (Dulic et al., 1992;Koff et al., 1992) (Figure 1.10).
Both cyclin D and cyclin E activated CDKs target the retinoblastoma family of proteins (pRb,
p107 and p130) which function to inhibit the cell cycle by binding to members of the E2F family
(Dyson, 1998). The E2F family is a group of transcription factors that allow for transcription of
genes important in G1-S transition and required for DNA synthesis (Dyson, 1998;Nevins, 1998).
Phosphorylation of Rb is a multi-step process that is initiated by the cyclin D dependent kinases
and completed by cyclin E-CDK2 complexes (Sherr, 1994;Lundberg and Weinberg, 1998). Once
sufficiently phosphorylated, Rb is released from E2F which allows for E2F-dependent gene
transcription (Adams, 2001) and entry in to the S phase (Figure 1.10).
Importantly, E2F initiates transcription of cyclin E, resulting in a positive feedback loop and
ensuring the irreversible commitment to passage through the G1-S checkpoint (Knoblich et al.,
1994;Geng et al., 1996). Cyclins A and B become important in later stages of the cycle by
promoting G1/S (cyclin A) and G2/M transition (cyclins A and B) (Bailly et al., 1992;Sherr,
1996). Cyclin A in complex with CDK2 drives cells through the S phase in conjunction with
PCNA (proliferating cell nuclear antigen), a protein involved in DNA replication and widely
used as a marker of cell proliferation (Figure 1.10). Entry into the G2 phase is associated with
26
cyclin A-CDK1 interaction which is followed by a switch to cyclin B- CDK1 interaction, as the
cell transitions from the G2 to M phase. Interaction between cyclin B and CDK1 is then
maintained and drives cells through the M phase (Figure 1.10) (Sherr, 1996). Additionally,
Ki67, a protein of unknown function, is expressed throughout the active phases of the mitotic
cell cycle, and has become a widely used marker of proliferation (Endl and Gerdes, 2000).
Although D and E cyclins both target the retinoblastoma family, they have many different
properties. Whereas, specific cell types often express different D-type cyclins and either CDK4
or 6, cyclin E1,E2 and CDK2 expression is not cell type specific (Geng et al., 2001).
Furthermore, unlike the mitogen mediated expression of D type cyclins, expression of cyclin E is
controlled by an autonomous mechanism and peaks sharply at the G1/S border (Dulic et al.,
1992;Koff et al., 1992). Additionally, cyclin E-CDK2 complexes have wider substrate
specificities than the cyclin D dependent kinases that primarily target Rb. These include
phosphorylation of Rb, histone H1, p27 and a variety of other proteins (Sherr and Roberts,
2004;Moroy and Geisen, 2004). Therefore, once activated, cyclin E-CDK2 complexes not only
complete the deactivation of Rb to initiate E2F regulated gene transcription, but they also initiate
a series of additional processes including DNA replication, centrosome duplication, and histone
biosynthesis. (Sherr et al., 2004;Moroy et al., 2004) (Figure 1.11)
Cyclins D and E have also been found to function in processes that are independent of their
catalytic functions. For example, cyclin D-CDK complexes also bind and sequester CDK
inhibitors, freeing the cyclin E-CDK2 complexes from their inhibition (Sherr and Roberts, 1995),
and cyclin E has been found to be required for the assembly of the pre-initiation complex at
origins of DNA replication in quiescent cells entering the cell cycle (Coverley et al., 2002) and in
the process of endoreduplication (MacAuley et al., 1998;Parisi et al., 2003) (Figure 1.11).
1.3.2 Cell Cycle Inhibitors
In mammalian cells, the cell cycle is inhibited by two groups of CDK inhibitors, the INK4 (p15,
p16, p18, and p19) and the Cip/Kip (p21, p27, and p57) family. INK4 (inhibitor of CDK4)
family members bind CDK4 and CDK6 to inhibit the G1-S transition by preventing their
association with D type cyclins, while Cip/Kip proteins bind a variety of cyclins, CDKs or cyclin
bound CDKs, and can inhibit the cell cycle at various points (Sherr and Roberts, 1999) (Figure
1.10).
27
Figure 1-11: Function of cyclin E1
A schematic of the CDK2 dependent and independent functions of cyclin E1. Cyclin E1 activates CDK2
phosphorylation of Rb, CDC25A phosphatase, and p27. This leads to G/S phase transition and a feed forward loop.
The cyclin E1 activated kinase activity of CDK2 is also involved in DNA replication, centrosome duplication, and
histone biosynthesis. CDK2 independent functions of cyclin E1 include roles in endoreduplication, re-entry from
G0-G1, and malignant transformation.
28
P21, originally discovered as a CDK inhibitor and as a mediator of p53 tumor suppression, is
now known to be involved in numerous roles geared toward regulating cell viability, including
cell cycle arrest in response to DNA damage, promotion of differentiation and enforcement of
cellular senescence. The cell cycle arrest function of p21 is reliant on its nuclear expression
where it binds to CDKs. Additionally p21 can bind and prevent the function of PCNA, a protein
required for DNA synthesis, thereby inhibiting cell cycle progression (Sherr et al., 1999).
In addition to its anti-proliferative functions, p21 can also act in a cell cycle promoting manner.
p21 mediates cyclin D-CDK assembly and aids in the transport the complex from the cytoplasm
into the nucleus (Labaer et al., 1997;Cheng et al., 1999;Sherr et al., 1999). The incorporation of
p21 into the cyclinD-CDK4/6 complex does not prevent the kinase activity, but instead further
stabilizes the cyclin-CDK complex within the nucleus, preventing its translocation back to the
cytoplasm, and thus promoting the progression through the G1 phase. Additionally, p21 can also
promote cell viability through an anti-apoptotic mechanism which is achieved by the binding of
p21 to caspase-3 and preventing its activation (Suzuki et al., 1998;Asada et al., 1999).
The p21 related protein, p27 was originally discovered as the mediator of G1 arrest induced by
TGF- or contact inhibition (Polyak et al., 1994). Although p27 can bind and inhibit a number of
CDKs, it was found to preferentially bind to cyclin E/CDK2, inhibiting the CDK2 activity by
interfering with the catalytic cleft and preventing ATP binding (Russo et al., 1996). In addition,
p27 has been linked to the maintenance of cell quiescence associated with mitogen starvation,
and in a number of cell types it is down-regulated upon growth factor stimulation (Sgambato et
al., 2000;Besson et al., 2006). Like p21, p27 can also aid in complex formation of D-type cyclins
with CDK4/6 (Sgambato et al., 2000). This interaction functionally sequesters the Cip/Kip
inhibitors, and subsequently frees the cyclin E-CDK2 complexes from their inhibition, allowing
CDK2 activity to occur. In contrast mitogen withdrawal leads to decreased cyclin D expression,
thereby freeing p21 and p27, enabling them to inhibit cyclin E-CDK2 and arrest the cell cycle
(Sherr et al., 1999). Additional functions of p27 continue to be discovered. Over the past few
years studies have revealed a role for p27 in cell differentiation, cell migration, transcriptional
regulation and apoptosis (Besson et al., 2008) (Figure 1.12).
29
Figure 1-12: Regulation and function of p27
The function and stability of p27 is regulated by subcellular localization, protein interaction and phosphorylation
status. p27 interacts with CDKs in the nucleus to prevent cell cycle progression. P27 is exported to the cytoplasm
following Ser10 phosphorylation and interaction with the exportin CRM1. In the cytoplasm p27 can interact with D
type cyclins and CDKs and aid in cyclin D-CDK assembly. Alternatively p27 can function in cell migration through
its interaction with RhoA. Phosphorylation of p27 at alternative sites can lead to either its stability or degradation by
promoting the interaction of p27 with various protein complexes. Known phosphorylation sites are depicted by the
colored circles.
30
1.3.2.1 Regulation by Phosphorylation
The function and stability of p21 and p27 is highly regulated by subcellular localization, protein
interaction and phosphorylation status. The cell cycle inhibitory role of both p21 and p27 occurs
in the nucleus of the cell through interaction with CDKs, but many of their secondary functions
occur in the cytoplasm. For example the cyclin D-CDK assembly role occurs in the cytoplasm
(Sgambato et al., 2000).
Modulation of the p27 phosphorylation status can modify the protein binding domains leading to
changes in binding partners, and can alter the subcellular localization by revealing or hiding
nuclear localization and export sequences (Figure 1.12). Phosphorylation of p27 on Tyr
(74/88/89) alters the ability of p27 to interact and inhibit CDK2 whereas phosphorylation of p27
at Th187 by CDK2 provides a recognition motif for E3 ubiquitin ligases and targets p27 for
ubiquitination and proteosomal degradation, thus allowing further cyclinE-CDK2 complexes to
be activated (Besson et al., 2008). Subcellular localization of p27 is regulated by
phosphorylation at Ser10, Thr157 or Thr198. Phosphorylation at Ser10, the most common site of
p27 phosphorylation, reveals a binding site for CRM1/exportin, promoting p27 export from the
nucleus to the cytoplasm (Rodier et al., 2001;Connor et al., 2003;Besson et al., 2006). Further
phosphorylation of p27 at Thr157 or Thr198 site interferes with the nuclear localization sequence
effectively averting nuclear entry while concomitantly supporting its association with 14-3-3
which provides additional protein stability (Fujita et al., 2002;Sekimoto et al., 2004). P21 is
equally influenced by phosphorylation. Phosphorylation at various sites modulates the ability of
p21 to interact with cyclin-CDK complexes, PCNA, ubiquitination machinery and the
proteosome. In addition, phosphorylation of p21 can lead to its cytoplasmic translocation and
retention (Child and Mann, 2006).
1.3.2.2 Cell cycle regulators in cancer
Perturbation in p21 and p27 expression and function has been linked to several pathologies
associated with hyper-proliferation, including numerous cancers. Surprisingly however,
mutations of either p21 or p27 in human cancers are very rare (Slingerland and Pagano, 2000).
Knock out mouse models have verified that p21 and p27 have tumor suppressor roles with loss
of either inhibitor resulting in a predisposition to tumourogenesis. Surprisingly, overexpression
31
of cytoplasmic p21 or p27 is also a marker of poor prognosis for many cancers (Slingerland et
al., 2000;Besson et al., 2008). It has been suggested that increased p21 and p27 in the nucleus
may have tumor suppressive function by interacting with CDK, but that increased expression in
the cytoplasm independent of CDKs may be oncogenic (Besson et al., 2008). Additionally,
nuclear p21 has also been found to be tumor promoting by increasing the levels of cyclin D1 in
the nucleus (Liu et al., 2007).
Overexpression of D and E type cyclins also commonly occurs in many human cancers, where
expression is believed to accelerate G1 progression (Sherr, 1996). In contrast, cells lacking D
and E type cyclins are resistant to oncogenic transformation, as they have decreased ability to re-
enter the cell cycle from quiescence, and have difficulty responding to mitogenic stimulation
(Sherr et al., 2004;Kozar et al., 2004).
Although a great deal is known about general cell cycle regulation little is known regarding how
the cell cycle is regulated in the placenta: Which regulators are expressed and with which
functions are they associated? Which proteins interact, and how do these events change with
placental maturation? These questions are of great importance to our understanding of
placentation and even more important as a basis in which to compare placental pathologies in
which proliferation is altered.
1.3.3 Regulation of Proliferation and the Cell Cycle in the Placenta
Proliferation of the trophoblast during placental development is highly organized, being
restricted to the single layer of cytotrophoblast cells in the floating villi, and concentrated in the
proximal portion of the anchoring columns (Figure 1.4).
Progenitor cytotrophoblast cells in the floating villi proliferate, and give rise to daughter cells
that either aid in expansion of the chorionic villi during early placental development, or
differentiate and fuse with the outer syncytium to replenish the materials of the aging outer layer.
Exponential growth of the placenta early in the first trimester requires that a large percentage of
the cytotrophoblast population maintain their proliferative capacity. As gestation progresses the
rate of placental growth slows and the ratio of proliferative cytotrophoblast cells in the placenta
decreases, remaining predominantly to replenish the overlying syncytium of the floating villi and
allow for continual trophoblast turnover.
32
In the anchoring villi, proliferation of the trophoblast is critical to the establishment of extensive
cell columns that effectively anchor the placenta to the maternal wall. Cell cycle regulation in
this compartment is therefore extremely important, as an adequate number of EVT cells must be
produced and differentiate to become invasive, as to ensure sufficient spiral artery remodeling.
Interestingly, acquisition of an invasive phenotype is associated with a switch from a mitotic to
an endoreduplicative phenotype (Sherr, 1996;Zybina et al., 2004), a form of cell cycling that
entails continuous multiplication of the genome in the absence of mitotic division, resulting in a
polyploid cell. This process has been shown to be regulated in part by the E type cyclins in a
process that is CDK independent (Sherr, 1996;Geng et al., 2003;Parisi et al., 2003). Genomic
content in human EVT cells, found deep in the deciduas, can reach up to 8c-16c, and, in mice,
the DNA content of invasive trophoblast giant cells can reach up to 1000N (Barlow and
Sherman, 1972;Zybina et al., 2002). The occurrence of endoreduplication has been shown to be
an essential aspect of placentation, as defects in the ability of the trophoblast to endoreduplicate
in mice (a phenotype associated with cyclin E null mice) lead to placental associated embryonic
lethality (Geng et al., 2003;Parisi et al., 2003). Although the functional relevance of reaching a
polyploid state is not well understood, it has been proposed that endoreduplication acts as a
mechanism to allow sufficient gene transcription and assure that metastatic transformation is
prevented during normal pregnancy (Zybina and Zybina, 2005).
Abnormalities in cell proliferation in either the floating or anchoring villi can lead to improper
placental function and clinical aspects of disease. Excessive proliferation in the floating villi can
enhance the rate of trophoblast turnover and subsequently increase the amount of syncytial
material entering the maternal blood stream. Elevated levels of placental debris in the maternal
circulation have been shown to be a significant contributor to the pathogenesis of preeclampsia
(Levine et al., 2004). Likewise insufficient production of villous cytotrophoblast can lead to a
deficiency in syncytial renewal and impact upon the capacity of the syncytium to uptake the
necessary gas and nutrient requirements of the growing fetus, a phenomenon associated with
intrauterine growth restriction (IUGR).
In the anchoring villi the balance between cell proliferation and trophoblast differentiation is also
critical. Decreased proliferation, premature differentiation or arrest of the EVT in an immature
state may result in an insufficient number of the migratory and invasive trophoblast cell type,
resulting in shallow invasion of the endometrium and consequently relatively few trophoblast
33
cells reaching the spiral arteries. Poor remodeling of the spiral arteries prevents the influx of
oxygen needed for proper development, a feature common to a number of placental pathologies,
including preeclampsia and IUGR. On the other hand, if proliferation and differentiation of the
extra villous trophoblast occur in excess, an increased degree of invasiveness can prevail, a
critical factor in the pathogenesis of placental acretia, molar pregnancy and choriocarcinomas.
The trophoblast biology in placental pathology will be further discussed in section 1.4 and
section 1.5 of the Introduction.
Change in the proliferative status of the placenta is driven largely by the environmental
surroundings experienced by the placenta over gestation. In the first trimester, the low oxygen
environment promotes trophoblast proliferation (Genbacev et al., 1996;Burton, 2009).
Proliferation is also supported by growth factors, and cytokines including vascular endothelial
growth factor, epidermal growth factor, Activin, and TGF3 among others. Elevated oxygen
levels, such as that experienced by the placenta in the late first trimester, and transcription factors
such as glial cell missing 1 (GCM1) inhibit villous and extra villous trophoblast proliferation and
promote differentiation (Baczyk et al., 2009). Although the oxygen and growth factor milieu are
capable of promoting or inhibiting the proliferative capacity of the trophoblast, little is known
regarding the specific cell cycle machinery targeted by these factors.
Although a number of papers have reported on the expression of cell cycle regulators in the
placenta, the studies have been based primarily on immunohistochemical analysis and have
resulted in a number of conflicting findings. To date, it has been reported that cyclin D1, cyclin
D3, cyclin E1, p21 and p27 are expressed by the cells of the placenta, and that their expression
often changes between the 1st and 3
rd trimester. In contrast it was found that cyclin D2 was not
expressed by the trophoblast (DeLoia et al., 1997). The localization of the individual molecules
and the pattern by which their expression changes over development remains ill defined.
For example DeLoia 1997 reported that expression of cyclin D1 was exclusive to the villous core
and to the extravillous trophoblast and that its expression was increased with advancing
gestation; whereas others have reported that cyclin D1 expression is predominant in the
cytotrophoblast layer and that cyclin D1 expression decreases towards term (DeLoia et al.,
1997;Genbacev et al., 2000;De Falco et al., 2004). Similarly cyclin E1 was shown to be
expressed predominantly in the cytotrophoblast layer in two reports, but in the
34
syncytiotrophoblast layer by another study (DeLoia et al., 1997;Bamberger et al.,
1999;Genbacev et al., 2000). Conflicting studies regarding subcellular localization of protein
expression have also been reported. For example, cyclin D3 was observed as cytoplasmic in a
study by Genbacev et al and nuclear by De Loia et al. Since contradictory observations have
been made for p21 and p27 where their expression was reported in the either the CT and EVT or
in the ST (Bamberger et al., 1999;Genbacev et al., 2000;Korgun et al., 2006). These
contradictions may be resolved by increased samples sizes and the use of multiple analytical
methods in future studies.
To date the majority of studies examining the regulation of the cell cycle in the placenta have
been based on immunohistological derived data. Quantitative assessment of mRNA and protein
expression is necessary to better comprehend cell cycle regulation in the placenta. In addition it
would be of value to examine multiple molecules in the same study to gain a more complete
understanding of the dynamics of the G1 phase in placental development. The balance between
the cyclins and the CDK inhibitors is critical to the decision of cell fate. It would therefore be of
great importance to determine the relationship between the cyclins and inhibitors with respect to
their co-expression and interaction in the trophoblast cells. A more in depth analysis, linking
mRNA, relative protein expression, localization, protein interaction, and environmental
influence, in a multi-parametric analysis, discriminating between early and late first trimester
may clarify the current knowledge and provide a better understanding of how proliferation and
the G1 phase of the cell cycle are governed in the human placenta.
1.3.4 Role of Bcl-2 Family Members in Cell Fate
Recent evidence has revealed a functional role for several Bcl-2 family members in regulating
cell cycle progression. Overexpression of anti-apoptotic multi domain members, Bcl-2, BCL-xL,
and BCL-w, has been shown to inhibit passage through the cell cycle by arresting cells at G0/G1
(inhibiting cell cycle entry) and by delaying the progression to S phase (prolongs G0 or G1)
(Jamil et al., 2005;Zinkel et al., 2006). Furthermore, this effect has been associated with
increased levels of p27. The mechanism linking Bcl-2/Bcl-xL to p27 overexpression has not
been elucidated; however it has been hypothesized that it may be a result of caspase inhibition
(Zinkel et al., 2006). The pro-survival Mcl-1 is also anti-proliferative; however, unlike Bcl-2 or
Bcl-xL, its cell cycle function is manifested in the S and G2 phases. Mcl-1 can bind and inhibit
35
PCNA or enter the nucleus and bind CDK1 thereby preventing its activity (Fujise et al.,
2000;Jamil et al., 2005). On the other hand, Bax, a pro-apoptotic family member, appears to
confer an advanced rate of the cell cycle by mechanisms that are currently unknown (Brady et
al., 1996;Knudson et al., 2001). Interestingly, this has been associated with decreased levels of
p27 conferring increased CDK2 activity (Zinkel et al., 2006). Only one study has reported on
Mtd with respect to the cell cycle. Interestingly, this in vitro study found that the Mtd promoter
could be activated by the E2F1/3 transcription factor (Rodriguez et al., 2006), suggesting that
Mtd expression may be initiated at the G1/S boundary and have an effect on the cell cycle.
However, whether Mtd has a direct role in cell cycle regulation remains to be established and
warrants further studies. As depicted in Figure 1.13, anti-apoptotic molecules generally exhibit
anti-proliferative properties while pro-apoptotic molecules appear to promote cell cycle
progression (Figure 1.13). This would suggest that like the pro-apoptotic molecule Bax, Mtd
may function to advance the cell cycle forward.
Caspase involvement in the cell cycle is highly cell type specific, involving particular caspases
and specific target substrates (Lamkanfi et al., 2007;Timmer et al., 2007). The main caspases
involved in cell cycle regulation appear to be caspase-3, 8 and, to a lesser extent caspase 6, and
their target substrates include p21, p27, Wee and NF-AT. Caspase-8 has been found to promote
the proliferation of T cells through the cleavage of the CDK1 inhibitor Wee (Alam et al., 1999).
Caspase-8 is also involved in trophoblast cell differentiation in the placenta (Huppertz et al.,
2004;Launay et al., 2005). So far, caspase-3 has been found to promote cell cycle progression in
lymphoid cells, forebrain cells and keratinocytes. In lymphoid cells this has been attributed to
caspase cleavage of p27. In contrast, Caspase-3 has been found to inhibit proliferation of B cells
through a mechanism involving cleavage of p21 (Waga et al., 1994;Woo et al., 2003). No study
has investigated the cleavage of p21 and p27 by caspase-3 in the human placenta during
development.
1.4 Placental Pathology
A number of placental pathologies are associated with an altered balance between proliferation
and cell death of the trophoblast cells. These changes influence the capacity of the trophoblast to
undergo cell turnover or invasion, two important events in normal placental function. Thus,
research directed at examining the mechanisms governing trophoblast cell cycle and apoptosis
36
Ray et al., Placenta, 2008
Figure 1-13: Dual role of Bcl-2 family members in cell death and proliferation
Anti-apoptotic molecules Mcl-1, Bcl-2 and Bcl-xL prevent apoptosis and exhibit anti-proliferative properties while
pro-apoptotic molecules Mtd, Bax, and Bak facilitate apoptosis and appear to promote cell cycle progression. Figure
modified from Placenta, Ray et at, 2008.
37
will provide insight into the underlying mechanisms contributing to placental disease. Our
research is aimed at investigating the molecules involved in trophoblast cell fate, in the hope of
uncovering potential targets for prevention, detection, and treatment.
1.4.1 Preeclampsia
Preeclampsia (PE) is one of the most commonly faced complications of pregnancy seen by
physicians in the obstetric field, affecting 5-7% of all pregnancies. This disorder, unique to
human pregnancy, is also the leading cause of fetal and maternal morbidity and mortality
worldwide. Although a great deal of research has been conducted to comprehend its
pathophysiology, preeclampsia remains difficult to predict and diagnose at an early stage.
Additionally, few therapies are available with delivery often being the only option.
1.4.1.1 Clinical Detection and Classification of Preeclampsia
Preeclampsia is a heterogeneous, multi-faceted disorder that is characterized more by its
symptoms then its pathophysiology. Its diagnosis is based primarily on the sudden onset of
maternal hypertension (>160 mmHg systolic pressure or >110 mmHg diastolic pressure), and
proteinuria (dipstick of 3+ on two random urine samples collected at least 4 hours apart) in the
second half of pregnancy ( 2002). Cases may also present with persistent cerebral symptoms
(altered mental status, headaches, blurred vision, or blindness), swelling, edema, epigastric or
right-quadrant pain with nausea or vomiting, thrombocytopenia (platelet count of <100,000 l),
abnormal liver enzymes, or seizures (Sibai et al., 2005). Importantly, the current criteria can only
be used reliably to diagnose women after the 20th
week of gestation, when the disorder has
already manifested.
Since the disorder is heterogeneous, the extent or pathogenesis of preeclampsia may vary,
resulting in very different patient outcomes. Outcome is usually favorable for both mother and
fetus if the symptoms are mild and present after the 36th week of gestation (Hauth et al.,
2000;Sibai, 2003), while patients who develop symptoms prior to the 33rd
week are at higher risk
of fetal and maternal complications (Sibai, 2003;Habli et al., 2007), such as pre-term birth,
placental abruption, reduced amniotic fluid for the fetus, and fetal growth restriction.
Complications to the mother also occur, including liver failure, seizures, stroke and even death
(Sibai et al., 2005). Although the symptoms of preeclampsia dissipate with the completion of
38
pregnancy, the effects of the disease persist, and are associated with increased incidence of
cardiovascular problems in both the fetus and the mother in later life (Barker, 2003;Wilson et al.,
2003;Kajantie et al., 2009). Patients who experience hypertension with elevated liver enzymes
and low platelets are currently categorized in an independent category termed HELLP syndrome
(hemolysis with elevated liver enzymes and low platelets). This is an extreme form of
preeclampsia with increased risk to the mother and child. The extent of preeclampsia has been
theorized to reflect different underlying mechanisms potentiating the disease, possibly arising
from both fetal and/or maternal origins. Therefore, determining the molecular differences
between the classes of preeclampsia may aid in their differential diagnosis and treatment.
Of note, women who are obese, have preexisting hypertension or diabetes, have undergone
assisted reproduction, carry multiple fetuses, are above the age of 40 or who have had
preeclampsia in a previous pregnancy are at a higher risk of developing preeclampsia in the
current pregnancy. Many of these risk factors are on the rise in our society, foreshadowing a
potential increase in the incidence of preeclampsia in the future. Not only will this translate to an
increased number of woman and children that suffer, but it will put a financial strain on the
health care system. This amplifies the need for a better understanding of the biology of the
disease in order to better prevent, diagnose, and treat the syndrome.
1.4.1.2 The Preeclamptic Placenta and Trophoblast Biology: Cell proliferation
and Trophoblast Turnover
Although the etiology of preeclampsia remains unknown, evidence confirms that the placenta
plays a central role in its pathogenesis, as removal of the placenta remains the only effective
treatment for the disease. Furthermore, preeclampsia can arise in the absence of a fetus or uterus
(if placental material persists after pregnancy, or in the case of an ectopic pregnancy), and it is
more common in cases of increased placental volumes, such as in multiple gestation pregnancies.
However, even though the placenta is the key component in the pathogenesis of the disorder, it is
accepted that preeclampsia is a syndrome of vascular endothelial dysfunction and excessive
systemic inflammation.
Normal pregnancy is associated with shedding of syncytial particles into the maternal blood
stream as part of the natural syncytial renewal, and this is associated with a low state of
inflammation in the pregnant woman (Sargent et al., 2003;Aly et al., 2004). In preeclamptic
39
women the inflammatory response is exaggerated (Redman et al., 1999;Sargent et al., 2003). It is
believed that factors originating from the placenta irritate the maternal endothelium and cause
the clinical symptoms. Research has attempted to decipher the factor or factors released by the
placenta that stimulates the endothelial dysfunction and the heightened inflammatory response in
preeclampsia. Angiogenic factors including sFlt1 (Nevo et al., 2008) and endoglin, syncytial
microvillous membrane particles, trophoblast specific proteins (cytokeratin), and increased
concentration of fetal proteins and free fetal DNA (Zhong et al., 2001) have been suggested as
potential stimulators (Levine et al., 2004). The exaggerated inflammatory response seen in
preeclamptic women is believed to result from either the excessive release of placental factors
into the maternal circulation (placental origin), or from a sensitivity of the endothelial lining in
the maternal vasculature, decreasing its tolerance to the normal load of placental debris
(susceptible women). In cases of placental origin these factors may arise from an increased
placental deportation into the maternal circulation. This has been supported by studies that have
found increased placental debris in the maternal serum of preeclamptic patients (Johansen et al.,
1999;Ishihara et al., 2002;Sargent et al., 2003). Moreover, this material has been shown at 16-18
weeks, preceding the classical symptoms of preeclampsia (Levine et al., 2004). The underlying
reason for the increased placental debris seen in preeclamptic women however, is currently
unclear.
It is widely believed that the development of preeclampsia is initiated in the first trimester of
pregnancy when the process of trophoblast differentiation begins. It has been suggested that the
entire event of trophoblast turnover, from trophoblast proliferation through fusion to extrusion, is
increased in preeclampsia (Huppertz et al., 2004). However, while studies have investigated the
apoptotic component of this path, fewer studies have been done to determine if fusion or
cytotrophoblast proliferation is altered in preeclampsia. The few studies that have looked at the
proliferative status in preeclamptic placentae have shown, based on ki67 or BrdU staining, an
overall increase in cytotrophoblast proliferation (Arnholdt et al., 1991;Brown et al., 2005).
However, the mechanisms leading to the hyperplasia seen in preeclampsia have been overlooked.
Understanding these underlying events in trophoblast turnover could aid in distinguishing the
differences between placental and maternal origin of preeclampsia. In addition this line of
investigation also has potential to lead to novel diagnostic and therapeutic discovery.
40
1.4.1.3 The Preeclamptic Placenta and Trophoblast Biology: Spiral Artery
Remodeling and Oxygenation
Preeclamptic placentae are also associated with altered development of the extravillous
trophoblast cells and anchoring column formation. The EVT cells of preeclamptic cases are
characterized as immature compared to normal placentae. They express markers associated with
a proliferative phenotype and display decreased markers of trophoblast differentiation
(Arkwright et al., 1993;Redline and Patterson, 1995;Zhou et al., 1998;Caniggia et al., 1999).
Moreover, they remain hyperproliferative, with limited migration into superficial decidua
(Redline et al., 1995). The shallow trophoblast invasion in preeclampsia is also associated with
an over-expression of HIF-1, a regulator of hypoxia, and TGF3, an inhibitor of trophoblast
differentiation (Caniggia et al., 1999). Importantly, both the low oxygen environment and
elevated expression of TGF3 have been shown to promote the maintenance of the proliferative
phenotype (Genbacev et al., 1996;Caniggia et al., 1999;Caniggia et al., 2000). In addition,
preeclampsia has been associated with increased EVT cell death at the feto-maternal interface
preventing adequate cell invasion (Genbacev et al., 1999;DiFederico et al., 1999). These
phenomena lead to reduced decidual invasion, and as a result fewer maternal spiral arteries are
remodeled (Gerretsen et al., 1981;Zhou et al., 1997). Furthermore, those maternal arteries that
are remodeled do not reach as deep as those in normal pregnancy, and in many cases the smooth
muscle is not fully eroded. This subsequently results in the persistence of high-resistance
vasculature that remains sensitive to endocrine stimuli. It has been suggested that contractility of
the uterine arteries may persist causing the placenta to experience interval changes in oxygen or
ischemia reperfusion (a hypoxia reoxygenation) type event. Not surprisingly, hypoxia
reoxygenation and oxidative stress are among the major drivers of preeclampsia (Jaffe et al.,
1997;Hubel, 1999;Burton and Jauniaux, 2004). In addition, the narrow, high-resistance
vasculature, is often unable to deliver an adequate blood supply to the fetoplacental unit.
Consequently, placentae from preeclamptic women are often associated with oxygen and nutrient
deprivation to the placenta and fetus (Kingdom and Kaufmann, 1997). Furthermore, since low
oxygen is known to promote proliferation and reduced invasion in early placental development,
this environment may reinforce the maintenance of the hyperproliferative premature state.
Importantly, the high velocity perfusion, caused by insufficient spiral artery remodeling, has
been proposed as a mechanism leading to increased syncytial shedding in preeclampsia (Crocker,
2007).
41
Due to its complexity and heterogeneity, preeclampsia has presented as a challenging disorder to
diagnose and treat, with the current management consisting of supportive care and expedited
delivery. Even though a substantial amount of research has been done in the field, we are just
beginning to uncover some of the mechanisms underlying the disorder. To date, there have been
a number of reports focusing on the factors governing the invasive pathway of the trophoblast;
however, the mechanisms governing regulation of cell proliferation and cell cycle regulation in
the trophoblast have not been well established. It is of great importance that the molecular
mechanisms underlying the regulation of the cell cycle during normal and abnormal placentation
be determined. Understanding these events will allow for earlier detection and possible avenues
of treatment. Providing easier and more effective treatment strategies will alleviate the cost to
heath care, and most importantly assure a better wellness of life to the mothers and newborn
infants.
1.4.2 IUGR
Intra uterine growth restriction (IUGR) describes the failure of a fetus to reach its growth
potential. Importantly, birth weight is the strongest known indicator of perinatal morbidity and
mortality and, as such, IUGR presents as a serious complication of pregnancy (Pollack and
Divon, 1992). The pathology of IUGR is closely related to preeclampsia, sharing similar risk
factors and perinatal outcomes‟, however, the two are physiologically different disorders (Villar
et al., 2006). The distinction between the two conditions has recently become an important issue
in the obstetric field as it is crucial to the development of proper diagnostic and treatment
strategies.
1.4.2.1 IUGR classification and etiology
The complication in prediction, treatment and management of IUGR stems from the fact that the
disorder can arise from a number of factors. Simply, IUGR can result from fetal, placental, or
maternal causes. Fetal factors include genetic disorders, comprised mainly of chromosomal
abnormalities, as well as fetal infection and congenital malformations (Pollack et al., 1992).
Maternal contribution stems from factors that influence the ability of the mother to produce and
deliver an adequate source of oxygen and nutrition to the growing fetus. These include maternal
nutritional deprivation, disorders that are associated with hypoxia (eg: asthma, cyctic fibrosis,
lung or heart diseases), and vascular pathologies (preeclampsia, diabetes, chronic hypertenstion).
42
Environmental factors such as cigarette smoking and alcohol consumption also predispose the
fetus to intra uterine growth restriction as they impair oxygen and nutrient delivery (Pollack et
al., 1992).
Unexplained cases of IUGR (those not due to congenital malformation, structural defects,
maternal smoking or under-nutrition, or occur secondary to preeclampsia or gestational
hypertension), are often attributed to placental insufficiency (Villar et al., 2006). Importantly,
placental insufficiency is recognized as the most common cause of IUGR among healthy non-
smoking women with adequate nutritional status (Villar et al., 2006). These cases of IUGR arise
from defects that affect placental development, structure and function. In many cases IUGR is
associated with poor trophoblast invasion and remodeling of the maternal spiral arteries, which
affects the utero-placental circulation and subsequently gas and nutrient bioavailability. Reduced
oxygenation can be determined based on normal umbilical arterial blood flow by Doppler
analysis, where absent end diastolic flow and reverse end-diastolic flow are defined as abnormal,
and are associated with high fetal risk of IUGR (Ferrazzi et al., 2002). Poor placental
differentiation including decreased branching of the villous tree and altered composition of the
villous membranes are also seen in cases of IUGR. These defects can result in deficient placental
transport and impaired gas and nutrient up-take (Cetin et al., 2004;Cetin and Alvino, 2009).
Ultimately, these structural and functional changes result in insufficient nutrient supply and
absorption, resulting in reduced tissue deposition to the fetus (Cetin et al., 2004).
Although classification of IUGR is under debate (Pollack et al., 1992;Maulik, 2006), the most
commonly used criteria is based on a birth weight below the 10th
percentile when corrected for
gestational age and fetal sex, as well as Doppler analysis of the utero-placental circulation.
Additionally, babies whose birth weight is below the 5th
percentile are considered to be severely
IUGR.
The criteria for classification of IUGR have been controversial due to population diversity, and
variation in methods of gestational age and fetal growth measurement among institutions
(Pollack et al., 1992). This has resulted in the inadvertent incorporation of babies that are small
for gestational age (SGA) but relatively healthy, into the pathological arm of numerous studies.
Additionally, the time of onset of the disorder greatly impacts the outcome of the fetus and
should be taken into consideration within studies. IUGR at term may be apparent only as fetal
43
distress requiring a cesarean section and lead to a favorable neonatal outcome. In contrast, early-
onset IUGR before 33 weeks may be so severe as to cause fetal death in-utero, or require
immediate delivery to avoid fetal demise (Villar et al., 2006). These severe cases of IUGR are
often delivered preterm and are thus associated with secondary handicaps and require intensive
care. In addition, IUGR can also occur as a secondary disorder to preeclampsia. It has thus been
suggested that studies take precaution to differentiate between the individual groups. Recently,
an international study of 24,678 pregnancies compared the risk factors and perinatal outcomes
(fetal death, preterm delivery, and length of stay in ICU) of preeclampsia, gestational
hypertension and IUGR, and concluded that although preeclampsia and gestational hypertension
showed many similarities, unexplained (due to placental insufficiency) IUGR presented as a
different entity from preeclampsia (Villar et al., 2006). This paper suggests that the underlying
causes of preeclampsia, IUGR and IUGR secondary to preeclampsia are likely very different,
and stresses the importance of treating these groups as separate disorders. This highlights the
importance of classifying these pathologies of pregnancy separately in scientific studies.
Babies that develop IUGR have an increased risk of developing cardiovascular disease,
metabolic syndrome, and diabetes in adult life (Barker, 1998). In addition, neurological
development problems in both preterm and term IUGR infants is significantly increased (Pollack
et al., 1992). Due to the heterogeneity of the disorder, IUGR presents as a complex and difficult
disorder to handle clinically, with current management of IUGR being largely patient specific
(Cetin et al., 2004). It is therefore important that diagnostic tools be developed to advance the
efficiency and effectiveness of individualized care. Lastly, it has become increasing apparent that
the distinction between preeclampsia and IUGR is of great importance. Molecular
characterization of the two conditions will provide a better understanding of their biological
differences and provide insight into designing improved diagnostic, prevention and treatment
strategies.
1.4.3 Mtd and the Bcl-2 family in preeclampsia
The rate of apoptosis and trophoblast turnover is increased in preeclampsia relative to normal age
matched control placentae, indicating that molecules involved in these pathways may be
candidate markers of placental disease. Interestingly however, the expression of most Bcl-2
family members including Bcl-2, Bcl-xL, Bax and Bak in preeclampsia and preeclampsia
44
associated with IUGR remain unchanged compared to controls (Allaire et al., 2000;Levy et al.,
2002). In contrast, the expression of both Mtd and Mcl-1 are both altered in preeclampsia,
underscoring a unique relationship of these Bcl-2 family members in placental pathology. In
preeclampsia, the full length Mcl-1 protein is cleaved into a pro-apoptotic fragment and the
levels of both Mtd-L and Mtd-P are increased (Soleymanlou et al., 2005b;Soleymanlou et al.,
2007). Hence, in preeclampsia, the Mtd/Mcl-1 rheostat is tilted towards the production of pro-
apoptotic “killer” isoforms. One of our objectives, therefore, has been to decipher the role of Mtd
in this pathological condition. Recently we have discovered that in preeclamptic samples Mtd is
not only abundant in the apoptotic syncytial knots as we have previously shown, but that Mtd
expression also occurs in the proliferative cells of the cytotrophoblast layer (Soleymanlou et al.,
2005b;Ray et al., 2008). This may suggest that the increased Mtd/Bok expression seen in
preeclampsia may contribute to both the increased apoptosis and hyperproliferative nature of the
disorder.
Preeclampsia is associated with an increased expression of the p53 transcription factor (Heazell
et al., 2008b) which has been shown to be a transcriptional regulator of Mtd, and regulates
apoptosis and proliferation (Yakovlev et al., 2004). Preeclampsia is also associated with low
oxygenation and increased levels of Hypoxia Inducible Factor-1 (HIF-1) which has also been
shown to increase the expression of Mtd expression (Hubel, 1999;Hung et al., 2001;Soleymanlou
et al., 2005b).
1.5 Complete Molar Pregnancy
Complete molar pregnancy is a devastating condition whereby abnormal placental tissue
develops in the absence of a fetus. Not only is the pregnancy emotionally devastating, but it is
also associated with the development of further pathologies including preeclampsia and
choriocarcinoma. Although research has been conducted to comprehend the disease, the
pathophysiology of complete molar pregnancy remains unclear. Further research is required to
understand how molar pregnancy develops and how it persists, in order to better prevent and
treat the disease.
Molar pregnancy is not uncommon, occurring in approximately 1 in every 1500 pregnancies in
Europe and North America (Steigrad, 2003) and 1 in 200 in Asia (Seckl et al., 2000;Tham et al.,
2003). Although there are no known risk factors for developing molar pregnancy, the disorder
45
has been shown to be increased in women over 45, and in teenagers (Palmer, 1994;Paradinas et
al., 1996;Steigrad, 2003). Furthermore, developing a hydatidiform mole does put the woman at
an increased risk of developing a future molar pregnancy; 5-40 times more likely than a person
who has not developed the disease previously (Palmer, 1994;Paradinas et al., 1996;Steigrad,
2003). Most importantly, molar pregnancies are predisposed to malignant transformation with 8-
30% of patients developing gestational trophoblastic diseases (GTD) requiring chemotherapy
(Kurman, 1991a;Mazur and Kurman, 1994). Choriocarcinoma (metastatic cancer) one of the
most aggressive types of cancer affecting women, occurs in 2-3% of patients following a molar
pregnancy, an incidence 2000-4000 times greater than that following a normal pregnancy (Seckl
et al., 2000;Li et al., 2002). Due to the devastating condition faced by women with molar
pregnancy and the high risk of GTD following the disease, it is important that precise detection
and diagnosis of molar pregnancy be available.
1.5.1 Clinical Detection and Classification of Molar Pregnancy
Until recently complete hydatidiform mole was diagnosed around 16-18 weeks of gestation with
symptoms including vaginal bleeding, passage of grape-like structures, abnormal growth of the
uterus, abdominal pain, severe nausea and vomiting, and detection of high hCG levels (human
chorionic ganadotropin) (Matsui et al., 2003;Slim and Mehio, 2007). Currently, with the use of
high resolution ultrasonography complete molar pregnancies are now diagnosed in the first
trimester (after the eighth week of gestation) (Kim et al., 2006). Since complete molar pregnancy
has no fetal component, these „pregnancies‟ are clinically terminated upon identification.
Persistent trophoblastic disease can occur following removal of the placental tissue. These cases
are identified by persistent or rising levels of hCG following molar evacuation. To date there are
no reliable markers to indicate that a mole will become persistent.
Molar pathology is classified as either partial or complete moles, a distinction made in 1977.
Complete hydatidiform moles (CHM) are typically diploid with all 46 chromosomes paternally
derived (Slim et al., 2007). It is believed that the androgenic mole arises from fertilization of an
anuclear „empty‟ ovum by either two sperm (dispermy) producing a diploid mole of either 46XX
or 46XY karyotype, or more commonly from fertilization of an empty ovum by a single sperm
that undergoes division after egg penetration (monospermy), producing a diploid mole of
karyotype XX (Slim et al., 2007). Either event results in a conceptus of paternal genomic origin,
46
which lacks the genetic contribution regulated by maternal imprinting. Both monospermic and
dispermic moles appear to have similar malignant potential (Li et al., 2002). Interestingly, recent
reports estimate that only 80% of CHM are diploid androgenic (60% monospermic, 20%
dispermic) with the remaining 20% arising from biparental genomic contribution (Slim et al.,
2007). Biparental moles are believed to develop from a defect in the maternal locus in the region
of 19q13.4 (Panichkul et al., 2005) and are consequently improperly imprinted .
In contrast partial hydatidiform moles (PM) are typically of triploid karyotype (XXX, XXY, or
XYY) arising from dispermy of a normal ovum (diantric) (Szulman and Surti, 1984). This results
in the addition of a second set of paternal chromosomes (two chromosome sets of paternal origin
and a third set of maternal origin) increasing the total genetic material and effectively doubling
the amount of paternally imprinted genes. Unlike complete moles, partial moles give rise to both
embryonic and extraembryonic tissue, however the fetus does not fully develop in these cases.
Although the placental portion of a partial mole is similar to that of a complete mole, partial
moles tend to be less invasive, with the majority of invasive moles being diploid (CHM) (Wake
et al., 1984).
1.5.2 Trophoblast Biology of the Complete Molar Placenta: Morphological
characteristics and Histopathology
Typically the complete mole (of 16-18 weeks) has been characterized by hydropic degeneration
of the placental villi and the absence of fetal tissue (embryo, cord, and amniotic membranes).
The villi are enlarged and surrounded by areas of excessive proliferation, with inner cistern
formation and lack of vascular structure. With diagnosis now occurring in the first trimester (8-
12 weeks of gestation), recent papers have begun to report on a new set of morphological
identification landmarks (Sebire, 2010). For example, the comparison of molar tissue between 8-
12 and 16-18 weeks has identified the presence of a primitive vasculature that later disappears
(Kim et al., 2006). Importantly the study of early gestation moles has provided morphological
clues regarding the development of the disorder, and suggests that further evaluation of molar
development spanning into late gestation would provide an even deeper insight in to its
pathogenesis. However, few second trimester specimens exist to be studied, since molar
pregnancies are artificially stopped once identified. Nevertheless, in rare molar cases placental
development is continued, if the mole develops in conjunction with a twin pregnancy. In these
rare cases, the molar placenta develops independently alongside a genetically normal twin
47
conceptus. These pregnancies are extremely high risk, and only with extreme caution are they
maintained to the earliest point of safe delivery.
Although new insights are being made through the study of molar tissue morphology, molecular
studies are required to gain a deeper understanding of the underlying mechanisms regulating this
type of placental development.
1.5.3 Trophoblast Biology of the Complete Molar Placenta: Molecular
Characteristics
Placentae from molar pregnancies exhibit excessive trophoblast proliferation and apoptosis. In
addition, Immunohistochemical studies have reported on abnormalities regarding the expression
of a variety of cell cycle regulating molecules in molar pregnancy. Although primarily
observational, studies agree that the highly proliferative phenotype of molar placentae is
associated with increased Ki67 and cyclin E expression (Kim et al., 2000;Olvera et al.,
2001;Bamberger et al., 2003). Furthermore, an increase in CDK2 and E2F-1 is also seen in molar
pregnancy (Olvera et al., 2001). p27 has been shown to be expressed by the molar placenta;
however, the literature is conflicting in regards to how it compares to normal placenta of the
same gestation (Olvera et al., 2001;Fukunaga, 2004). Surprisingly, p21 is upregulated in molar
pregnancy despite the hyperproliferative nature of the disorder (Cheung et al., 1998).
The study of proliferation markers in the mole has been shown to be helpful in differentiating
complete vs partial moles and distinguishing moles from spontaneous miscarriage (Xue et al.,
2005). However, research to date has suggested, that the proliferative indices of the mole have no
predictive value with respect to malignant transformation (Cheung et al., 1998). In contrast,
studies indicate that the incidence of apoptosis negatively correlates with progression to GTD
(Wong et al., 1999). In addition, it has also been theorized that increased stromal apoptosis may
be associated with inadequate vascularization contributing to the improper development of the
villi (Kim et al., 2006). It is therefore of great importance to determine the molecules involved in
regulating apoptosis in the molar placenta.
Surprisingly, very few reports have focused on apoptosis in molar pregnancy. Apoptosis,
typically detected by TUNEL recognition, has been found to occur at higher levels in complete
hydatidiform mole compared to partial mole, spontaneous abortion or normal pregnancy (Qiao et
48
al., 1998;Halperin et al., 2000;Chiu et al., 2001;Kim et al., 2006). Interestingly molar pregnancy
has also been found to be associated with increased levels of p53, a phospho-protein that
regulates both apoptosis and proliferation (Qiao et al., 1998;Halperin et al., 2000;Chiu et al.,
2001). Importantly, p53 regulates the expression of many Bcl-2 family members. This includes
the down-regulation of Bcl-2, and the up-regulation of both Bax, and Mtd (Miyashita et al.,
1994;Yakovlev et al., 2004). Although p53 is increased in the molar pathology, no significant
difference in Bax expression and only slightly lower Bcl-2 levels have been detected (Qiao et al.,
1998;Wong et al., 1999;Chiu et al., 2001). Furthermore, neither expression of Bcl-2 nor Bax has
been associated with progression to GTD. Additionally, no significant differences have been
detected at the protein level for apoptosis activating caspase 8 or caspase 10, however caspase 3
activity was shown to be increased in the stromal region of molar tissue (Chiu et al., 2001;Fong
et al., 2006;Kim et al., 2006).
Surprisingly, no study to date has investigated the expression of Mtd in molar pregnancy. Its
exploration however seems intuitive, given that Mtd is highly expressed in the placenta and that
molar pregnancy is associated with a high incidence of developing preeclampsia (Soleymanlou et
al., 2005b). Furthermore, molar pregnancy exhibits increased expression of HIF-1, and E2F-1,
two transcription factors that are hypothesized to induce Mtd expression.
Although cell cycle and apoptotic regulating molecules have been reported in the hydatidiform
mole, these studies have been primarily of descriptive nature and clinically focused. In addition
they have been predominantly restricted to molar tissue of the first trimester. In the future
additional molecular based studies on the mechanisms driving molar progression are needed, to
provide useful knowledge towards the development of diagnostic and treatment strategies.
Importantly the study of cell cycle progression and cell death in the molar placenta will provide
insight into the overall understanding of cell cycle regulation during trophoblast proliferation and
the mechanisms that determine trophoblast cell fate.
1.6 Thesis Hypothesis and Objectives
In humans, cellular proliferation, differentiation and death accompany early placental
development of the trophoblast lineage, the cells forming the placenta. Abnormality at any stage
49
of this development, due to altered proliferation, differentiation or cell death may lead to
improper placental function and subsequent pregnancy related complications.
Members of the Bcl-2 family are central to nearly all pathways governing apoptotic cell death.
Additionally, an increasing body of evidence suggests that a number of anti-apoptotic Bcl-2
members are also involved in the regulation of the cell cycle. Mtd, a pro-apoptotic member of the
Bcl-2 family, is expressed primarily in the placenta and is increased in pregnancies complicated
by preeclampsia. In addition it has been reported that the Mtd promoter is activated at the G1/S
boundary by the E2F1/3 transcription factor, indirectly implicating a role for Mtd during the
early stages of cell cycle progression.
Placental pathologies including preeclampsia, intra uterine growth restriction, and molar
pregnancy are characterized by an immature proliferative trophoblast phenotype accompanied by
excessive cell death. We have previously found Mtd to be associated with the increase in
trophoblast cell death in preeclamptic placentae, however, the relationship between Mtd
expression and the hyperproliferative nature of preeclampsia has not yet been studied.
Furthermore, the molecular mechanisms regulating cell cycle progression and cell fate in normal
and pathological placentation remains unclear.
My overall objective was to investigate the role of Mtd in placentation, focusing specifically on
its role in the G1 phase of the cell cycle. Secondly I aimed to distinguish the factors involved in
promoting the cell cycle in normal placental development and to establish the underlying
molecular defects associated with trophoblast proliferation in placental pathology.
My overall hypothesis was that the expression of Matador is associated with cell proliferation
and apoptosis in normal placental development and in pathological conditions, and that altered
cell cycle regulation at the G1 phase leads to hyperproliferation of the trophoblast and
contributes to placental pathologies including preeclampsia, IUGR, and molar pregnancy
The first objective, presented in chapter 2 of the thesis, was to establish whether Mtd was
directly involved in G1/S phase transition in the trophoblast and to investigate the mechanisms
by which Mtd differentially regulated proliferation and apoptosis. This chapter uncovered a
direct effect of Mtd-L on cyclin E1 expression in proliferative trophoblast cells in normal
placental development.
50
This led to the second objective (Chapter 4) which examined how the G1 phase CDK activator,
cyclin E1 and the CDK inhibitor, p27 were regulated in normal trophoblast development and in
preeclampsia.
Finally, the fifth chapter examined the expression of Mtd in molar pregnancy. Conditions of
molar pregnancy have a high incidence of developing preeclampsia, and exhibit increased
expression of Hif-1 and E2F-1, two transcription factors that are hypothesized to induce Mtd
expression. Therefore, the final objective presented in this thesis (chapter 5) was to determine
whether molar tissue displayed increased levels of Mtd associated with apoptosis and altered
regulation of molecules involved in the G1 phase of the cell cycle.
51
2 Materials and Methods
2.1 Placental Tissue Collection
Informed consent was obtained from each individual patient. Tissue collections were approved
by the Mount Sinai Hospital's Review Committee on the Use of Human Subjects and carried out
in accordance with the participating institutions' ethics guidelines, and in accordance with the
guidelines in The Declaration of Helsinki. First-trimester human placental tissue (5-7 weeks of
gestation, n=30; 9-14 weeks of gestation, n=35) were obtained immediately following elective
termination of pregnancies by dilatation and curettage, or suction evacuation.
2.1.1 Placental samples for studies on preeclamptic pathology
Preeclamptic placentae were selected to represent classic severe early onset preeclampsia (PE;
n=47), late onset preeclampsia (LPE; n=20), and severe preterm Intra Uterine Growth Restricted
(IUGR; n=28) cases, according to the clinical and pathological criteria set out by the American
College of Obstetrics and Gynecology (ACOG 2002; Abuhamad, 2008). Placentae from IUGR
pregnancies were documented with absence or reversal of end diastolic velocity in the umbilical
artery without signs of preeclampsia, and were of fetal weight less than 5th
percentile. Preterm
normotensive age-matched control placentae (AMC; n=52) were selected as age-matched healthy
pregnancies with normally grown fetuses that did not have signs of placental dysfunction. Term
samples (TC; n=51) were obtained immediately following delivery and were within healthy
physiologic range for all maternal and fetal clinical parameters. Birth weight, gestational age,
laboratory values and clinical observations relevant to the health of the mother were taken from
the clinical records. Patients with diabetes, infections and kidney disease were excluded. Due to
organ heterogeneity, multiple specimens were sampled from central and peripheral regions of the
placentae. Placenta samples with calcification, necrosis and visually ischemic areas were also
excluded from the collection. Normal preterm deliveries were due to either multiple pregnancy
without discordancy, preterm labour due to incompetent cervix and premature preterm rupture of
membrane. Term controls were included as pregnancies delivered preterm are by definition
abnormal and may be associated with placental abnormality (Hansen et al., 2000;Redline, 2008).
Tissues were snap frozen or prepared in paraformaldehyde (PFA) and processed for
histochemical analysis. Clinical parameters for severe early preeclamptic, late preeclamptic,
IUGR, preterm age-matched, and term placentae are summarized in Table 2.1.
52
Table 2-1: Clinical data for preeclamptic, intra uterine growth restricted, and control cases
Values are expressed as averages +/- SEM. (A.G.A: appropriate for gestational age, IUGR
intrauterine growth restricted. CS: caesarian section, VD: vaginal delivery)
53
2.1.2 Samples for studies on molar twin pathology
The study presented in the Chapter 5 includes two molar twin sets consisting of a complete
hydatidiform mole as well as a co-existing genetically normal placenta with fetus. Clinical
parameters for the molar cases are summarized in Table 2.2.
The group of control twin sets included placentae from normal twins without discordancy and
without signs of growth restriction in either of the twins. The twin sets were selected as age-
matched healthy pregnancies with normally grown fetuses that did not have signs of placental
dysfunction or preeclampsia. None of the twin pregnancies had evidence of twin to twin
transfusion syndrome (TTTS) and none of the control twins had evidence of infection, anomalies
or abnormal chromosomes. Chorionicity was determined by first trimester ultrasound scan and
confirmed after delivery by histopathological examination of the placenta and membranes. The
placentas of the monochorionic twins were sampled from the area of the cord insertion of each
twin and lateral to it, avoiding the area between the cord insertions in order to reduce the chances
of sampling from shared cotyledons. The clinical characteristics of the twin pregnancies are
shown in Table 2.2. Birth weight, gestational age, laboratory values and clinical observations
relevant to the health of the mother were taken from the clinical records. Placenta samples with
calcification, necrosis and visually ischemic areas were also excluded from the collection.
Tissues were snap frozen or prepared in PFA and processed for histochemical analysis.
2.1.3 Samples for laser capture microdissection
Samples for laser capture microdissection were prepared immediately after collection. Fresh
tissue was washed in phosphate-buffered saline (PBS), saturated in cryoprotect solution (66%
OCT in 33% sucrose solution) and embedded in OCT (optimal cutting temperature) compound
(Tissue-Tek®, Sakura Finetek, Torrance, CA), snap frozen and kept at -800C until further use.
Cryosections, (7µm thick) were cut using RNAse-free blades and mounted on to uncoated,
uncharged slides (Superfrost, Fisher Scientific, Ottawa, Ontario).
54
Table 2-2: Clinical data for molar twin pregnancies and age matched control twin cases
Values are expressed as averages +/- SEM. (CS: caesarian section, VD: vaginal delivery)
55
2.2 First trimester Villous Explant Culture
First trimester placental tissue were collected following elective termination, rinsed and stored in
ice-cold PBS, and processed within 2 hours of collection. Endometrial tissue and fetal
membranes were dissected out. Small fragments of placental villi (25-40 mg wet weight) were
carefully dissected, and placed in either serum-free DMEM/F12 media (GIBCO BRL, Grand
Island, New York, USA) supplemented with 100 g/mL streptomycin, 100 U/mL penicillin, or
placed on Millicel-CM culture dish inserts (Millipore, Bedford, MA) previously coated
with 150
µl of undiluted Matrigel (Collaborative Biomedical Products, Bedford, MA). Explants were then
incubated at 37oC in standard condition (5% CO2 in 95% air, 20% O2, 150 mmHg) overnight.
Treatment was performed the following day.
2.2.1 Mtd antisense knockdown
Chorionic villous explant culture and Mtd knock-down was performed using phosphorothiolated
(all positions) sense (S) and antisense (AS) oligonucleotides designed against the Mtd-L
transcript NM_032515 (S-L: 50-CATGGAGGTGCTGCGG-30, AS-L: 50-
CCGCAGCACCTCCATG-30) as previously described (Caniggia et al., 2000;Soleymanlou et
al., 2005b). Villous explants were incubated at 3% O2 (92% N2 and 5% CO2, 21 mmHg) in the
presence or absence of S or AS oligos (10 mM) or DMEM/F12 alone for 72 hours. Explants
from eight different first trimester placentae (6-8weeks) run in triplicate, were used for the
antisense knockdown experiments.
2.2.2 TGF treatment
To study the effect of TGF on trophoblast cell cycle, cultured explants were treated with either
10ng/ml of TGF1 or TGF3 (R&D Systems) or left untreated. Villous explants were then either
maintained at 37oC in either standard tissue culture conditions (5% CO2 in 95% air, 20% O2,
150 mmHg) or in an atmosphere of 3% (92% N2 and 5% CO2, 21 mmHg) or 8% (87%
N2 and
5% CO2, 57 mmHg) O2 for 24 h at 37°C. Explants from a single placenta were prepared in
triplicate for each treatment condition. Experiments from 3 different first trimester placentae (6-
8weeks) were conducted for analysis in each study.
56
2.3 Laser Capture Microdissection
Prior to laser capture microdissection, immunostaining for Ki67 was performed initially to
identify areas of proliferation. Frozen sections were fixed in 4% PFA, treated with 0.3%
hydrogen peroxide (H2O2), blocked with 5% horse serum, and incubated with anti-Ki67 antibody
(dilution 1:100) for 1 hour at room temp. Laser capture microdissection was performed on
adjacent cryosections, using the Arcturus Pixcell II system (Arcturus Engineering, Mountain
View, CA) according to the manufacturer‟s protocol. Sections were overlaid with a thermoplastic
membrane (CapSure LCM Caps, Arcturus, Mount view, CA), and cells were captured by focal
melting of the membrane through laser activation. Three individual caps were used for each
placental sample, specifically isolating either, the villous trophoblast layer comprising both
cytotrophoblast and syncytiotrophoblast cells; the proliferative (Ki67 positive) region of
proximal column; or the non-proliferative (Ki67 negative) cells of the distal anchoring columns.
Approximately 2000-5000 cells were captured onto each cap.
2.4 RNA Analysis
RNA was extracted from frozen placental tissue or collected cells, using either an Rneasy Mini
Kit (Qiagen, Valencia, CA) or a Trizol extraction method. RNA from LCM samples was
extracted using PicoPure™ RNA Isolation Kit (Arcturus). All samples were treated with DNase I
to remove genomic DNA. 1 g of purified RNA was then used to synthesize cDNA using
random hexamers (Applied Biosystems, Foster City, CA), (denature 5 minutes at 65oC;
amplification 10min at 25oC, 120 minutes at 37
oC, and five minutes at 95°C). Analysis was done
using the DNA Engine Opticon®2 System (MJ Research, Waltham, MA). Mtd was quantified
using the SYBR Green I dye DyNamoTM
HS kit (MJ Research) based on the manufacturer‟s
protocol using isoform specific primers (Mtd-L: Forward 5‟-GCCTGGCTGAGGTGTGC-3‟,
Mtd-P: Forward 5‟-GCGGGAGAGGCGATGA, Reverse (both L and P) 5‟-
TGCAGAGAAGATGTGGCCA-3‟). Taqman Universal MasterMix and specific Taqman
primers and probe for cyclin E1, p27, cyclin D1, cyclin D3, and 18S were purchased from ABI
as Assays-on-DemandTM
for human genes (Applied Biosystems, Foster City, CA). Data were
normalized against expression of 18S ribosomal RNA using the well established 2-CT
formula
as previously described (Livak and Schmittgen, 2001).
57
2.5 Antibodies
Rabbit polyclonal antibody generated against a peptide mapping within an internal region of Mtd
(137-151) of human origin (NM_032515) was raised in the Caniggia laboratory. Rabbit serum
collected prior to Mtd peptide inoculation (pre-immune serum) was used as negative control, to
identifiy non-specific bands by Western blotting (WB 1:500). Remaining antibodies were
purchased from Cell Signaling Technology, Beverly, MA: rabbit polyclonal Mtd/Bok (4521) [IF
1:50]; rabbit monoclonal Cytochrome c (136F3) [WB 1:200]; mouse monoclonal cyclin D1
(DCS6) #2926 [IF 1:300(pc), WB 1:1000], mouse monoclonal cyclin D3 (DCS22) #2936 [IF
1:300, WB 1:1000], and rabbit monoclonal p21 (12D1) #2947 [IF 1:200, WB 1:1000]. The
following antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA: rabbit
polyclonal Mtd/Bok (H151) [IF 1:400], mouse monoclonal Cyclin E1 (HE12) sc-247 [IF 1:400,
WB 1:1500], goat polyclonal Lamin A (C20) sc-6214 [WB 1:200], mouse monoclonal GFP (B-
2) sc-9996 [WB 1:500], rabbit polyclonal p27 (N-20) sc-527 [IF 1:300, WB 1:500], rabbit
polyclonal phospho-p27 (Ser10)-R sc-12939-R [IF 1:400, WB 1:200]; rabbit polyclonal
phospho-p27 (Thr-198) sc-130603 [IF 1:400, WB 1:200]; rabbit polyclonal CDK2 (M2) sc-163
[WB 1:200]; and goat polyclonal Actin (I-19) sc-1616 [WB 1:15,000]. Multiple bands were
observed by Western blotting for the p27 (N-20) antibody. These bands were verified by
preabsorption with a p27 blocking peptide (N-20P at 5X the dilution of the p27 antibody).
Antibodies were also purchased from Vector Laboratories, Burlingame, CA: mouse monoclonal
Ki67 (clone MM1) [IF 1:100]; and Sigma-Aldrich, St.Louis, MO: mouse monoclonal -Tubulin
(clone DM 1A) [WB 1:2000]; DakoCytomation, Denmark A/S: mouse monoclonal proliferating
cell nuclear antigen (PCNA; clone PC10) [WB 1:500]; Abcam, Cambridge, MA: mouse
monoclonal E-Cadherin (HECD-1) [IF 1:200]; Roche, Mannheim, Germany: mouse monoclonal
BrdU [IF 1:100]; and R & D systems, rabbit polyclonal phosphor-p27 (Th157) [WB 1:2000].
Normal rabbit and mouse IgG (sc-2027 and sc-2025 respectively) were purchased from Santa
Cruz Biotechnology and used as negative control. Secondary antibodies were purchased from
Santa Cruz Biotechnology: horseradish peroxidase-conjugated or biotinylated anti-
rabbit/mouse/goat IgG [WB 1:5000]; or Molecular Probes, Eugene, OR: Alexa Fluor 488 anti-
goat/rabbit, Alexa Fluor 594 anti-mouse/rabbit [IF 1:200].
58
2.6 Western Blot Analysis
Western blot analysis was performed on 30g of total protein separated on 12%, or 14% (wt/vol)
SDS-PAGE gels and transferred to PVDF membranes. Non-specific binding was blocked by a 60
minute incubation in 5% (wt/vol) nonfat dry milk in Tris-buffered saline containing 0.1%
(vol/vol) Tween-20 (TBST). Following manufacturer‟s protocol the membranes were incubated
overnight at 4oC with primary antibody diluted in either 5% (wt/vol) nonfat dry milk or 5%
(wt/vol) bovine serum albumin (BSA). Membranes were then incubated with secondary
antibodies (horseradish peroxidase-labeled) prepared in 5% (wt/vol) nonfat dry milk for 60
minutes at room temperature and visualized by enhanced chemiluminescence (Western
LightningTM
Chemiluminescence Reagent Plus, Perkin Elmer, Shelton CT, USA) exposure to x-
ray film (Kodak). All western blots were confirmed for equal loading using 0.1% (w/v) Ponceau
S solution. For quantification purposes, bands of interest were scanned using CanoScanLiDE90
image scanner (Canon Canada Inc. Mississauga, ON) and analyzed using Image Quant software
5.0 (Molecular Dynamics).
2.7 Immuno-precipitation
For interaction studies, 300g of total protein was incubated with either 2g of p27 antibody or
normal rabbit IgG at 4oC overnight. 30l of protein A agarose beads sc-2001 (Santa cruz, Santa
cruz, CA) were then added to each sample for 2 hours at 4oC. The samples were then spun down
and the pelleted beads subjected to a series of washes in RIPA, and 1 X PBS and a final 50l
volume of 2X SDS sample loading buffer (125mM Tris-HCL (pH6.8), 4% (w/v) SDS, 20%
glycerol, 0.005% (w/v) bromophenol blue, 10% -mercaptoethanol), and heated for 5 minutes at
95oC. 25l of the resulting supernatant was subjected to SDS-PAGE followed by western
blotting.
2.8 Peroxidase Based Immunohistochemistry
Placental tissue were rinsed in PBS, dehydrated in 70% Ethanol and fixed for 2-4 hours at 4oC in
4% (vol/vol) paraformaldehyde and embedded in paraffin. Sections of 7m width were cut.
Every 10th
section was stained with hematoxylin and eosin in order to verify the quality of the
tissue and to aid in the selection of the most representative sections. Sections were de-
paraffinized in 3 five minute immersions in xylene, re-hydrated in a serial gradient of alcohol
59
solutions (two minute immersions in 100, 100, 100, 95, 90, 85, 80, 75, 70, and 50% ethanol),
followed by a five minute immersion in double distilled water (ddH2O), and washed in
phosphate buffered saline (PBS). Sodium Citrate antigen retrieval was performed using 10mM
sodium citrate pH6.0 (Cyclin E1: slides in sodium citrate solution were subjected to 5 minutes at
power 4 in a microwave and left to incubate for 15 minutes, this was followed by a an additional
3 minutes at power 4 in the microwave and a 20-30 minute cooling period; Ki67; slides were
boiled in sodium citrate by pressure cooker for 10 minutes) and endogenous peroxidase enzyme
activity was quenched with 3% (vol/vol) hydrogen peroxide in methanol for 30 minutes. Non-
specific binding sites were blocked using 5% (vol/vol) normal horse serum (NHS) and 1%
(wt/vol) BSA in Tris-buffer for 60 minutes at room temperature. Slides were incubated overnight
at 4oC with primary antibody. The following day the slides were incubated with biotinylated
secondary antibody (1:200) for 60 minutes at room temperature and subsequently incubated with
avidin-biotin complex for 60 minutes. Following a final wash slides were subjected to 0.075%
(wt/vol) 3,3-diaminobenzidine tetraaminobiphenyl (DAB) in PBS (pH 7.6) containing 0.002%
(vol/vol) H2O2, initiating a reaction that produced a brownish product. The reaction was stopped
in PBS and the slides were then counterstained with hematoxylin. Prior to mounting the slides
were dehydrated in an ascending ethanol series, and cleared in xylene. In control experiments,
primary antibodies were replaced with non-immune IgG.
2.9 Immunofluorescence (IF) Staining
Sections were de-paraffinized in 3 five minute immersions in xylene, re-hydrated in a serial
gradient of alcohol solutions (two minute immersions in 100, 100, 100, 95, 90, 85, 80, 75, 70,
and 50% ethanol), followed by a five minute immersion in ddH2O, and washed in PBS. Antigen
retrieval was performed using 10mM sodium citrate pH6.0 (in sodium citrate solution slides
were subjected to 5 minutes at power 4 in a microwave and left to incubate for 15 minutes, this
was followed by a an additional 3 minutes at power 4 in the microwave and a 20-30 minute
cooling period). Slides were then washed in PBS followed by a 10min incubation with Sudan
Black (0.1% sudan black in 70% EtOH) to quench endogenous fluorescence typical of red blood
cells. After additional PBS washes sections were pre-incubated in 5% horse serum diluted in
antibody diluent (0.04% sodium azide and 0.008% gelatin in PBS), for 60 minutes at room
temperature to block non-specific binding. Slides were then incubated with primary antibodies
diluted in antibody diluent overnight at 4ºC. The following day sections were washed in PBS and
60
incubated with fluorescence conjugated 2o antibodies [Alexa Fluor®488 (donkey anti-mouse, or
donkey anti-rabbit), Alexa Fluor®594 (donkey anti-mouse, or donkey anti-rabbit)] diluted in
antibody diluent for 60 minutes at room temperature in a covered container. Slides were then
washed in PBS, submerged for ten minutes in 0.4% DAPI (4‟.6-diamidino-2-phenylindole) for
nuclear detection and mounted in 50% glycerol solution. For dual labeled slides, samples were
incubated simultaneously with two 1o antibodies raised in different species. 2
o antibodies were
conjugated to fluoroforms of different wavelengths (specified above) and applied separately for
60 minutes each. For negative controls, primary antibody was replaced by corresponding
concentration of mouse or rabbit non-immune IgG. Fluorescence images were viewed using 20x
regular and 40x and 100x oil immersion objective lens (NA 1.35) and collected using
DeltaVision Deconvolution microscope (Applied Precision, LLC, Issaquah, WA).
Positive and negative cell counts were based on the presence or absence of immunoreactivity of
the Ki67 and Mtd antibody. Cell counts were recorded as a percentage of the total cell number in
the field where the total cell number was taken as the number of nuclei (DAPI stained) in the
trophoblast layer of floating villi or within anchoring columns. For Ki67 and Cyclin E1
quantification in normal and pathological samples, placental sections were stained using a
peroxidase-based method and cell counts were recorded as a percentage of either Ki67 or cyclin
E1 positive trophoblast cells per field. Five fields of view were analyzed per sample.
2.10 TUNEL (Terminal Deoxynucleotidyl Transferase-dUTP-Nick End Labeling)
Paraffin sections were dewaxed in xylene, rehydrated in descending grades of ethanol and rinsed
in PBS. Tissue sections were then pre-treated with 10g/ml proteinase K (invitrogen) in PBS for
10 minutes and treated with 3% H2O2 in methanol to quench endogenous peroxidase activity.
Following PBS washes, sections were incubated with TdT solution (Amersham, Piscataway NJ)
[1X One-Phor-All buffer (100mM Tris-acetate, 100uM magnesium acetate, 500mM potassium
acetate), biotin-16-dUTP (Fermentas), 1M dATP (Fermentas), and 210units/ml of TdT
(terminal deoxynucleotide transferase) enzyme in 0.1% triton X-100 in ddH2O) at 37oC for 1.5
hours. For peroxidase based staining sections were then subjected to ABC reagent/solution
(Vector laboratories) followed by multiples washes in 0.1% Triton-X in PBS, and finally DAB
substrate (Vector laboratories). Sections were counterstained with Harris hematoxylin (Sigma-
61
Alderich) and subjected to acid ethanol (1% HCL in 70% Ethanol) and sodium bicarbonate (1%
in H2O) treatment prior to mounting. Images were captured with a LEICA DC 200 imager. For
dual fluorescence labeled staining, 1o antibody against Mtd was applied following TdT
enzymatic reaction and slides were left overnight at 4oC. Secondary antibody application and
mounting were performed as described for IF staining.
2.11 Cell Line Culture and Analysis
Human choriocarcinoma JEG-3 cells (ATCC, Manassas VA) were grown in EMEM media
(ATCC, Manassas VA) supplemented with 10% (vol/vol) fetal bovine serum (non-heat
inactivated) in standard conditions, 20% oxygen (5% CO2 in 95% air).
2.11.1 SNP (Sodium nitroprusside) treatment
JEG-3 cells were seeded into a 6 well plate (2 X 105 cells per well) or 96 well plate (5000
cells/well) and treated after 16 hours with SNP (Sodium nitroprusside, Sigma) (dose range
between 1mM and 10mM).
2.11.2 Trypan Blue Exclusion Assay
Cells were harvested by trypsinization, centrifuged, resuspended in media, and diluted 1:10 in
0.4% Trypan blue solution (Invitrogen Corp., Carlsbad, CA, USA). Blue vs. white cells were
counted on a hematocytometer. Data is represented as an average of three independent
experiments.
2.11.3 Cell Viability (MTT)
Cell viability was determined by the 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT, Sigma) dye-reduction assay. JEG-3 cells seeded into a 96 well plate (5000 cells
per well) were subjected to 30ul of MTT dye (5mg/ml) per well, at 0, 6, 12, 24, and 48 hours.
The cells were then incubated at 370C for 4 hours. The media was subsequently aspirated and
100ul of dimethyl-sulfoxide (DMSO) were added to each well. Absorbance (relative optical
density) was measured at 570nM with an uQuant microplate spectrophotometer (Bio-Tek
Instruments, Winooski, VT). Data is presented as an average of four experiments.
62
2.11.4 Cell Fractionation
JEG-3 cells were grown to 70% confluence and collected in lysis buffer (0.3M sucrose, 1M
EDTA pH8, 5mM Mops, 5mM KH2PO4, 0.1%BSA – pH to 7.4 using KOH) for fractionation.
Cells were lysed using a dounce homogenizer, and differentially centrifuged to attain cell
fractions: 5 min at 600g to precipitate nuclei followed by 12 min at 10,000 g to pellet light
membrane fraction. Resulting supernatant containing the cytoplasmic fraction was lyophilized
and all fractions were resuspended in RIPA buffer. Verification of fraction specificity was
assessed by Western blot detection of specific subcellular markers: -tubulin (cytoplasm),
cytochrome c (mitochondria), and lamin A (nuclear membrane).
2.11.5 Localization of Mtd to Mitochondria
3.0x105 JEG-3 cells were seeded on sterile glass cover slips and allowed to adhere overnight.
Cells were then incubated with 100nM MitoTracker Red CMXRos (M-7512) (Molecular Probes,
Eugene, OR), a dye taken up by active mitochondria, for 15 minutes at 37oC, and fixed in 3.7%
formaldehyde. Incubation with primary antibody against Mtd was done overnight and secondary
detection was performed as described above for embedded sections.
2.11.6 siRNA Treatment
JEG-3 cells were seeded into a six-well plate at a density of 1x105 cells per well until optimal
confluency for siRNA transfections experiments (>70%) was reached. Cells were then washed
with 1xPBS and 1.5mL of fresh media was added to each well. Subsequently, cells were
transfected with siRNA duplexes (30M) against Mtd and a scramble sequence, purchased from
Ambion (Applied Biosystems, Austin, TX, USA), employing a liposome-based reagent:
lipofactamine2000
(Invitrogen, Carlsband, CA, USA). Sequences were re-suspended in nuclease
free water for a final stock concentration of 50mM. siRNA duplexes and lipofactamine2000
were
first incubated separately for 5 minutes at room temperature, in 0.250mL of OPTI-MEM
(Invitrogen, Carlsband, CA & GIBCO BRL, Grand Island, NY, USA) and then incubated
together for 30 minutes. Treatment was performed over a 48hr time course at standard conditions
and fresh media was added to the cells 6-8hrs following transfection.
63
2.11.7 BrdU Incorporation
BrdU (5-Bromo-2‟-deoxy-uridine) labeling was performed in accordance with the
manufacturer‟s protocol: Roche Applied Sciences (made in: Mannheim, Germany; distributed in
Indianapolis, IN USA). Cells were plated on coverslips and subjected to BrdU (1:1000) for either
20 or 45 minutes following either doxycycline (0.25ug/ml) or Mtd-L siRNA (30uM) treatments.
Cells were then fixed for 20 minutes at -20oC in ethanol fixative (70 ml ethanol, 30 ml of 50mM
glycine, pH to 2.0). Fixed cells were then washed with PBS, blocked with 5% horse serum and
subjected to anti-BrdU antibody for 30 minutes at 37oC. Cells were then washed and 2
o anti-
mouse antibody was applied for 1 hour at room temperature. DAPI staining and mounting were
performed as described for IF staining.
2.11.8 TGF treatment
For experimental procedure cell lines were cultured in either standard conditions (5% CO2 in
95% air) or an atmosphere of 3% O2/92% N2/5% CO2, in the presence or absence of 10 ng/ml of
TGF1 or TGF3 for 24hours.
2.12 Construction of Stable Cell Line Expressing GFP-hMtdL
Human embryonic kidney (HEK) cell line stably expressing GFP-hMtd-L was generated as
follows: plasmid encoding GFP-hMtd-L was stably introduced into Flp-In T-Rex-293 cell line
(Invitrogen as described in the manufacturer‟s protocol. Briefly, the human Mtd-L gene was
amplified from full-length cDNA hMtd-L (Open Biosystems) by PCR using the forward primer
5‟-ggcgcgccagaggtgctgcggcgctcctcg-3‟ and the reverse primer 5‟-cagagagatgacccggatcccg-3‟.
The PCR was digested by AscI/BamHI and cloned into pcDNA5/FRT/TO/GFP (a kind gift from
Dr. Gingras, SLRI at Mount Sinai Hospital, Toronto). The resulting plasmid was verified by
digest and sequencing and finally co-transfected along with pOG44 (Invitrogen) into Flp-In T-
Rex-293 cells to induce a site-specific integration event. Hygromycin-resistant clones were
obtained under 100-ug/ml hygromycin selection. Mtd-L was induced in the stable transfected
cell line with 0.05–2.5 ug/ml of doxycycline.
2.13 Statistical analysis
Statistical analyses were performed using GraphPad Prism 4 software (San Diego, CA). For
comparison of data between multiple groups we used one-way ANOVA Kruskal-Wallis test with
64
Dunns post-hoc test. For comparison between 2 groups we used Mann-Whitney U test.
Significance was defined as P< 0.05. Results are expressed as the mean standard error of the
mean (SE) or box and whisker plots showing medians and interquartile ranges.
65
3 Pro-apoptotic Mtd/Bok Regulates Trophoblast Cell
Proliferation during Human Placental Development and in
Preeclampsia
Note: The content presented in this chapter was first published in Cell Death and Differentiation
(please refer to reference Ray,J.E., Garcia,J., Jurisicova,A., and Caniggia,I. 2009). Contents of
the publication have been reproduced in this data chapter with official permission from the
publisher.
3.1 Abstract
We have previously reported that Mtd/Bok, a pro-apoptotic member of the Bcl-2 family,
regulates human trophoblast apoptosis and that its levels are elevated in severe preeclamptic
pregnancy. Herein we demonstrate that Mtd is also involved in the regulation of proliferation in
normal placentae. Mtd was found in proliferating trophoblast cells during early placental
development, and co-localized with cyclin E1, a G1/S phase cell cycle regulator. The main
isoform of Mtd associated with trophoblast proliferation was Mtd-L, the full length isoform,
which preferentially localized to the nuclear compartment in proliferating cells while during
apoptosis it switched localization to the cytoplasm where it associated with mitochondria.
Antisense specific knock-down of Mtd-L in early first trimester villous explants, as well as loss
and gain of function studies in HEK293 cell line, revealed a direct effect of Mtd-L on cyclin E1
expression and cell cycle progression. We conclude that Mtd-L functions to regulate trophoblast
cell proliferation during early placentation. Of clinical relevance, we hypothesize that the
elevated levels of Mtd found in preeclampsia, may contribute to the increased trophoblast
proliferation accompanying this disorder.
66
3.2 Introduction
In humans, early placental development is defined by an intricate balance between cellular
proliferation, differentiation and death of the trophoblast lineage, the cells forming the placenta
(Smith et al., 1997;Lea et al., 1999;Levy and Nelson, 2000). These cellular events are closely
linked and likely regulated by many of the same molecules.
Early development takes place in a relatively hypoxic environment (~20 mmHg), whereby low
oxygen acts as a key regulator of early trophoblast differentiation (Rodesch et al., 1992;Jaffe et
al., 1997;Burton et al., 1999). In this environment trophoblast proliferation is abundant, whereas
the rate of trophoblast cell death is low (Smith et al., 2000). By 10-12 weeks of gestation, the
oxygen levels increase to ~55 mmHg. This is accompanied by changes in the expression pattern
of a number of gene products (Genbacev et al., 1997;Soleymanlou et al., 2005a), and is
associated with a decrease in trophoblast proliferation and an increased susceptibility to cell
death (Genbacev et al., 1997;Smith et al., 2000). This fine tuning of trophoblast turnover is
governed by a variety of molecules expressed by the placenta, including those comprising the
Bcl-2 family (Ray et al., 2008;Heazell et al., 2008a).
The Bcl-2 family of molecules, classically known for their involvement in the regulation of
apoptosis, include both cell death suppressors (Bcl-2, Bcl-xL, Mcl-1, A1) and cell death inducers
containing either three Bcl-2 homology (BH) domains (Bax, Bak, and Mtd/Bok) or inducers with
only a single (BH3) domain (Hrk, Bim, Bad, Bik, Noxa and Puma). Members of this gene family
act through a complex network of homo- and hetero-dimers with limited specificity. The pro-
apoptotic multi-domain members are believed to regulate apoptosis by forming channels in the
outer membrane of mitochondria leading to the release of pro-apoptogenic factors (Green and
Reed, 1998;Finucane et al., 1999;Soleymanlou et al., 2005b). In recent years it has become
apparent that a number of Bcl-2 family members also regulate cell cycle progression (Bonnefoy-
Berard et al., 2004;Zinkel et al., 2006;Maddika et al., 2007;Ray et al., 2008). Interestingly, while
anti-apoptotic multi domain members slow down progression through the cell cycle, pro-
apoptotic molecules appear to promote cell cycle progression (Brady et al., 1996;Fujise et al.,
2000;Knudson et al., 2001;Bonnefoy-Berard et al., 2004;Jamil et al., 2005;Zinkel et al.,
2006;Maddika et al., 2007).
67
Mtd is a pro-apoptotic Bcl-2 family member that is highly expressed in reproductive tissues (Hsu
et al., 1997;Soleymanlou et al., 2005b). Mtd, is alternatively spliced and encodes three protein
isoforms Mtd-L, Mtd-S, and Mtd-P, with the L and P isoforms predominating in the human
placenta (Hsu and Hsueh, 2000;Soleymanlou et al., 2005b). Similar to Bax, all isoforms of Mtd
contain three BH domains and a transmembrane domain which facilitate pro-apoptotic activity
via mitochondrial depolarization (Soleymanlou et al., 2005b). Previously, we reported that Mtd-
L and Mtd-P expression is high during early placental development (Soleymanlou et al., 2005b).
As this period is characterized by intense trophoblast cell proliferation and little trophoblast cell
death, we hypothesize that Mtd, in addition to its classical role in apoptosis, may have a function
in regulating trophoblast cell proliferation. Interestingly, in vitro studies have shown that the Mtd
promoter can be activated at the G1/S boundary by the E2F1/3 transcription factor (Rodriguez et
al., 2006), providing indirect evidence that Mtd may contribute to regulation of cell cycle
progression. However, the mode by which Mtd exerts its function in the cell cycle remains to be
established.
Herein we report that Mtd is expressed in proliferative trophoblast cells during early placental
development, where it plays a direct role in regulating cyclin E1 expression and promoting G1 to
S phase transition. Furthermore, the dual role of Mtd in apoptosis and proliferation was
associated with a change in sub-cellular localization. Our results indicate that, in addition to its
role in apoptosis, Mtd may be involved in the regulation of trophoblast cell proliferation during
the early stages of human placentation.
3.3 Results
3.3.1 Mtd expression in proliferating trophoblast cells
To determine the pattern of Mtd protein expression in proliferating cells during early gestation,
we performed dual labeled immunofluorescence (IF) analysis with antibodies against Mtd and
Ki67, a common marker of proliferation (Endl et al., 2000) (Figure 3.1). Mtd localization
displayed a unique spatial and temporal pattern of expression. At 5-7 weeks of gestation Mtd was
observed primarily in the cytotrophoblast layer, displaying a prevalent nuclear localization
(Figure 3.1a), whereas weak staining for Mtd was apparent in the syncytiotrophoblast layer
(Figure 3.1a). By 10-13weeks Mtd expression became restricted primarily to the apical border
of the syncytiotrophoblast, with weak nuclear and cytoplasmic expression in the trophoblast
68
Figure 3-1: Mtd expression in proliferating trophoblast cells.
Spatial localization of Mtd and Ki67 in placental sections from the first trimester a: representative early first
trimester (5-8 weeks) floating villous, b: representative late first trimester (9-12 weeks) floating villous, c: early first
trimester anchoring villi (6 weeks), and d: late first trimester anchoring villi (11 weeks). Immunopositivity for Mtd
(green), Ki67 (red), and nuclei labeled with DAPI (blue). Merged images show co-localization of Mtd, Ki67 and
DAPI (overlap of red and blue: pink; overlap of green and blue: light blue; overlap of red, green and blue: white).
Lower panels show boxed region at high magnification. (CT: cytotrophoblast; ST: syncytiotrophoblast; PC:
proximal column; DC: distal column; arrow heads: nuclear positivity, arrows: cytoplasmic positivity). Panel e:
negative controls; first trimester floating and anchoring placental sections (6 weeks) immunostained with
mouse/rabbit IgG.
69
layers (Figure 3.1b). Ki67 expression was restricted to the nuclei of the cytotrophoblast cells and
decreased with advancing gestation (Figure 3.1). Localization of Mtd to Ki67 positive cells
within the cytotrophoblast layer was abundant at 5-7weeks (Figure 3.1a), with 59% of
cytotrophoblast cells expressing both proteins (Table 3.1). However, Mtd expression was not
exclusive to Ki67 positive cells. At 10-13 weeks co-localization of Mtd and Ki67 was less
frequent (Figure 3.1b) with the majority of the cells (57%) being Mtd and Ki67 negative (Table
3.1).
We next investigated whether Mtd localized to Ki67 positive extravillous trophoblast cells
(EVT) forming the anchoring columns. Mtd and Ki67 expression was similarly distributed
throughout the villous column during early first trimester (Figure 3.1c), with co-localization
occurring in 60% of cells (Table 3.1). In contrast, in late first trimester (Figure 3.1d), only 27%
of EVT showed co-localization (Table 3.1). Interestingly, Mtd displayed both nuclear and
cytoplasmic expression at 5-7 weeks (Figure 3.1c), whereas, its localization became
predominantly cytoplasmic by 10-13 weeks (Figure 3.1d).
3.3.2 Mtd localizes to villous trophoblast cells in the G1-phase of the cell cycle
Although Ki67 can be used to indicate cell proliferation it does not discriminate between the
various stages within the cell cycle. To investigate whether Mtd was expressed in the G1 phase of
the cell cycle we tested the co-localization of Mtd with cyclin E1, a cyclin specific to the late G1
phase (Lew et al., 1991;Sherr et al., 2004). Similar to Ki67, cyclin E1 expression was restricted
to the nuclei of cytotrophoblast and EVT within floating villi (Figure 3.2a) and anchoring
columns (data not shown) throughout gestation. Dual labeling of Mtd with cyclin E1 revealed
that Mtd was present during the G1 phase of the cell cycle in early first trimester cytotrophoblast
cells of floating and anchoring villi (Figure 3.2a, data not shown), with co-expression
decreasing in the late first trimester (data not shown). To confirm that Mtd expression was
localized to cytotrophoblast cells during early first trimester, we performed co-expression studies
of Mtd with the cytotrophoblast marker, E-cadherin (Brown et al., 2005)(Figure 3.2b). Of note,
Mtd expression was not restricted to cyclin E1 positive cells; as cells that were presumably
undergoing mitosis, as shown by chromosomal patterning (Figure 3.2c) and Ki67 (Figure 3.2c,
bottom panel), also displayed Mtd expression in the nucleus.
70
Table 3-1 Expression of Ki67 and Mtd in trophoblast cells
Expression of Ki67 and Mtd in trophoblast cells from floating and anchoring villi during first
trimester of gestation (values are reported as percentage)
71
Figure 3-2: Association of Mtd with cyclin E1.
a-b: Spatial localization of Mtd (green) co-localized with a: cyclin E1 (red), or b: E-cadherin (red) in an early first
trimester (6week) floating villous. Nuclei are detected by DAPI (blue). Lower panels show boxed region at high
magnification. a: Arrowheads: co-localization of Mtd with cyclin E1. Upper-right panel; negative control. b:
Arrowheads: representative cells positive for both Mtd and E-cadherin. c: Mtd in mitotic cytotrophoblast cells; Mtd
(green), Ki67 (red) and Top panels: Merged images show co-localization of Mtd, and DAPI (overlap of green and
blue: light blue). Lower panels: Merged images show co-localization of Mtd, Ki67 and DAPI. Middle and right
hand panels show boxed region at high magnification. (CT: cytotrophoblast; S: stroma; ST: syncytiotrophoblast,
arrows: mitotic cells).
72
3.3.3 Mtd expression can occur independently of cell death during early
placentation
We next assessed the association of Mtd expression with the incidence of cell death during the
first trimester using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
(TUNEL), cleaved caspase-3 staining, and analyses of nuclear morphology (Figure 3.3a-d). Cell
death, assessed by these parameters was sporadic and increased with advancing gestation. The
occasional cells that were positive for either TUNEL, cleaved caspase-3, or displayed
fragmented nuclei in early gestation, were found in the stroma (Figure 3.3b), the
syncytiotrophoblast (Figure 3.3a), the syncytial sprouts (Figure 3.3c, upper panel) and the
distal portion of the anchoring villi (Figure 3.3c, lower panel). As expected, Mtd could be
detected in the occasional apoptotic cells as identified by apoptotic blebbing (Figure 3.3c upper
panels), and in the distal portion of the anchoring column (Figure 3.3c, lower panels). No sign
of apoptosis was evident in the cytotrophoblast cells (Figure 3.3).
Interestingly, punctuate expression of Mtd was observed in the nuclei and to a lower extent in the
cytoplasm of proliferating cells (Figure 3.3d) whereas in apoptotic cells Mtd appeared
aggregated and accumulated in the cytoplasm (Figure 3.3c right panels).
3.3.4 Mtd-L is the predominant isoform expressed in proliferative trophoblast
cells
Transcript expression of Mtd-L and Mtd-P, the primary isoforms of Mtd expressed by the
placenta (Soleymanlou et al., 2005b), was examined in proliferating versus non-proliferative
trophoblast cells using Laser Capture Microdissection (LCM), as isoform specific antibodies
toward Mtd-L and Mtd-P were unavailable.
Placental sections from 5-7 and 10-13 weeks were stained for Ki67 to identify proliferative
trophoblast cell populations (data not shown). Adjacent sections were then used to isolate
proliferative EVT, non-proliferative EVT and villous trophoblast cells by LCM (Figure 3.4a).
Both Mtd-L and Mtd-P transcripts were expressed in all three trophoblast populations examined
however; Mtd-L was expressed with Ct (threshold cycle) values ranging between 24-29
(moderate abundance), whereas Mtd-P only exhibited Ct values ranging between 31-35 (very
low abundance) (Figure 3.4b). Expression of Mtd-L in the villous trophoblast layers exhibited
an increase from early to late first trimester and in the anchoring columns Mtd-L mRNA
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Figure 3-3: Apoptosis in early first trimester placental sections.
a: TUNEL staining in a 6 week placental section. TUNEL (red) DAPI (blue). Arrow: TUNEL positive ST cells. b:
Immunohistochemical staining for cleaved caspase-3 in a 6 week placental section. Cleaved caspase-3 (red) DAPI
(blue). Right panel: higher magnification of the boxed area. Arrow: fragmented pieces of nuclei in the cleaved
caspase-3 positive trophoblast cell. Arrowhead: nuclei of adjacent trophoblast cell showing no sign of apoptosis. c:
Mtd expression in apoptotic trophoblast in syncytial knots (upper panel) and distal EVT (lower panel) from early
first trimester (7 week placenta). Mtd (green), DAPI (blue). Middle and right panels: high magnification of the
boxed areas. Arrow: fragmented pieces of nuclei in the Mtd positive cell. Right panel showing clumped Mtd
expression in the cytoplasm of an apoptotic cells. d: Mtd localization to cytotrophoblast cells that express the
proliferative marker ki67. Mtd (green), Ki67 (red), DAPI (blue). Middle and right panels: high magnification of the
boxed area. Arrowhead: punctuate expression of Mtd. (CT: cytotrophoblast; S: stroma; ST: syncytiotrophoblast;
distal EVT: distal portion of extravillous trophoblast column).
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Figure 3-4: Mtd isoform mRNA expression in trophoblast subpopulations.
a: Laser capture microdissection of the non-proliferative, distal portion of an anchoring column (7 week placenta).
(PC: proximal column, DC: distal column). b: Expression of Mtd L and P isoforms by qPCR. Threshold cycle was
taken as the point where the curve contacted the dashed line c: Graphical representation of Mtd-L mRNA in villous
(open bars), proliferative EVT in proximal column (hatched bars) and non-proliferative EVT distal column (black
bars) in first trimester placental sections. N=6 for samples from 5-8 weeks, n=5 for samples from 9-12 weeks. Data
did not reach statistical significance P<0.05 Kruskal-Wallis test.
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expression shifted from the proliferative EVT at 5-7 weeks to the non-proliferative EVT cell
subpopulation at 10-13 weeks; although this did not reach significance (Figure 3.4c).
3.3.5 Mtd isoforms are differentially localized within proliferative JEG-3 cells
Sub-cellular localization of specific Mtd isoforms was investigated in human choriocarcinoma
JEG-3 cell line under normal cell culture conditions. Expression of Mtd-L and Mtd-P isoforms in
this cell line was confirmed by qPCR where Mtd-L was the predominant isoform expressed
(Figure 3.5a). Sub-cellular fractionation demonstrated that Mtd-L was predominantly localized
to the nuclear and light membrane organelle fractions whereas Mtd-P was found in the light
membrane organelle and cytoplasmic fractions (Figure 3.5b).
To determine if Mtd localizes to the mitochondria, we subjected JEG cells to Mitotracker, a
specific mitochondrial tracer dye, and assessed for Mtd co-localization by immunofluorescence
(Figure 3.5c-e). It was confirmed that a subset of Mtd localized to the mitochondria in healthy
(Figure 3.5d), mitotic (Figure 3.5e) and in apoptotic cells (data not shown). To verify that a
similar localization of Mtd is found in vivo we examined first trimester human placental sections
dual labeled with antibodies against Mtd and markers of various cellular organelles. No co-
localization was seen between Mtd and the nuclear envelope, the endoplasmic reticulum, the
golgi apparatus, the cell membrane or lysosomes (data not shown). However, consistent with our
cell line studies, a subset of Mtd co-localized with mitochondria in the cytotrophoblast cells (data
not shown).
3.3.6 SNP-induced apoptosis promotes mitochondrial localization of Mtd in JEG-
3 cells
To determine if Mtd preferentially localized to the mitochondria during apoptosis, we assessed
Mtd localization in JEG-3 cells following apoptotic induction with SNP (Sodium nitroprusside),
a nitric oxide donor recognized to induce apoptosis in the JEG-3 cell line (Soleymanlou et al.,
2007). Cell death and viability were assessed over 48 hours by trypan blue exclusion and MTT
assays (Figure 3.6). In the untreated control group cell proliferation was maximal between 24
and 48 hours (Figure 3.6) with a death rate below 5% [3.86±2.12, 2.94±1.26]. Two days
exposure to 2.5mM and 5mM SNP inhibited proliferation (Figure 3.6) and triggered cell death
[2.5mM: 23.65±4.07% and 5mM: 73.82±6.57]. Nuclear morphology was
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Figure 3-5: Subcellular localization of Mtd isoforms in JEG-3 cells.
a: mRNA expression of Mtd-L (filled bar) and Mtd-P (hatched bar) in JEG-3 cells grown in standard conditions for
48 hours. b: Fractionation of JEG-3 cells grown in standard conditions for 48 hours. Upper panel: Western blot for
Mtd. Lower panel: verification of cellular fractions: tubulin (cytoplasmic), cytochrome c (mitochondrial), and lamin
A (nuclear membrane). c-e: JEG-3 cell grown in standard conditions for 24 hours labeled with mitotracker (red) and
immuno-stained for Mtd (green), DAPI (blue). Mitotic cells denoted by an asterix. Co-localization of Mtd and
mitochondria: yellow (overlap of red and green) d: JEG-3 cell at high magnification displaying normal nuclear
morphology e: high magnification of JEG-3 cells in mitosis. *P<0.01, Mann Whitney U test.
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Figure 3-6: Cell viability of SNP treated JEG-3 cells.
a: Percentage of cell death measured by trypan blue exclusion in JEG-3 cells subjected to increasing doses of SNP
(1mM to 10mM) over 24 hours. b: Percentage of cell death measured by trypan blue exclusion in JEG-3 cells
untreated (open bar) and subjected to 2.5mM (hatched bar) and 5mM (filled bar) SNP treatment over 48 hours. c:
Assessment of cell viability by MTT assay in JEG-3 cells subjected to SNP treatment over 48 hours: untreated
(circle), 2.5mM SNP (square unfilled), 5mM SNP (triangle), 10mM SNP (square filled). In all cases bars represent
standard error over three independent experiments.
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assessed 24 hours following treatment. Mitotic structures were readily seen in untreated
conditions (Figure 3.5c,e, Figure 3.7a), whereas nuclear blebbing was frequently observed in
both SNP treated conditions (Figure 3.7b,c). Mtd was assessed by fluorescence
immunocytochemistry, in JEG-3 cells treated with mitotracker. Mtd could be seen in both the
nuclear and cytoplasmic compartment of cells in the early stages of apoptosis, displaying nuclear
condensation and reformation (Figure 3.7a-c, top right panels). With progressive degree of
apoptosis, evidenced by increased nuclear transformation and blebbing, Mtd expression became
less nuclear and predominantly cytoplasmic where it appeared aggregated and localized to
mitochondria (Figure 3.7b, lower panel. 3.7c, middle panel). At late stages Mtd could be
detected in the cytoplasm but its localization to mitochondria could not be found as the
mitochondria were no longer capable of uptaking the tracer dye (Figure 3.7c, bottom panel).
3.3.7 Inhibition of Mtd-L suppresses cyclin E1 expression
In order to determine the functional significance of Mtd in trophoblast cell cycle during the first
trimester we evaluated the consequences of inhibiting Mtd-L on cyclin E1 expression in first
trimester human placental explants using an antisense knockdown approach. This approach has
been previously used in our laboratory with a knockdown efficiency in explants of 40-60%
(Soleymanlou et al., 2005b). The inhibition of Mtd-L resulted in a 31.5% and 31.3% reduction of
both cyclin E1 mRNA and protein expression, respectively (Figure 3.8a,b). In addition this was
accompanied by a marked decrease in PCNA expression, a marker of S phase (Figure 3.8b,
middle panel). Interestingly, knockdown of Mtd-L had no effect on cyclin E1 expression in
explants from late first trimester of gestation (data not shown).
The Caniggia lab has previously reported that overexpression of Mtd-L and Mtd-P results in
cellular apoptosis (Soleymanlou et al., 2005b). In order to establish a role for Mtd in cellular
proliferation, we used a doxycycline-inducible Mtd-L expression system in HEK293 cells,
generated in the lab; which allowed for controlled Mtd-L expression (Figure 3.8c). Induction of
Mtd-L using 0.25ug/ml doxycycline over 36hours did not affect the rate of cell death as
determined by trypan blue exclusion (data not shown) and it was associated with 32% increase
in cyclin E1 protein expression (Figure 3.8c). This was accompanied by an increase in BrdU
incorporation by these cells (Figure 3.8d). Conversely, an siRNA-mediated reduction of Mtd-L
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Figure 3-7: Localization of Mtd to mitochondria in apoptotic JEG-3 cells
JEG-3 cells were left untreated (a), treated with 2.5mM SNP (b) or 5mM SNP (c) for 24 hours. Cells were labeled
with mitotracker (red) and immuno-stained for Mtd (green). Co-localization of Mtd and mitochondria: yellow
(overlap of red and green). Arrowhead denotes apoptotic cells; Star denotes area of apoptotic cells; Asterix denote
mitotic cells. 100x images show apoptotic cells representative from each treatment group. a: occasional apoptotic
cell in untreated group displaying early stages of apoptosis and co-localization of Mtd with mitotracker. b: cells
showing the occasional mitotic structure and increased levels of cell death c: numerous apoptotic cells displaying a
range from early (condensed, blebbing nuclei, top right panel) to blebbing and fragmented nuclei (middle panel) and
apoptotic bodies (lower panel).
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Figure 3-8: The effect of Mtd-L interference on cyclin E1.
a,b: Expression of cyclin E1 (mRNA: n=4; protein: n=4) in early first trimester explants treated with Mtd-L Sense
(S, non-silencing) oligonucleotides or Mtd-L antisense oligonucleotides (AS, silencing) or untreated (C) in 3% O2
for 72 hours. a: Relative transcript levels of cyclin E1 as detected by qPCR and corrected against 18S. *P<0.05,
Mann Whitney U test. b: Expression levels of cyclin E1 protein (upper panel) and PCNA protein (middle panel).
Confirmation of equal loading by Ponceau detection of total proteins (lower bands). c-d: Induction of GFP-hMtdL in
Flp-In T-Rex-293 cell line with increasing concentrations of doxycycline (0.05, 0.25, 0.5, 1.25, and 2.5ug/ml) over a
36hour period (n=3). c: Top panel: Level of hMtdL expression detected by GFP. Middle panel: Expression of cyclin
E1 and bottom panel, control actin. HEY ovarian cancer cell lysate was included as a negative control for GFP and a
positive control for cyclin E1. d: BrdU incorporation in hMtdL Flp-In T-Rex-293 cell line following treatment with
or without 0.25ug/ml doxycycline over a 36hour period. e-g: knockdown of MtdL in HEK293 cells using siRNA
strategy (n=3). e: Fold changes in MtdL transcript levels relative to scrambled sequence (SS) as detected by qPCR.
Statistical significance was assessed by Kruskal-Wallis or Mann Whitney U test; a,b,c: P<0.05. f: Expression levels
of cyclin E1 protein and actin. g: BrdU incorporation in HEK293 following knockdown of MtdL using 30uM
siRNA for 48hours. Negative control; Immunostaining with mouse IgG in HEK cells subjected to BrdU.
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(75%) in HEK293 cells lead to a decrease in both cyclin E1 protein expression (41%) and BrdU
incorporation (Figure 3.8f, g).
3.4 Discussion
In recent years it has become apparent that members of the Bcl-2 family are involved in
regulating cell fate in a variety of systems (Bonnefoy-Berard et al., 2004;Zinkel et al.,
2006;Maddika et al., 2007). Data presented herein demonstrate that Mtd, a pro-apoptotic
molecule of the Bcl-2 family, is involved in the regulation of both proliferation and cell death in
the human placenta in physiological and pathological conditions. In particular we demonstrate
that 1) Mtd is expressed in proliferating trophoblast cells during early placental development,
where it acts on the G1 phase of the cell cycle by directly regulating the expression of cyclin E1,
2) that Mtd-L is the isoform responsible for this effect on cell proliferation, and 3) that Mtd
localizes to the nuclear compartment in proliferating cells while during apoptosis it switches
localization to the cytoplasm where it interacts with mitochondria.
Both anti-apoptotic and pro-apoptotic members of the Bcl-2 family have been shown to
participate directly in cell cycle regulation independent of their apoptotic function (Bonnefoy-
Berard et al., 2004;Zinkel et al., 2006;Maddika et al., 2007). Anti-apoptotic members, Bcl-2,
BCL-xL, BCL-w, and Mcl-1, have been shown to have inhibitory effects on passage through the
cell cycle whereas the pro-apoptotic member, Bax, promotes cell proliferation by conferring cell
cycle advancement (Brady et al., 1996;Knudson et al., 2001;Zinkel et al., 2006). Although the
Mtd promoter has been found to be activated at the G1/S boundary in vitro (Rodriguez et al.,
2006), we present for the first time, direct evidence that the Mtd protein is expressed in cycling
cells in vivo. In the human placenta the expression of Bax associates predominantly with the
apoptotic index (De Falco et al., 2001), suggesting that Bax may not be involved in trophoblast
cell cycle regulation. This underscores a unique function for Mtd as a pro-apoptotic regulator of
cell cycle progression in the human placenta.
Our study found that nuclear expression of Mtd closely associated with the proliferative index
throughout human placental development. In the early first trimester Mtd localized primarily to
the nucleus in Ki67- and cyclin E1- positive cytotrophoblast cells that displayed normal nuclear
morphology with no sign of apoptosis, whereas past the 9th
week of gestation, as the percentage
of proliferative cells decreased, expression of Mtd switched to the apical border of the
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syncytiotrophoblast layer. The effect of Mtd on cyclin E1 was also associated with this temporal
regulation, occurring only in early first trimester explants. Nonetheless, the apoptotic role of Mtd
has been shown to be maintained past the first trimester (Soleymanlou et al., 2005b). Taken
together, these data suggest that a change in Mtd function may take place late in the first
trimester, away from a cell cycle regulatory role, and that this is accompanied by a change in
sub-cellular localization. This switch in localization and function is likely regulated by the
increase in oxygen concentration experienced by the placenta at this time. This is supported by
previous studies that have found hypoxia response elements in the promoter region of Mtd (Gao
et al., 2005;Soleymanlou et al., 2005b), and have shown Mtd to be decreased in higher oxygen
conditions (Gao et al., 2005;Soleymanlou et al., 2005b).
Since the inhibition of Mtd leads to a decrease in cyclin E1 we hypothesize that Mtd may
function during the G1/S transition. In accordance with our observations a recent study
performed with a mouse fibroblast cell line showed expression of the Mtd transcript to be
increased at mid to late G1 phase, following overexpression of G1/S phase transition factors
(Rodriguez et al., 2006). Moreover the decrease in PCNA and BrdU incorporation following Mtd
knockdown and increased BrdU incorporation following doxycycline-induced Mtd
overexpression, indicates that the effect on cyclin E1 expression results in altered cell cycle
progression to the S phase. In addition, we also observed Mtd expression in mitotic cells and in
Ki67 negative cells. It is possible that like Mcl-1, which functions at both the G0/G1 and at the
G2/M border (Fujise et al., 2000;Jamil et al., 2005), Mtd may play different roles at distinct
phases of the cell cycle(Fujise et al., 2000;Jamil et al., 2005).
Our data also suggest that the role of Mtd in cell fate is likely dependent on conformation and
location of the protein. This mode of regulation has previously been shown for Bax, a Bcl-2
family member structurally similar to Mtd (Hsu et al., 1997;Inohara et al., 1998). Whereas Bax is
cytoplasmic and monomeric in proliferating cells it‟s apoptotic function depends upon its
oligomerization and translocation to the mitochondria (Wolter et al., 1997;Antonsson et al.,
2000;Antonsson et al., 2001;Dejean et al., 2005). Similarly, we observed a diffuse, punctuate
expression pattern of Mtd in proliferative trophoblast cells, whereas cells undergoing apoptosis
displayed a clumped or aggregated pattern of Mtd expression, consistent with oligomerization.
Furthermore, Mtd has previously been reported to form oligomers under apoptotic stimuli in
HEK293 cells (Gao et al., 2005).
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In addition, Mtd expression was both cytoplasmic and nuclear in proliferating cells, whereas its
expression became cytoplasmic and localized to the mitochondria in cells undergoing apoptosis.
Mtd has been shown to interact with the exportin Crm1 (Bartholomeusz et al., 2006), supporting
the idea that Mtd can travel between the nuclear and the cytoplasmic compartments to exert its
cellular function. It is therefore plausible that in trophoblast cells, low levels of Mtd remain
monomeric and locate to the nucleus, where their function is linked to cell cycle regulation,
whereas cytoplasmic accumulation of Mtd promotes oligomerization, localization to the
mitochondria, and a functional switch towards its apoptotic role.
Interestingly, Mtd was also observed in a sub-set of mitochondria in proliferating JEG-3 cells.
This may be explained by previous work showing that in MCF-7 cells, Mtd loosely associates
with the mitochondria and that upon apoptotic stimulation, Mtd becomes tightly integrated into
the mitochondrial membrane (Gao et al., 2005).
Mtd is expressed as three isoforms in total human placental lysate, the principle isoforms being
Mtd-L and Mtd-P (Soleymanlou et al., 2005b). We postulate that the Mtd-L isoform has a dual
role in both cell proliferation and death while Mtd-P may have primarily a “killing” role. This is
supported by our fractionation studies that revealed Mtd-L to be the predominant isoform
expressed in the nuclear compartment of proliferating JEG-3 cells, where it would have direct
access to interaction with cell cycle regulating molecules, and by our knockdown and
overexpression experiments where Mtd-L was seen to have a direct effect on cyclin E1
expression. In contrast, both Mtd-L and Mtd-P were located in the mitochondrial fraction of
JEG-3 cells and their overexpression has been shown to result in apoptosis (Soleymanlou et al.,
2005b).
Based on a putative working model (Figure 3.9) we postulate that during early placental
development, Mtd-L is upregulated by the low oxygen environment, and that it localizes to the
nucleus of the cytotrophoblast cells where it promotes proliferation by aiding in the G1 to S
transition. Introduction of apoptotic stimuli may lead to cytoplasmic accumulation of Mtd, and
result in Mtd oligomerization and interaction with the mitochondria, thus producing a pro-
apoptotic response. In conclusion, Mtd appears to play an important role in the proliferative and
apoptotic pathways that mediate trophoblast cell fate. Of clinical relevance, improper regulation
of Mtd, as seen in preeclampsia, may play a role in altering the homeostasis of trophoblast
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layers, contributing to the increased rate of proliferation and apoptosis associated with this
pathology.
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Figure 3-9: Putative model of Mtd function in the trophoblast
Under low oxygen conditions, as seen in early placental development, Mtd remains monomeric and localizes to the
nucleus of cytotrophoblast cells where it promotes proliferation by aiding in the G1 to S transition. Under conditions
of oxidative stress, Mtd accumulates in the cytoplasm where it interacts with the mitochondria. Mtd pore formation
in the mitochondria leads to release of apoptogenic molecules in to the cytoplasm, thus activating of the apoptotic
cascade and cell death.
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4 Altered trophoblast proliferation in preeclampsia is
associated with increased cyclin E1expression and
abnormal regulation of the cell cycle inhibitor p27
4.1 ABSTRACT
While knowledge concerning molecular pathways regulating human placental development has
increased dramatically, regulation of trophoblast cell cycle remains poorly understood. Recently
we reported that Mtd, a pro-apoptotic member of the Bcl-2 family, promotes the expression of
cyclin E1, an activator of G1/S phase transition of the cell cycle. Moreover, we have previously
shown Mtd to be elevated in the preeclamptic pathology where it is involved in apoptosis of the
trophoblast. Herein we demonstrate that Mtd is also expressed in proliferating trophoblast cells
in preeclampsia. In addition we investigate the regulation of cyclin E1, and the CDK inhibitor,
p27. During normal development levels of cyclin E1 decreased with gestational age as p27
increased. In contrast, the protein levels of both cyclin E1 and p27 were elevated in severe early
onset preeclampsia (SPE), compared to age matched controls (AMC). In addition, the majority of
p27 was phosphorylated at the Ser10 site and localized predominantly to the cytoplasm of cyclin
E1 positive cytotrophoblast cells in SPE. This phenomenon was specific to early onset severe
preeclampsia, as placentae from late onset preeclamptic (LPE) and intra uterine growth restricted
(IUGR) pregnancies did not show differences in expression of either cyclin E1 or p27. We have
previously reported on both increased TGF3 and a hypoxic environment in preeclamptic
placentae, factors that drive trophoblast differentiation. In this study it was found that the
combined treatment of TGF3 and 3%O2 lead to an increase of cyclin E1 and p27 expression in
cultured placental explants and the JEG choriocarcinoma cell line. Our data suggest that the
elevated levels of Mtd and TGF3, in addition to the hypoxic environment in preeclamptic
placentae, may contribute to the increased trophoblast proliferation accompanying early onset
severe preeclampsia.
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4.2 Introduction
Preeclampsia, a serious pregnancy related disorder currently faced by the obstetrician, affects 3-
8% of pregnancies, and if left untreated can lead to fetal and maternal death and morbidity
(Roberts and Cooper, 2001). Currently, diagnosis of this disorder is based on the onset of the
maternal symptoms, including the sudden onset of maternal hypertension and proteinurea, which
typically do not manifest until the second or third trimester once the disease has already been
established. In addition, the etiology of preeclampsia remains unknown. It is well recognized that
the placenta plays a central role in its pathogenesis, as its removal at delivery is the only
treatment known to resolve the maternal symptoms. It is therefore important that effort be put
forward to decipher the underlying cellular and molecular defects of this placental disorder so
that early diagnosis and treatment can be improved.
Physiologically, the placenta has a likeness to a controlled cancer; in the early stages of
pregnancy its growth is exponential and the tissue is highly invasive. Extravillous trophoblast
cells differentiate, from proliferative to invasive cells, that invade the uterine wall to obtain
access to oxygen and nutrients for the developing fetus (Graham et al., 1991;Kurman, 1991b).
Meanwhile the nutrient exchanging syncytiotrophoblast, bathed in the maternal blood, is
constantly sloughed off and replenished through fusion of the underlying proliferative
cytotrophoblast cells (Kaufmann, 1982). The extensive proliferation of cytotrophoblast cells in
the early stages of pregnancy allows for considerable expansion of the organ, facilitating the
increase in surface area and the nutrient absorbing capacity of the syncytiotrophoblast.
In cases of preeclampsia however, the placenta exhibits excessive trophoblast cell turnover, a
process involving increased trophoblast proliferation, fusion and extrusion (DiFederico et al.,
1999;Allaire et al., 2000;Leung et al., 2001;Huppertz et al., 2004). Consequently, this leads to
release of excessive placental debris in to the maternal circulation (Johansen et al., 1999;Redman
and Sargent, 2000), causing maternal endothelial dysfunction and the maternal symptoms of
preeclampsia. In addition, preeclampsia has been associated with a deficiency of the extra villous
trophoblast cells to differentiate into cells capable of adequately infiltrating the uterine arteries.
This results in poor placental perfusion, and insufficient nutrient delivery to the fetus. Of note,
preeclampsia can manifest in either early (<34 wks) or late pregnancy (>34wks) and in many
cases is accompanied by intrauterine growth restriction of the fetus. However, the molecular
88
differences defining the individual types of preeclampsia are still being uncovered and are
currently not fully understood.
Studies have reported that trophoblast cells in preeclamptic placentae are arrested to an immature
hyperproliferative phenotype, that may account for the increased cell turnover and poorly
invasive properties (Arnholdt et al., 1991;Brown et al., 2005). The precise mechanisms leading
to the increased proliferation and turnover in preeclampsia however, have yet to be identified.
The Caniggia lab has reported that Mtd-L and Mtd-P expression levels are significantly
increased in preeclamptic placentae and that this is associated with increased trophoblast cell
death (Soleymanlou et al., 2005b). Moreover, we have previously identified a role for Mtd-L in
the regulation of cyclin E1, an activator of cyclin-dependent kinase-2 (CDK2) and promoter of
the G1 to S transition, in normal placental development (Ray et al., 2009). However, the
relationship between Mtd expression and the hyperproliferative nature of preeclamptic
trophoblast cells remains unexplored. During normal placentation the regulation of trophoblast
proliferation and differentiation is also mediated in part by the oxygen status of the
microenvironment as well as the associated changes in growth factor expression including
members of the TGF family (Jaffe et al., 1997;Genbacev et al., 1997;Caniggia et al.,
1999;Caniggia et al., 2000;Caniggia et al., 2002). Importantly, preeclampsia is associated with
placental hypoxia and a hyperactive TGF pathway (Gerretsen et al., 1981;Caniggia et al.,
1999;Hung et al., 2002;Soleymanlou et al., 2005a). Additionally, the Caniggia lab has previously
shown that cyclin E1 is upregulated by TGF3 (unpublished data).
Herein we investigate whether Mtd is associated with trophoblast proliferation in placentae from
severe early onset preeclampsia. In addition we examine the regulation of cyclin E1 and p27, a
classic CDK2 inhibitor, with respect to the proliferative capacity of the trophoblast cells in the
human placenta. Here we reveal that Mtd was expressed in proliferative trophoblast cells in
placentae from pregnancies complicated with severe early onset preeclampsia. In addition we
show that this disorder was associated with increased expression of both cyclin E1 and p27,
which may lead to excessive trophoblast cell production and possibly improper trophoblast
differentiation. Furthermore, we demonstrate that the low oxygenation and increased TGF
associated with preeclampsia may potentiate altered cell cycling through increased cyclin E1
expression and cytoplasmic localization of p27.
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4.3 Results
4.3.1 Mtd expression in proliferating trophoblast cells in preeclampsia
We have previously reported that Mtd expression is elevated in placentae from severe
preeclamptic pregnancies and that this is associated with increased trophoblast cell death,
characteristic of this disease (Soleymanlou et al., 2005b). To determine whether the increase in
Mtd was also associated with the hyper-proliferative nature of the trophoblast cells we tested
whether Mtd was expressed in Ki67 and cyclin E1 positive cells in placental samples from
severe early onset preeclampsia. In addition we tested whether Ki67 and cyclin E1 levels were
elevated in placentae from severe early onset preeclampsia (PE) relative to age matched (AMC)
and term controls (TC). As anticipated, preeclampsia displayed greater levels of Ki67 (% of Ki67
positive trophoblast cells, PE: 8.6% vs AMC: 3.7% and TC: 1.7%; p< 0.05), cyclin E1 (% of
cyclin E1 positive trophoblast cells, PE: 13% vs AMC: 6.3% and TC: 2.5%; p< 0.05), and Mtd
compared to age-matched and term controls (Figure 4.1a,b, Figure 4.2a,b). Furthermore, dual
labeling of Mtd with Ki67 and cyclin E1 was evident to a greater extent in preeclamptic samples
compared to either age-matched (Figure 4.1a,b, Figure 4.2a,b) or term controls (data not
shown). Interestingly, nuclear expression of Mtd in preeclamptic samples was predominant in
Ki67 or cyclin E1 positive cells whereas in the syncytial knots the expression was mainly
cytoplasmic. In contrast, comparable levels of Mtd were seen in both the nuclear and
cytoplasmic regions of age-matched (Figure 4.1a,b and Figure 4.2a,b bottom panels) and term
control sections (data not shown). Co-expression of Mtd with E-cadherin confirmed that
cytotrophoblast cells were the predominant sites of nuclear Mtd expression in preeclamptic
placentae (Figure 4.2c).
4.3.2 Cyclin E1 and the CDK inhibitor p27 show opposing expression during
normal placentation
Since Mtd appears to impact the cell cycle at the level of cyclin E1, we performed experiments to
gain insight into how cyclin E1 may be regulated and contribute to normal placental
development. In general, the cell cycle is initiated by expression of G1 phase cyclins in response
to a variety of pro-proliferative cues in the environment (Sherr et al., 2004). Since the placenta
experiences a physiological change in oxygenation (Jauniaux et al., 2000) and growth factor
milieu in the late first trimester of normal pregnancy (Caniggia et al., 2000), we evaluated the
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Figure 4-1: Localization of Mtd to proliferative cells in preeclamptic placentae.
a: Spatial localization of Mtd-L and Ki67 in placental tissue from preeclamptic (PE) and age-matched control
(AMC) placentae. Mtd (green), Ki67 (red) and DAPI (blue). Top panels: floating villi from a preeclamptic placenta
at 29 weeks of gestation, bottom panels: floating villi from an age-matched control placenta (34wks). Left hand
panels: 20X magnification; middle and right panels: 100X magnification. Merged panels show co-localization
(yellow/white) of Mtd with Ki67 with nuclei detected by DAPI staining. CT: cytotrophoblast cells; SK: syncytial
knots; arrows: co-localization of Mtd and Ki67 in the nuclear compartment of cells in the trophoblast layer. b:
quantification of the percentage of trophoblast cells positive for Ki67 in PE (n=5), AMC (n=5) and term controls
(TC) (n=5). Statistical significance was assessed by Kruskal-Wallis with Dunn‟s post hoc test; a,b,c: P<0.05.
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Figure 4-2: Localization of Mtd to cyclin E1 positive cytotrophoblast cells in preeclamptic placentae.
a: Spatial localization of Mtd and Cyclin E1 in floating villi from a PE placenta at 26 weeks of gestation (top pane)
and from an AMC placenta of 32wks (bottom panel). Mtd (green), Cyclin E1 (red) and DAPI (blue). Arrowhead: co-
localization of Mtd and cyclin E1 in the nuclear compartment of cells in the trophoblast layer. b: quantification of
percentage of trophoblast cells positive for Cyclin E1 in PE (n=5), AMC (n=5) and term controls (TC) (n=5).
Statistical significance was assessed by Kruskal-Wallis with Dunn‟s post hoc test; a,b,c: P<0.05. c: Spatial
localization of Mtd and the cytotrophoblast marker E-cadherin in placental tissue from preeclamptic (32 weeks) and
age-matched control placentae (32 weeks). Mtd (green), E-cadherin (red) and DAPI (blue). Left hand panels: 20X
magnification; middle and right panels: 100X magnification. Open arrowhead: cytotrophoblast cell co-expressing
Mtd and E-cadherin. (CT: cytotrophoblast cells; SK: syncytial knots);
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protein expression of cyclin E1 in total placental lysates from gestational windows of 5-8weeks,
9-14 weeks and term (Figure 4.3). Cyclin E1 protein expression was found to be significantly
increased during early first trimester compared to term; paralleling the mitotic index previously
published for the placenta (Figure 4.3a). Increased expression of cyclin E1 was associated with
an elevated expression of its binding partner, cyclin dependent kinase 2 (CDK2) (Figure 4.3c),
as well as PCNA, an S phase marker (Figure 4.3d). CDK inhibitors are important negative
regulators of the cell cycle. In particular, the cip/kip inhibitor p27, was found to preferentially
bind to cyclin E/CDK2, inhibiting the CDK2 activity by interfering with the catalytic cleft
(Russo et al., 1996). Hence, we next assessed the expression of p27 in the placenta over
gestation. In contrast to cyclin E1, protein levels of p27 increased significantly with each
advancing window of gestational age (Figure 4.3b).
We next assessed the cellular localization of cyclin E1 and p27 during normal placental
development by dual labeled florescence immunohistochemistry. Trophoblast cells in the
floating and anchoring villi expressed both cyclin E1 and p27 (Figure 4.4). In the trophoblast
layer of the floating villi, expression of cyclin E1 was restricted to the nuclei of the
cytotrophoblast cells throughout gestation (Figure 4.4a,b). Additionally cytoplasmic expression
of cyclin E1 could be observed in the endothelial cells of the villous vessels (Figure 4.4a,b).
Expression of p27 occurred in both the cytotrophoblast and syncytiotrophoblast layer as well as
in cells of the stroma in the floating villi throughout development (Figure 4.4a,b). However,
during the early first trimester expression of p27 was predominantly nuclear (Figure 4.4a) while
in the late first trimester and term, its expression became both cytoplasmic and nuclear (Figure
4.4b, data not shown).
In the anchoring columns both cyclin E1 and p27 were expressed in the extra villous trophoblast
(EVT) throughout the column (Figure 4.4c,d). Although the EVT of the proximal site of the
anchoring column exhibit proliferative capabilities, the intensity of cyclin E1 expression in the
late first trimester was increased toward the distal portion of the column (Figure 4.4d), where
the cells have been shown to be more differentiated. In contrast to cyclin E1, expression of p27
expression remained consistent throughout the column in both early and late first trimester
samples (Figure 4.4c,d). Co-localization of nuclear p27 with cyclin E1 in the EVT was reduced
in the late first trimester compared to the early first trimester as evidenced by the decrease in
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Figure 4-3: Cyclin E1 and p27 expression during placental development
a-d: Representative Western blot and densitometric analysis for cyclin E1(a), p27(b) CDK2 (c), and PCNA (d) over
gestation; (cyclin E1: 5-8 weeks, n=14; 9-14 weeks, n=19; term, n=11) (p27: 5-8 weeks, n=20; 9-14 weeks, n=26;
term, n=17) (CDK2: 5-8 weeks, n=15; 9-14 weeks, n=14; term, n=10) (PCNA: 5-8 weeks, n=9; 9-14 weeks, n=9;
term, n=7). 5-8weeks (filled bars), 9-14 weeks (unfilled bars) and term (hatched bars); Data were normalized against
actin. Statistical significance was determined as P<0.05 Kruskal-Wallis followed by Dunn‟s test.
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Figure 4-4: Spatial localization of cyclin E1 and p27 in human placentae from first trimester and term
a,b: Immunohistochemical dual staining of p27-green and cyclin E1-red in an early first trimester (5weeks), and
term (37week) floating placental villi. Nuclei are visualized by DAPI labeled chromatin (blue). Middle and right
panels: high magnification of the boxed areas. Arrow: cytotrophoblast cell expressing both cyclin E1 and p27
Arrowhead: endothelial cells expressing nuclear p27 and cytoplasmic cyclin E1 (CT: cytotrophoblast; ST:
syncytiotrophoblast; S: stroma; V: vessel). c,d: spatial localization of p27-green, and cyclin E1-red in extra villous
trophoblast of (c) an early first trimester (5week) and (d) a late first trimester (11 week) anchoring villi. (S: stroma;
PC: proximal column; DC: distal column). e: Immunoprecipitation of p27 in total placental tissue lysates over
gestation followed by immunoblotting for cyclin E1 and 27. Input lane (lane 10) is 30ug of total term placental
lysate (same sample as lane 9).
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combined immuno-reactivity (yellow) (Figure 4.4c,d bottom panels). Immunoprecipitation
studies confirmed the interaction between cyclin E1 and p27 throughout placental development
and further demonstrated that the interaction between p27 and cyclin E1 decreased from early
first trimester to late first trimester and term (Figure 4.4e).
4.3.3 Expression of cyclin E1 and p27 is altered in severe early onset
preeclamptic placentae compared to age matched and term controls
Since we observed an increase in cyclin E1 expression in preeclampsia and since trophoblast
cells of preeclamptic placentae are poorly differentiated and maintained in a proliferative
immature state, we examined the overall expression pattern of cyclin E1 and p27 in this
pathology. In line with our immunohistochemical analysis, preeclamptic placentae displayed
significantly up-regulated levels of cyclin E1 protein compared to age matched and term controls
(P<0.05, P<0.01 respectively) with a 2.5 fold increase in protein level compared to term (Figure
4.5b). Similarly, fluorescence immunohistochemical analysis revealed that placentae from
preeclamptic patients display elevated levels of cyclin E1 expression in the cytotrophoblast cells
of the floating villi compared to age matched controls (Figure 4.5e). Cyclins have a half life of
20-30 mins with their protein levels typically paralleling that of their transcript. Surprisingly,
cyclin E1 mRNA was not significantly different in preeclamptic samples compared to controls
(Figure 4.5a), suggesting that regulation of cyclin E1 may be altered by posttranslational
regulation in preeclampsia.
We next assessed whether the increase in trophoblast proliferation seen in preeclampsia was
associated with a decrease in expression of the cell cycle inhibitor p27, similar to that seen in
early placental development. Unexpectedly, levels of p27 were elevated in preeclamptic
placentae compared to age matched and normal term controls (P<0.01, P<0.001) (Figure 4.5d).
In comparison to age matched control sections, p27 expression in PE was predominantly
cytoplasmic in the trophoblast layer and mainly nuclear in the endothelial cells surrounding the
villous vessels (Figure 4.5f). In addition, co-localization studies revealed that p27 expression
was primarily cytoplasmic in cyclin E1 positive PE cytotrophoblast cells (Figure 4.6b).
However, immunoprecipitation of p27 resulted in an increased association of p27 with cyclin E1
in preeclamptic samples compared to controls, a finding that may reflect the overall abundance
of p27 in the preeclamptic samples (Figure 4.6a). No interaction was evident between p27 and
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Figure 4-5: mRNA and protein expression of cyclin E1 and p27 in placentae from pregnancies associated with
severe early onset preeclampsia.
a,c: qRT-PCR analysis of cyclin E1 and p27 mRNA from total placental tissue lysates from severe early-onset
preeclampsia (PE, filled bars n=10), normotensive age-matched (AMC, unfilled bars, n=7), and term control
samples (hatched bars, n=9). Data were normalized against expression of 18S ribosomal RNA using the well
established 2-CT
formula; fold changes are relative to term control levels. b,d: Representative Western blot and
densitometric analysis for cyclin E1 and p27 protein in placental lysates from severe early onset preeclampsia, age-
matched and term control samples (cyclin E1: PE n=14, AMC n=10, TC n=10) (p27: PE n=28, AMC n=17, TC
n=19). Samples are corrected by actin and normalized to term controls. Statistical significance was determined as
P<0.05, Kruskal-Wallis with Dunn‟s post hoc test. e,f: Spatial localization of cyclin E1 (red) or p27 (green) in
severe early onset preeclamptic, and age-matched control placental sections. Nuclei are visualized by DAPI labeled
chromatin (blue). Right hand panels: high magnification of the boxed areas. e: Arrow: expression of cyclin E1 in
cytotrophoblast cells; Arrowhead; syncytiotrophoblast cells negative for cyclin E1 f: Fetal blood cells are visible by
autofluorescence in red. Arrow: expression of p27 in cytotrophoblast and syncytiotrophoblast cells; Arrowhead; p27
expression in endothelial cells (CT: cytotrophoblast; ST: syncytiotrophoblast; SK: syncytial knot; V: vessel).
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Figure 4-6: Cyclin E1 and p27 interaction in preeclamptic pregnancies
a: Immunoprecipitation of p27 in severe early onset preeclampsia (PE), age matched controls (AMC) and Term total
placental tissue lysates followed by immunoblotting for cyclin E1, cyclin D1, and p27. Input lane (lane 10) is 30ug
of total term placental lysate (from same sample as lane 9). b: Spatial localization of cyclin E1 (red) with total p27
(green) in severe early onset preeclamptic (PE-34weeks), and age-matched control (AMC-32week) placental
sections. Nuclei are visualized by DAPI labeled chromatin (blue). Right hand and lower panels: high magnification
of the boxed area with and without DAPI visualization. Arrow: cytotrophoblast cells expressing both cyclin E1 and
p27 in a preeclamptic placentae; Arrowhead cytotrophoblast cell expressing cyclin E1 and low level of p27 (CT:
cytotrophoblast; SK: syncytial knot).
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cyclin D1. Interestingly, no change in p27 transcription could be observed in preeclamptic vs age
matched or term control samples (Figure 4.5c), suggesting that the increase of p27 seen in
preeclampsia may occur at the level of post translational regulation. No differences in cyclin E1
or p27 expression were detected for mRNA or protein between age matched and term controls
(Figure 4.5/4.6, and data not shown).
4.3.4 Post translation regulation of p27 is altered in preeclampsia
Localization and functional regulation of p27 is highly regulated through phosphorylation.
Phosphorylation of p27 at the Ser10 residue can lead to its export from the nuclear compartment
into the cytoplasm, where p27 can no longer inhibit cyclin E1-CDK2 mediated G1/S transition
(Besson et al., 2006). In addition, phosphorylation of p27 at the Thr157 or Thr198 sites result in
the protein‟s stabilization and persistence in the cytoplasm, which may account for the increased
levels of total p27 seen in preeclampsia (Fujita et al., 2002;Liang et al., 2007). Using a p27 Ser10
phospho-specific antibody (p-p27 Ser10) we determined that phosphorylation of p27 at the Ser10
site was upregulated in placentae of preeclamptic patients compared to age matched or term
controls (Figure 4.7a). In addition, immunofluorescent studies confirmed that Ser10
phosphorylation of p27 did confer cytoplasmic retention of the protein, and it revealed that the
majority of the cytotrophoblast cells expressing p-p27 Ser10 were cyclin E1 positive (Figure
4.7d). Interestingly, our studies demonstrated no appreciable difference in phosphorylation at
either the Thr157 or Thr198 p27 residue in preeclampsia compared to control samples (Figure
4.7b,c).
4.3.5 Regulation of cyclin E1 and p27 are altered in several placental
pathologies
To examine whether the observed increase in cyclin E1 and p27 was specific to the severe early
onset form of preeclampsia we assessed cyclin E1 and p27 protein expression in placentae from
two related placental pathologies; late onset preeclampsia (LPE) and intra uterine growth
restricted (IUGR) pregnancies. In contrast to the early onset severe form of preeclampsia, neither
LPE nor IUGR samples displayed significantly elevated levels of cyclin E1 or p27 expression
compared to their respective age matched controls (Figures 4.8a,b; 4.9a,b). However in LPE
there was a trend toward an increase in p27 compared to term (Figure 4.8b) and cyclin E1 levels
were increased in preterm IUGR cases relative to the term control samples (Figure 4.9a).
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Figure 4-7: Phosphorylation of p27 in severe early onset preeclampsia
a: Representative Western blot and densitometric analysis of p27 phosphorylated at Ser10, normalized to Ponceau
(upper graph), and relative to total p27 levels (lower graph), in placental lysates from severe early-onset
preeclampsia (PE, filled bars, n=14), normotensive age-matched (AMC, unfilled bars, n=10), and term control
samples (hatched bars, n=10). b,c: Representative Western blot and densitometric analysis of p27 protein
phosphorylated at (b) Thr157 and (c) Thr198 in placental lysates from severe early-onset preeclampsia (PE, filled
bars), normotensive age-matched (AMC, unfilled bars), and term control samples (hatched bars). Protein expression
was normalized to actin for densitometric analysis. d: Spatial localization of cyclin E1 (red) with p-p27 Ser-10
(green), in sever early onset preeclamptic (30weeks), and age-matched control (24week) placental sections. Nuclei
are visualized by DAPI labeled chromatin (blue). Right hand panels: high magnification of the boxed areas.
Arrowhead: Cytotrophoblast cells expressing nuclear cyclin E1 and cytoplasmic phospho-p27-ser-10; Arrow;
trophoblast cells negative for cyclin E1 and p-p27 Ser10. Statistical significance was determined as P<0.05 Kruskal-
Wallis followed by Dunn‟s test.
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Figure 4-8: Expression of cyclin E1 and p27 in pregnancies complicated by late onset preeclampsia.
a-c: Representative Western blot and densitometric analysis of (a) cyclin E1, (b) total p27, and (c) p-p27 Ser10,
normalized to Ponceau (upper graph), and relative to total p27 levels (lower graph), in placental lysates from late-
onset preeclampsia (LPE, filled bars, n=13) and term control samples (TC, unfilled bars, n=11). Protein expression
was normalized to Ponceau for densitometric analysis. Statistical significance was determined as P<0.05 Mann
Whitney U test. d,e: Spatial localization of cyclin E1 (red) with (d) total p27 (green) or (e) p-p27 Ser10 (green), in
late onset preeclamptic (36week), and term (38,42 week) placental sections. Nuclei are visualized by DAPI labeled
chromatin (blue). Middle and right hand panels: high magnification of the boxed areas with or without DAPI
visualization. Arrowhead: Trophoblast cells expressing nuclear p27; Arrow; trophoblast cells expressing cyclin E1.
(SK: syncytial knot; V: vessel; S: stroma).
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Figure 4-9: Expression of cyclin E1 and p27 in pregnancies exhibiting IUGR
a-c: Representative Western blot and densitometric analysis of (a) cyclin E (b) total p27, and (c) p-p27 Ser10 in
placental lysates from pregnancies complicated by intra uterine growth restriction (IUGR, filled bars, n=14),
normotensive age-matched (AMC, unfilled bars, n=8), and term control samples (TC, hatched bars, n=10). Protein
expression was normalized to actin expression for densitometric analysis. P<0.05 Kruskal-Wallis with Dunn‟s test.
d,e: Spatial localization of cyclin E1 (red) with (d) total p27 (green) or (e) p-p27 Ser10 (green), in IUGR (30,32
week), and age matched control (31,32 week) placental sections. Nuclei are visualized by DAPI labeled chromatin
(blue). Middle and right hand panels: high magnification of the boxed areas with or without DAPI. Arrowhead:
Trophoblast cells expressing cytoplasmic p27; Arrow; trophoblast cells expressing cyclin E1. (V: vessel; S: stroma).
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Interestingly, Immuno-localization studies of cyclin E1 exposed a more prominent pattern of
expression in the vasculature of both the LPE and IUGR pathologies compared to their
respective controls (Figure 4.8d,e; 4.9d,e). In addition, p27 expression was localized to a greater
extent in the nuclear cell compartment of the trophoblast cells in LPE compared to term
placentae (Figure 4.8d). In contrast, sections from IUGR placentae displayed a comparable
pattern of p27 to their AMC controls (Figure 4.9d). No observable differences in p-p27 Ser10
were detected by western blot or immunofluorescent analysis between LPE and term, or IUGR
and age matched control samples (Figure 4.8c,e; 4.9c,e).
4.3.6 Phosphorylation of p27 is increased in the early stages of normal placental
development
To understand the relevance of p27 phosphorylation at Ser10 in severe early onset preeclampsia,
we examined the expression of p-p27 Ser10 in normal placental development. Although no
overall abundance of p-p27 Ser10 was observed in placental lysates throughout gestation (Figure
4.10a upper graph), the ratio of p-p27 Ser10 to total p27 was found to be significantly higher
during early first trimester compared to term (Figure 4.10a lower graph).
Dual labeled florescence immunohistochemistry of p-p27 Ser10 with cyclin E1 revealed that
during normal placental development p-p27 Ser10 was predominantly expressed in the
cytoplasm of cyclin E1 positive cytotrophoblast of the floating villi (Figure 4.10b) and the
cyclin E1 positive extra villous trophoblast cells of the distal anchoring column (Figure 4.10c).
Furthermore, in late first trimester samples, expression of p-p27 Ser10 was reduced in the
cytotrophoblast and its expression became evident to a greater extent in the villous stroma
(Figure 4.10b).
4.3.7 TGF influences cyclin E1 and p27 expression in villous explants cultured
under varying oxygen conditions
We have previously shown that trophoblast differentiation in the late first trimester is driven by
the change in oxygen status and associated decrease in TGF3 expression (Everett and
MacDonald, 1979;Rodesch et al., 1992;Caniggia et al., 2000). We therefore tested whether
treatment with TGF1 or TGF3 under various oxygen conditions had an effect on the protein
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Figure 4-10: Expression Ser10 phosphorylated p27during placental development
a: Representative Western blot and densitometric analysis for p27 phosphorylated at Ser10 normalized to -actin
(upper graph), and relative to total p27 levels (lower graph), in placental lysates over gestation; (5-8 weeks, n=12; 9-
14 weeks, n=12; term, n=10). 5-8weeks (filled bars), 9-14 weeks (hatched bars) and term (unfilled bars); Statistical
significance was determined as P<0.01 Kruskal-Wallis with Dunn‟s test. b,c: Spatial localization of cyclin E1 (red)
with p-p27 Ser10 (green), in an early first trimester (5week), and late first trimester (12week) floating placental villi
(b) and anchoring villi (c). Nuclei are visualized by DAPI labeled chromatin (blue). Middle and right panels: high
magnification of the boxed areas. Arrow: cytotrophoblast cell expressing both cyclin E1 and p-p27 Ser10.
Arrowhead: cytotrophoblast cells expressing cyclin E1 alone (CT: cytotrophoblast; ST: syncytiotrophoblast; S:
stroma; PC: proximal column; DC: distal column).
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expression of cyclin E1 or p27 in the trophoblast using a first trimester explants model (Figure
4.11).
Interestingly, neither cyclin E1 nor p27 expression was increased when cultured in 3%
oxygenation conditions compared to 20%. Furthermore, neither treatment with TGF1 nor
TGF3 resulted in an increase in the expression of cyclin E1 or p27 under 20% oxygenation. In
contrast, the combined treatment of TGF in 3% oxygen culture conditions resulted in a trend
toward an increase in both cyclin E1 and p27 protein expression compared to untreated controls,
however this trend did not reach significance (Figure 4.11 top and middle panels). Interestingly
explants treated with either TGF1 or TGF3 displayed decreased levels of p-p27 Ser10 at each
oxygen level tested, when compared to untreated controls (Figure 4.11 bottom panels).
4.3.8 TGF influences cyclin E1 and p27 expression in JEG-3 choriocarcinoma
cell line cultured under varying oxygen conditions
JEG-3 choriocarcinoma trophoblast cell lines were employed as a cell model to further test the
effect of oxygen and TGF on the expression of cyclin E1 and p27 in the trophoblast. Similar to
our explant model, cells cultured at 3% oxygen and treated with TGF1 or TGF3 displayed a
trend toward an increase of both p27 and cyclin E1 compared to cells untreated (Figure 4.12a
top and middle panels). Interestingly, similar effects were seen between cells cultured at 3%
and those cultured at 20% oxygen (Figure 4.12). In contrast to the explant model, JEG
choriocarcinoma cells displayed increased phosphorylation of p27 at Ser10 when treated with
either TGF1 or TGF3 (Figure 4.12a bottom panels).
Subcellular localization of cyclin E1 and total p27 was assessed in TGF3 treated JEG cells by
dual labeled fluorescence immunohistochemistry. Compared to untreated control cells, those
treated with TGF3 displayed an observable increase in cyclin E1 positive cells and an increased
incidence of mitotic figures (Figure 4.12b). Furthermore, cyclin E1 was detected in association
with mitotic figures (ii), in structures consistent with the appearance of spindles (iv) and in nuclei
of cells with average and excessively large nuclei (i,iv). P27 showed an increase in both
cytoplasmic and nuclear expression upon TGF3 treatment (Figure 4.12b)
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Figure 4-11: Effect of TGF on cyclin E1 and p27 expression in first trimester villous explants exposed to
various oxygen conditions.
a: Representative Western blot analysis of cyclin E1, p27, and p27 phosphorylated at Ser10 from early first trimester
placental explants treated with or without TGF1 or TG3, cultured in 3%, 8% or 20% oxygen condition. b:
Protein expression was normalized to -actin for densitometric analysis. N=3 for each treatment group. P<0.05
Kruskal-Wallis with by Dunn‟s test. Data did not reach statistical significance.
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Figure 4-12: Effect of TGF on cyclin E1 and p27 expression and localization in JEG-3 choriocarcinoma
trophoblast cell line exposed to different oxygen conditions
a: Representative Western blots and densitometric analysis of cyclin E1, p27, and p27 phosphorylated at Ser10 in
JEG-3 trophoblast choriocarcinoma cells treated with or without TGF1 or TGF3, cultured in 3%, or 20% oxygen
condition. Three independent experiments were run in triplicate. Protein expression was normalized to -actin for
densitometric analysis (n=3) P<0.05 Kruskal-Wallis followed by Dunn‟s test. Data did not reach statistical
significance. b: subcellular localization of cyclin E1 (red) and p27 (green) in JEG-3 trophoblast choriocarcinoma
cells treated with or without TGF3, cultured in 3%, or 20% oxygen conditions. Nuclei are visualized by DAPI
labeled chromatin (blue). Middle and right panels: high magnification of the boxed areas.
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4.4 Discussion
Altered rates of trophoblast proliferation and the subsequent increase in cell turnover are factors
underling severe early onset preeclampsia. Surprisingly, little is known about the underlying
mechanisms leading to the hyperproliferative phenotype of the trophoblast in this pathology.
Recently we reported that Mtd is increased in preeclampsia and that Mtd has a positive effect on
cell cycle progression. Herein we established that Mtd is expressed in proliferative cells in the
cytotrophoblast layer and that levels of cyclin E1 and p27 protein are also elevated in placentae
from severe early onset preeclampsia. Secondly, that the inhibitory function of p27 is hampered
due to increased phosphorylation at its Ser10 site resulting in its nuclear export. We also
determined that the increase in TGF and oxidative stress associated with the preeclamptic
disorder may alter the levels of cyclin E1 and p27, and lastly that severe early onset preeclampsia
displays a molecular profile distinct from late onset preeclampsia or IUGR.
We have previously shown that both the Mtd-L and Mtd-P isoforms are increased in
preeclamptic placental tissue compared to age-matched and term controls (Soleymanlou et al.,
2005b). Preeclampsia is a placental disorder, characterized by hyper-proliferation of the
trophoblast cells and accompanied by excessive apoptosis and syncytial shedding. We therefore
hypothesized that Mtd may in part contribute to the increase of both cellular apoptosis and cell
proliferation in this pathology. We have previously shown that in preeclampsia, Mtd is
associated with mitochondrial depolarization and an increase in apoptosis (Soleymanlou et al.,
2005b), and in the current study we demonstrate co-localization of Mtd to the Ki67- and cyclin
E1-positive cytotrophoblast cells in preeclampsia, suggesting that the abundance of Mtd may
also contribute to the hyper-proliferative phenotype. This is further supported by our data
showing that in preeclampsia, the hyperproliferative phenotype of the trophoblast cells is
associated with increased Ki67 and cyclin E1 expression levels. It is likely that Mtd works in
concert with a variety of cell cycle regulating molecules including the Cip/Kip family of
inhibitors and the Notch proteins, which have also been found to be overexpressed in
preeclampsia (reviewed in Crocker and Heazell 2008)(Heazell et al., 2008a).
During normal placentation, the percentage of proliferative trophoblast cells has been shown to
decrease towards term, as the placenta prepares for the final stages of pregnancy (Olvera et al.,
2001). Our data demonstrated that this was associated with a decrease in expression of cell cycle
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advancing molecules cyclin E1, CDK2, and PCNA as well as an increase in the expression of the
cell cycle inhibitor, p27. Consistent with previous reports, we found that cyclin E1 was highly
expressed in the mitotically active cytotrophoblast layer and in the EVT of the anchoring
columns (DeLoia et al., 1997;Bamberger et al., 1999;Olvera et al., 2001). As cyclin E1 acts as
an important checkpoint required for the G1 to S transition during the cell cycle, it is likely that
the expression of cyclin E1 in the cytotrophoblast cells functions to promote cell proliferation
required for the extensive growth of this organ in the first trimester. Interaction between p27 and
cyclin E/CDK complexes has been shown to inhibit cyclinE/CDK2 activation thereby inhibiting
cell cycle progression (Sherr et al., 1995). The observed localization of p27 to the cyclin E1
positive trophoblast cells as well as the interaction of p27 with cyclin E1 at early gestation
therefore suggest that p27 plays a key role in regulating the activity of cyclinE1-CDK2 during
early placental development. Studies have also shown that p27 is elevated in quiescent cells and
that it is involved in the differentiation of a number of cell types (Sgambato et al., 2000;Besson
et al., 2008). It is therefore possible that p27 may function to arrest the cell cycle in the
cytotrophoblast as well as maintain quiescence in the syncytium. This idea is consistent with our
observation that p27 localized to nuclei of the proliferative cytotrophoblast layer as well as
nuclei of the adjacent non-proliferative syncytium. In support of our findings, McKenzie et al,
found that in vitro, differentiation from CT to ST coincided with a decrease in cyclin E1 and an
increase in p27 expression (McKenzie et al., 1998).
In our study, expression of cyclin E1 and p27 was also observed in the endothelium of the villous
vasculature, suggesting that these two molecules affect cell types at multiple levels of
placentation. Similarly, cyclin E knockout mice have been shown to display defects in yolk sac
vascularization, suggesting that cyclin E plays an important role in vasculogenesis (Parisi et al.,
2003). The cytoplasmic localization of cyclin E1 in the endothelial cells suggests that the role of
cyclin E1 may be independent of its role on G1-S phase progression in human placental
vasculature. Alternatively, the cytoplasmic localization may reflect a functional role in
centrosome duplication (Jackman et al., 2002). In addition to villous expression, cyclin E1 was
highly expressed in the more differentiated, non-proliferating cells of the distal columns. This
suggests that in the EVT, cyclin E1 may function in a role alternative to promoting cell
proliferation. This concept has been exemplified in the cyclin E1/E2 double knock out murine
model, which was found to be embryonic lethal due to defects in placental giant cells, the murine
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equivalent of human EVT (Geng et al., 2003;Parisi et al., 2003). Alike human EVT, giant cells
are migratory and are believed to function in maternal spiral artery remodeling during
implantation, and in hormone and cytokine production (Cross et al., 1994). In giant cells cyclin E
was shown to play an important role in endoreduplication, a process which allows for multiple
rounds of DNA synthesis to occur in the absence of cell division (Geng et al., 2003;Parisi et al.,
2003). Geng et al postulate that during endoreduplication cyclin E may aid in priming the
chromosomes for replication prior to S phase entry, a function independent of its classical role in
CDK activation. Similar to the murine model, invasive EVT in the human placenta also display
increased DNA content (referred to by Weier et al as aneuploidy), arising from an unknown but
comparable process (Weier et al., 2005). It is therefore plausible that cyclin E1 may have a
similar function in human trophoblast aneuploidy to that of endoreduplication in the murine
model, a line of study that warrants further investigation. In addition Ser10 phosphorylated p27
was also found in the cytoplasm of EVT cells in the distal portion of the anchoring columns. This
may reflect the role of p27 in cell migration, when localized to the cytoplasm (McAllister et al.,
2003). Hence, the wide range of regulatory roles attributed to cyclin E1 and p27 in the placenta
demonstrate their importance in placental development.
It is widely known that perturbation in the expression or function of various cyclins or CDK
inhibitors is linked to several pathologies associated with hyper-proliferation, including
numerous oncogenic diseases. It is therefore plausible that altered expression of cyclin E1 or p27
may result in pathology of the human placenta. This is supported by the cyclin E1/E2 double
knockout murine model that results in lethality due to placental defects as well as a handful of
studies based on Ki67 staining and BrdU incorporation that suggest that trophoblast cells in
preeclamptic placentae are arrested to an immature hyperproliferative phenotype (Arnholdt et al.,
1991;Redline et al., 1995;Geng et al., 2003;Parisi et al., 2003). Moreover, we have demonstrated
previously and in this study, that preeclampsia is associated with an increased incidence of Ki67
and cyclin E1 expression and that the cyclin E1 levels in preeclampsia were similar to that seen
in early development, supporting the theory that PE trophoblast cells are arrested to an immature
state (Ray et al., 2009).
The hyperproliferative phenotype of the preeclamptic trophoblast is an underlying factor
contributing to preeclampsia. The vascular endothelial dysfunction and a systemic inflammatory
response associated with preeclampsia are believed to result from the excessive debris (placental
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origin) and/or an enhanced sensitivity of the maternal vascular endothelium to a normal
circulating load (maternal origin), the former being associated with severe early onset
preeclampsia and the latter being associated with late onset preeclampsia (Johansen et al.,
1999;Redman et al., 2000). This theory has been supported by the presence of increased
placental material, including syncytial microvillous membrane particles, trophoblast specific
protein cytokeratin, fetal proteins and free fetal DNA, in the maternal circulation of preeclamptic
women, even prior to the onset of the classical symptoms. As well, it has been shown that
placental debris can directly contribute to the inflammatory response of endothelial
cells(Johansen et al., 1999;Redman et al., 2000;Zhong et al., 2001;Levine et al., 2004).
Elevated expression of p27 in severe early onset preeclampsia was unexpected, as
hyperproliferative disorders are often associated with decreased levels of p27. Interestingly, a
number of cancers, including breast, ovarian, colon and oesophageal cancer have also been found
to express increased levels of p27, while maintaining a hyperproliferative state (Besson et al.,
2008). In these cases p27 was seen to be translocated into the cytoplasm of the cell, where its
inhibitory role on cell cycle progression was hampered. Similarly, we observed that in placentae
from preeclamptic pregnancies p27 was predominantly cytoplasmic in cyclin E1 positive cells.
Although p27 can be transcriptionally regulated, the main mechanism of regulation occurs post-
translationally through phosphorylation at a number of serine and threonine sites (Vervoorts and
Luscher, 2008). Moreover, whereas no alteration in p27 mRNA was seen in the preeclamptic
samples compared to controls, these samples displayed significantly increased levels of p27
Ser10 phosphorylation. Phosphorylation at Ser10 has been shown to stabilize p27 by directing
p27 out of the nucleus and in to the cytoplasm through interaction with the exportin CRM1
(Rodier et al., 2001;Connor et al., 2003). This translocation effectively prevents its
phosphorylation at the Thr187 residue and the subsequent proteosomal degradation in the
nucleus (Vlach et al., 1997). Therefore, the cytoplasmic localization and increased expression of
p27 in preeclampsia likely result from the increased phosphorylation of p27 at the Ser10 residue.
Further stabilization of p27 in the cytoplasm can occur through its phosphorylation at Thr157 or
Thr198 or its incorporation into a protein complex such as the cyclinD1-CDK4/6 complex
(Polyak et al., 1994;Fujita et al., 2002;Liang et al., 2007). However, no significant increase in
p27 phosphorylation at either the Thr157 or Thr198 sites was found, nor did p27 interact with
cyclin D1 in the preeclamptic samples.
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Previous data have shown trophoblast proliferation to increase under low oxygen tension in vitro
and in pregnancy complications involving placental hypoxia, such as maternal anemia and
preeclampsia(Genbacev et al., 1997;Caniggia et al., 2000;Huppertz et al., 2003;Myatt, 2006).
However, our studies in placental explants and JEG cells suggested that low oxygen alone was
insufficient to cause an increase in cyclin E1 or p27.
Our data indicate that it is more likely that the upregulation of cyclin E1 and p27 expression seen
in severe early onset preeclampsia result from the combined effect of altered TGF
(transforming growth factor ), a family of growth factors, and oxidative stress associated with
the disorder. We have previously shown that placental hypoxia increases the expression of TGF
and that TGFs regulate cell proliferation, differentiation, migration and invasion in a number of
cell types including those in the placenta (Polyak et al., 1994;Irving and Lala, 1995;Massague,
1998;Caniggia et al., 1999;Ietta et al., 2006). In addition, the significance of the TGF3 pathway
in preeclampsia has been exemplified by the increase in soluble endoglin levels (a co-receptor of
TGF) and increased activation of the Smad pathway (downstream target of TGF) (Venkatesha
et al., 2006). Moreover, this theory is supported by our in vitro explant and cell line studies that
displayed increased cyclin E1 and p27 expression when treated with TGF under low oxygen
conditions.
In contrast to severe early onset preeclampsia, late onset preeclampsia and IUGR, showed little
appreciable difference in the level of cyclin E1 or p27 expression, compared to their respective
controls. However, placentae from late PE displayed a trend toward an increase in p27 which
may function to maintain proper regulation over cell proliferation in this pathology. This was
supported by the predominant localization of p27 to the nuclei of cyclin E1 positive cells in late
onset preeclampsia. Furthermore, unlike severe early onset preeclampsia that is instigated by the
increase in trophoblast turnover, late preeclampsia is believed to be a maternal rather than
placental disorder. There has been conflicting data as to whether IUGR is a hyperproliferative
disorder (Smith et al., 1998;Chen et al., 2002;Gurel et al., 2003;Jeschke et al., 2006). In contrast
to Jeschke et al 2006, who found the proliferation marker Ki67 to be decreased in IUGR, our
data show a trend toward an increase in cyclin E1 expression supporting the theory that the
IUGR placenta is hyperproliferative. Although late preeclampsia and IUGR are closely related to
severe early onset preeclampsia, sharing similar risk factors and perinatal outcomes, our data
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support the theory that these pathologies are physiologically different disorders (Villar et al.,
2006;Romero et al., 2008).
Proper trophoblast proliferation is vital for a healthy pregnancy. Insufficient trophoblast
proliferation and invasion during placentation can result in poor implantation and pregnancy
loss, or result in syndromes such as preeclampsia, and IUGR. It is therefore critical that we
understand the molecular basis governing cell cycle regulation in the placenta, so that we can
continue to uncover the mechanisms underlying these disorders. A greater understanding of the
trophoblast cell cycle, and the changes that occur in placental pathology, will inevitably aid in
the discovery of future therapeutic and diagnostic strategies.
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5 Dual role for Mtd in trophoblast proliferation and apoptosis in
molar pathology
5.1 Abstract
Complete hydatidiform molar pregnancy (CHM) is a devastating condition whereby placental
tissue develops in the absence of a fetus. Although it is established that molar pregnancy is
characterized by excessive trophoblast proliferation and death, the molecular basis governing its
pathogenesis is largely unknown and has been based primarily on descriptive analysis of molar
tissue from the first trimester. We have previously shown that Mtd, a pro-apoptotic member of
the Bcl-2 family, functions to promote both cell death and proliferation in the human placenta.
The objective of this study was to examine the expression of Mtd in the context of cell death and
proliferation in molar pregnancy using a second trimester twin model consisting of a molar and a
genetically normal co-twin placenta, unbiased by environmental conditions. Mtd was elevated in
the molar tissue compared to the co-existing twin placentae where it was associated with
increased TUNEL positive apoptotic cells as well as increased Ki67 and cyclin E1 positive
proliferative trophoblast cells. In addition, placentae from molar pregnancy exhibited decreased
expression of cyclin D1 and D3 protein despite the high level of cyclin E1 present. Cyclin E
function is inhibited by both p21 and p27 cell cycle inhibitors. Interestingly only p27 was
decreased in molar samples whereas p21 expression was significantly increased in comparison with
their co-twin and age-matched control twin sets.
We conclude that molar placentae from second trimester twin pregnancies are associated with a
disruption in the expression of G1 phase cell cycle regulating molecules. The excessive
trophoblast proliferation in these cases is likely due to elevated levels of cyclin E1 and is not
reliant on the presence of D type cyclins. In addition, the increased trophoblast proliferation may
be due to a decreased inhibition by p27, but not p21. These alterations are unrelated to
environmental conditions as placentae from their twin counterpart were unaffected.
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5.2 Introduction
Complete molar pregnancy, a devastating trophoblastic disease of androgenic chromosomal
origin, is typically characterized by a developing placenta in the absence of a fetus (Slim et al.,
2007). Once identified these pregnancies are therefore immediately terminated. This placental
disorder has a high rate of incidence in Asia occurring in 1:200 pregnant women while it
manifests in 1:1500 Caucasians (Seckl et al., 2000;Steigrad, 2003;Tham et al., 2003) However
the incidence of molar pregnancy is on the rise. Moreover, in many cases, complete molar
pregnancy will persist following evacuation, with 3-33 % of the cases developing into
choriocarcinoma requiring chemotherapy (Kurman, 1991a;Mazur et al., 1994). The malignant
transformation of molar pregnancy is thought to arise from the abnormally high proliferative and
sometimes invasive nature of the trophoblast cells, typical of the disease.
First trimester molar placentae have been shown to exhibit exuberant trophoblast cell
proliferation and excessive cell death in both the stromal and trophoblastic regions. Interestingly
the excessive proliferation found in molar tissue has been associated with elevated markers of
proliferation including Ki67 and cyclin E1 (Kale et al., 2001;Olvera et al., 2001), similar to that
seen in preeclampsia (Ray et al., 2009), and increased apoptosis has been assessed by TUNEL
(Qiao et al., 1998;Chiu et al., 2001). These studies however have been mostly descriptive, and
restricted primarily to immunohistochemical analysis. Absence of the cell cycle inhibitor, p57,
due to paternal imprinting has remained one of the most identifiable features of the complete
mole pathology (McConnell et al., 2009a;Kipp et al., 2010). However it has been shown that
molar tissue can develop in the presence of p57, as seen in two unique cases of molar pregnancy
that expressed p57 due to retention of a single maternal chromosome (Fisher et al.,
2004;McConnell et al., 2009b). It is therefore important to understand the cell cycle regulating
molecules, other than p57, that contribute to the disease. Thus far however, the molecular
alterations contributing to the pathogenesis of this disorder are not fully understood. As such, the
study of molecules involved in trophoblast cell fate in molar pregnancy will provide further
understanding of how improper placentation develops in this pathology, and may provide insight
towards future treatment strategies.
Although the Bcl-2 family of molecules have been shown to play significant roles in trophoblast
cell fate (Ray et al., 2008), few studies have reported on their expression in molar pregnancies
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(Qiao et al., 1998;Fong et al., 2005;Fong et al., 2006). Studies have found the ratio between pro-
apoptoic Bax and anti-apoptotic Bcl-2 to be elevated in compete molar tissue while others have
suggested a role for anti-apoptotic Mcl-1 in molar pathogenesis (Qiao et al., 1998;Fong et al.,
2005).
Mtd is a pro-apoptotic member of the Bcl-2 family, expressed primarily in tissues of
reproductive origin (Hsu et al., 1997;Soleymanlou et al., 2005b). In preeclampsia, a common
placental disorder complicating 3-5% of all pregnancies (Roberts et al., 2001), we found Mtd to
be elevated in the placenta and have a role in both the apoptotic and proliferative trophoblastic
features of the disease (Soleymanlou et al., 2005b;Ray et al., 2009). In apoptotic cells Mtd was
associated with mitochondrial depolarization and induction of caspase-3 activity, whereas in
proliferative cells Mtd was seen to effect cyclin E1 expression (Soleymanlou et al., 2005b;Ray et
al., 2009). Interestingly, molar pregnancies are associated with a high incidence of preeclampsia
(Soto-Wright et al., 1995;Koga et al., 2009) and may therefore exhibit overlapping dysregulatory
mechanisms with preeclampsia, including increased Mtd expression. However, no study to date
has addressed the expression or function of Mtd in molar pregnancy, despite its high abundance
in the placenta. We therefore postulate that Mtd may be elevated in molar tissue and contribute
to the abnormal balance of apoptosis and proliferation associated with molar pathogenesis.
In normal clinical circumstances the identification of complete molar tissue occurs early on and
results in immediate termination of the pregnancy. Hence, studies examining the molecules
involved in regulating trophoblast cell fate in complete molar pregnancy have been restricted to
tissue of the first trimester. In this chapter, we report on two rare cases of molar pregnancy,
consisting of a complete molar placenta with a co-existing twin; the molar tissue having
developed adjacent to the independent genetically normal placenta of a live fetus. These cases
are unique, as unlike typical moles that are terminated early, the pregnancies were carried on till
delivery which occurred preterm. Therefore, they allow us to perform molecular studies in molar
placental tissue that has been permitted to develop into the second trimester. In addition, these
cases provide twin placental controls, grown in an identical uterine environment for which to
compare the molecular findings.
Herein, we examine the expression of Mtd in the context of cell death and proliferation in two
twin molar cases. As we have shown Mtd to be involved in cell proliferation and apoptosis in
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normal and preeclamptic placentation, we studied the expression of Mtd in conjunction with
markers of apoptosis, as well as molecules involved in regulating the G1 phase of the cell cycle.
Importantly, comparisons were made between the molar tissue samples and the placentae of their
co-existing twins, as well as between twin controls from normal preterm pregnancies.
5.3 Results
5.3.1 Two cases of a twin pregnancy with a complete hydatidiform mole and
coexistent twin fetus
A twin gestation consisting of a complete hydatidiform mole and a co-existing twin fetus is a
rare event and presents extreme clinical risks. Often these pregnancies develop preeclampsia and
require preterm delivery (Soto-Wright et al., 1995;Koga et al., 2009;Massardier et al., 2009). In
addition they are also at increased risk of additional pregnancy-related complications including
the development of persistent gestational trophoblastic disease (GTD) (Massardier et al., 2009).
Case 1
Patient one, age 37, carried a twin pregnancy with complete hydatidiform mole. It was her first
pregnancy. During her pregnancy she exhibited hyperthyroidism and mild pregnancy induced
hypertension, but she did not develop preeclampsia. She delivered by vaginal delivery at 23
weeks due to chorioamnionitis and sepsis. A male fetus of 485g was born preterm and was not
viable (Table 2.2).
Case 2 (Figure 5.1)
Patient two, age 33, also carried a twin pregnancy with complete hydatidiform mole. It was her
fifth pregnancy and first incidence of molar pregnancy. She presented with severe preeclampsia
at 24 weeks and developed hyperthyroidism. Her pregnancy resulted in neonatal death of a 350g
female fetus at 25 weeks. The fetus was diagnosed as being preterm and growth restricted.
Patient two chose to have a full hysterectomy immediately following the pregnancy (Table 2.2).
In both cases of molar pregnancy the molar placentae demonstrated classic morphological,
anatomical, and histopathological signs of complete mole pathology. These included enlarged
villi with prominent swelling (Figure 5.1b,c), extensive circumferential trophoblastic
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Figure 5-1 Morphologic characteristics of placentae from the mole and its co-existing twin
a: Macroscopic appearance of the placentae from case 2, with molar placenta left of the hatched line and genetically
normal placenta with gestational membrane and umbilical cord associated with twin fetus at right. b: magnification
of the molar placenta with the swollen cystic villi bulging from beneath the membrane cover (arrows) c-f: Histologic
sections of the mole and twin placenta from case 2 stained with H&E c,d: section of placenta from the mole and the
co-twin presented at equal magnification, Molar villi (left panel) and multiple small mature villi of normal placenta
(right panel). e,f: high magnification of molar villi. Asterisk: dilated cystic villi. HT: hyperproliferative trophoblastic
layer. Arrow, thinned trophoblastic layer.
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hyperplasia (Figure 5.1e), as well as acellular cistern formation in the central portion of the villi
(Figure 5.1c,e,f) (Shih and Kurman, 2002). The molar villi also displayed no sign of mature
placental vasculature, a common characteristic of molar pathology (Slim et al., 2007;Kim et al.,
2009). In addition, both cases exhibited areas of the enlarged villi with a thinned layer of
trophoblast cells, a feature noted in molar pathology (Figure 5.1f). Both pregnancies were
severely compromised; ending preterm, and resulting in fetal death. The molecular finding from
the molar tissue was therefore compared to placentae from normal preterm (age-matched) twin
pregnancies in addition to the co-existing twins of the moles, to control for any abnormality
resulting in the placenta of the co-existing twin fetus.
5.3.2 Second trimester complete molar placentae display increased trophoblast
proliferation and apoptosis
Since increased trophoblast proliferation and apoptosis have been reported in complete
hydatidiform molar tissue from singleton pregnancies terminated in the first trimester, we first
set out to determine if second trimester molar tissue associated with a co-existent twin fetus
would show a similar phenotype. Proliferation was assessed by immunohistochemistry using an
antibody towards Ki67, a commonly used marker of cell proliferation (Endl et al., 2000) (Figure
5.2a-f).
In both molar twin cases (case 1 and case 2) the molar placenta exhibited an extreme level of
proliferation compared to both its co-existing twin and the control twin sets (Figure 5.2a-f). The
excessive Ki67 positive staining in the molar placenta was found in the trophoblast cells in both
the continuous trophoblast layers and the focal areas of cellular hyperplasia (Figure 5.2a,c). In
tissue sections from the genetically normal placenta from case 2, and from normal control twin
sets, Ki67 was expressed predominantly in the cytotrophoblast cells and in the occasional
mesenchymal cell (Figure 5.2d-f). Unexpectedly, the genetically normal placenta in the first
molar twin set was devoid of Ki67 detection (Figure 5.2b). No obvious differences in Ki67
expression were observed between twin placentae within the same pregnancy in the control twin
sets or between sets of control twins.
To determine if the two second trimester molar cases displayed excessive apoptosis,
characteristic of first trimester moles, we assessed the level of terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling (TUNEL) reactivity (Figure 5.3a-f). In contrast to
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Figure 5-2 Proliferative assessment of placentae from the mole and its co-existing twin
a-f: Immunolocalization of Ki67 in molar tissue (top, middle left), the placenta of its co-existing twin (top, middle
right), and in control twins (bottom panels). Brown staining represents positive Ki67 immunoreactivity.
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Figure 5-3 Apoptotic assessment of placentae from the mole and its co-existing twin by TUNEL staining
a-f: Labeling of apoptotic cells by enzymatic detection of DNA fragmentation using terminal transferase TUNEL
method in molar tissue (top, middle left), and in the placenta of its co-existing twin (top, middle right) and in control
twins (bottom panels). Brown staining represents positive TUNEL reactivity.
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the control twin sets, an increased number of apoptotic cells were found in both cases of molar
placentae, occurring mainly in the trophoblastic cells associated with the area of hyperplasia and
in the stromal region of the chorionic villi (Figure 5.3a,c). Limited apoptosis was detectable in
the control twins tested or in the genetically normal placenta form case 2, where the occasional
TUNEL positive cell was observed in the trophoblastic layer or syncytial knots (Figure 5.3d-f).
Interestingly, in the first molar case, an abundance of apoptosis was observed by TUNEL
reactivity in the genetically normal placentae of the co-twin (Figure 5.3b).
5.3.3 Pro-apoptotic Mtd is elevated in the molar placenta compared to its co-
existing twin and it is associated with apoptotic cells in the trophoblastic
and stromal areas
To determine if Mtd was associated with cell death in the molar tissue, we assessed the protein
expression of Mtd in conjunction with a number of molecules involved in apoptosis. Cleaved
caspase 8, cleaved caspase 3 and cleaved Parp1 are protein products commonly used to indicate
the presence of apoptosis, whereas Mcl-1 is an anti-apoptotic Bcl-2 family member known to
play an important role in trophoblast survival. Contrary to what we had anticipated, Mtd was the
only pro-apoptotic molecule tested that was found to be increased in the molar samples (Figure
5.4b-d). In contrast cleaved-Parp1, cleaved caspase-3, and cleaved caspase-8 were decreased or
unchanged in the molar placentae compared to either their co-existing twin or the normal twin
control sets (Figure 5.4a). In addition, no differences were observed for Mcl-1 between the
molar samples and the co-existing twin control (Figure 5.4b). We next assessed the transcript
level of Mtd-L to determine if the elevated levels of Mtd were due to increased gene
transcription. Mtd-L mRNA expression was increased in case 1 relative to its co-twin but similar
expression was observed between the molar and the control co-twin placentae for case 2 (Figure
5.4c).
Since Mtd is associated with cell death in the placenta, we performed co-localization studies of
Mtd and TUNEL to determine if Mtd was co-expressed in cells with fragmented DNA in the
molar pathology. As expected, Mtd expression was associated with TUNEL positive cells in the
molar placentae (Figure 5.4d upper panels). The placentae from the co-twin and normal twin
controls also displayed co-expression of Mtd and TUNEL however fewer TUNEL positive cells
were found in these samples (Figure 5.4d lower panels, data not shown). Interestingly cells
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Figure 5-4 Expression of apoptotic molecules in molar twins and control twins
a: Representative Western blot of apoptotic molecules: cleaved-Parp1, cleaved caspase-3, cleaved caspase-8, and b:
Mtd and anti apoptotic Mcl-1 in placental lysates from molar twin cases, and normal age matched twin controls.
Actin expression was used as an internal control. c: qRT-PCR analysis of MtdL mRNA from total placental tissue
lysates from molar twins (Mole, filled bar, n=2; co-existing twin, unfilled bar, n=2), and AMC twins (Con:A and
Con:B, hatched bars, n=7 twin sets). Data were normalized against expression of 18S ribosomal RNA using the well
established 2-CT
formula; fold changes are relative to AMC singleton pregnancy internal control group. Statistical
significance was assessed between control twins. P<0.05 Mann Whitney U test d: Spatial localization of Mtd
(green) with TUNEL (red) in placental sections from molar case 1 (left panels), and molar case 2 (right panels).
Arrow: cells positive for TUNEL reactivity
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that were TUNEL positive displayed predominantly cytoplasmic Mtd staining whereas Mtd was
primarily nuclear in cells that were TUNEL negative (Figure 5.4d).
5.3.4 Mtd localizes to the nuclei of proliferative trophoblast cells in molar
pathology
To determine if the pattern of Mtd expression seen in molar pregnancy was reflective of the role
of Mtd in proliferation, we performed dual labeled immunofluorescence analysis with antibodies
against Mtd and Ki67 (Figure 5.4 and 5.5). Similar to our previous reports, co-expression of
Mtd and Ki67 was localized to the cytotrophoblast cells in control sections (Ray et al., 2009)
(Figure 5.5b-d). An increased number of trophoblast cells in the molar tissue co-expressed Mtd
and Ki67 compared to the co-existing twin or normal twin control placentae (Figure 5.5a). In the
molar placentae Mtd-Ki67 co-localization could be detected underlying areas of excessive
syncytial accumulation, (Figure 5.6-i) throughout hyperproliferative sprouts (Figure5.6-ii.iii),
and in thinned trophoblastic areas (Figure 5.6-iv).
5.3.5 Mtd is associated with increased cyclin E1 in villous trophoblast cells of
molar placentae
Since the molar samples exhibited excessive proliferation, and displayed co-expression of Mtd
with Ki67 positive trophoblast cells, we tested the level of cyclin E1, a G1 phase cell cycle
regulator that we have previously reported to be associated with Mtd expression in the placenta
(Ray et al., 2009). In both molar sets, the samples from the molar tissue displayed higher levels
of cyclin E1 protein and mRNA expression compared to the placentae from their co-existing
twin controls (Figure 5.7a,b respectively). To test whether Mtd was co-localized with cyclin E1
in the molar pathology we performed dual labeled immunofluorescence with antibodies against
Mtd and cyclin E1 (Figure 5.7c). Similar to that seen in preeclampsia Mtd was expressed in
cyclin E1 positive trophoblast cells in the molar placentae (Figure 5.7c upper panels)(Ray et
al., 2009). Co-localization of Mtd and cyclin E1 was also observed occasionally in the placentae
of the co-existing twins and the normal twin controls, but to a lower extent (data not shown). In
addition cyclin E1 could be detected around the vasculature in the control sections (Figure 5.7c
lower panels), a characteristic absent in the molar pathology.
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Figure 5-5 Co-localization of Mtd with Ki67 expression in mole and twin placentae
Co-localization of Mtd (green) with Ki67 (red) in placental sections from molar case 2 (top left), and in the placenta
of its co-existing twin (top right), and in the placentae of a representative set of age matched control twins (bottom
panels). Right hand panels: high magnification of the boxed areas. Arrow: cytotrophoblast cells expressing both Mtd
and Ki67
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Figure 5-6 Mtd is expressed in proliferative trophoblast cells associated with various molar characteristics
Co-localization of Mtd (green) with Ki67 (red) in (i) areas of excessive syncytial accumulation, (ii-iii)
hyperproliferative sprouts, and (iv) in thinned areas of trophoblast. Middle and right hand panels: high magnification
of the boxed areas. Arrows: trophoblast cells expressing both Mtd and Ki67
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Figure 5-7 Cyclin E1 is overexpressed in molar placentae compared to twin controls
a: Representative Western blot of cyclin E1 in placental lysates from molar twin cases, and normal age matched
twin controls. Actin expression was used as an internal control. b: qRT-PCR analysis of cyclin E1 mRNA from total
placental tissue lysates from molar twin sets (Mole, filled bars, n=2; co-existing twin, unfilled bars, n=2), and AMC
twins (Con:A and Con:B, hatched bars, n=7 twin sets). Data were normalized against expression of 18S ribosomal
RNA using the well established 2-CT
formula; fold changes are relative to group of age matched singleton
pregnancy. Statistical significance was assessed between control twins. P<0.05 Mann Whitney U test c: Spatial
localization of Mtd (green) with cyclin E1 (red) in placental sections from molar case 2 (top panels), and in the
placenta of its co-existing twin (bottom panels). Right hand panels: high magnification of the boxed areas. Arrows:
cytotrophoblast cells expressing both Mtd and cyclin E1; Arrowheads: cyclin E1 expression in endothelium.
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5.3.6 Molar placentae exhibit decreased levels of cell cycle inhibitor p27
As shown in chapter 3, the CDK2 inhibitor, p27 is highly expressed in association with high
levels of cyclin E1 in placentae from severe early onset preeclamptic pregnancies. We therefore
investigated the expression of p27 in the molar pathology. In contrast to early onset severe
preeclampsia, lower levels of p27 protein were observed in molar placentae compared to their
co-existing twin or the normal twin controls (Figure 5.8a). Co-localization of p27 with cyclin E1
also demonstrated p27 expression to be decreased in the cyclin E1 positive cells in the molar
tissue in comparison to the controls (Figure 5.8b).
5.3.7 Molar placentae exhibit altered expression of molecules involved in
regulating the G1 phase of the cell cycle
Next we assessed the expression of D type cyclins in the placenta as well as the CDK inhibitor
p21, to determine whether the increased proliferation in the mole was facilitated by cell cycle
events in the G1 phase additional to the increase in cyclin E1. Both cyclin D1 and cyclin D3
were decreased at both protein and mRNA levels in the molar pathologies relative to the
placentae of the co-existing twin from case 2 and the control twin sets (Figure 5.9a,b).
Interestingly, expression of cyclin D1 and cyclin D3 in the molar placentae was predominantly
localized to the stroma (Figure 5.9c,d), whereas in control sections cyclin D1 was expressed
primarily in the cytoplasm of the syncytiotrophoblast (Figure 5.9c) and cyclin D3 was expressed
in the nuclear compartment of cells of the trophoblastic layer (Figure 5.9d). In contrast to the
expression of the D type cyclins or p27, p21 expression was increased in the molar samples
compared to the placentae of their co-existing twins (Figure 5.9a). Localization of p21 however
was restricted to the nuclei of trophoblast cells in both molar and control placental sections
(Figure 5.9d). Of note, the overall level of protein expression for cyclin D1, cyclin D3 and p21,
was low in case 1 (Figure 5.9a). No significant differences in cyclin D1, D3 or p21 expression
were seen within or between normal twin placental controls (Figure 5.9a).
5.4 Discussion
The placenta shares similar features to that of a controlled cancer, with highly proliferative and
invasive properties. However, unlike a cancer it is intrinsically regulated to regress and evade
pathological progression. In molar pregnancy, this regulation is altered, causing uncontrolled cell
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Figure 5-8 p27 expression in molar placentae and twin controls
a: Representative Western blot of p27 in placental lysates from molar twin cases, and normal age matched twin
controls. Actin expression was used as an internal control. b: Spatial localization of p27 (green) with cyclin E1 (red)
in placental sections from molar case 2 (top panels), and in the placenta of its co-existing twin (bottom panels).
Right hand panels: high magnification of the boxed areas. Arrow: cell expressing cyclin E1, Arrowhead:
cytotrophoblast cells expressing both p27 and cyclin E1.
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Figure 5-9 Expression of G1 phase cell cycle regulators in the molar and control twins
a: Representative Western blot of: cyclin D1, cyclin D3, and p21 in placental lysates from molar twin cases (M,Tw),
and AMC twins (A,B). Actin was used as an internal control. b: qRT-PCR analysis of cyclin D1 and cyclin D3 in
total placental tissue lysates from molar twin cases (Mole, filled bars, n=2; co-existing twin, unfilled bars, n=2), and
AMC twins (Con:A and Con:B, hatched bars, n=7 twin sets). Data were normalized against 18S using the well
established 2-CT
formula; fold changes are relative to singleton AMC group. Statistical significance was assessed
between control twins. P<0.05 Mann Whitney U test c,d: Spatial localization of c: cyclin D1 (red) or d: p21 (green)
with cyclin D3 (red), in placental sections from molar case 2 (top left), and in the placenta of its co-existing twin
(top right), and in a set of age matched control twins (bottom panels). Right hand panels: high magnification of the
boxed areas. c: Arrow: syncytiotrophoblast; Arrowhead: cytotrophoblast cells negative for cyclin D1. d: Arrow:
trophoblast cells positive for cyclin D3; arrowhead: trophoblast cells positive for p21.
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behavior, and in many cases leads to malignant transformation. This can result in persistent
trophoblastic disease and in the most severe cases choriocarcinoma. Herein, we examined
molecules known to play key roles in regulating normal trophoblast cell fate, to gain further
insight in to the pathogenesis of molar pregnancy. Specifically we discovered 1) that Mtd, a pro-
apoptotic Bcl-2 family member, is increased in molar placentae, 2) that the expression of Mtd in
molar tissue is associated with both hyperproliferative and apoptotic characteristics of placental
cells, in particular that the hyperproliferation of the trophoblast cells are associated with an
imbalance between low p27 and high cyclin E1 expression, and 3) that decreased expression of
cyclin D1 and cyclin D3 are associated with increased p21 expression in the trophoblast layers of
molar tissue.
This study reported on two unique cases of molar pregnancy consisting of a complete
hydatidiform mole and a co-existing fetus. These molar twin cases are extremely rare with a
frequency reported at 1 in 22,000 to 1 in 100,000 pregnancies (Vaisbuch et al., 2005). A number
of studies have reported on singleton molar pregnancy from the early first trimester, however,
due to the immediate evacuation of these placentae once identified, molar tissue at later
gestations have not been studied at the molecular level. Interestingly, the second trimester molar
cases presented in this study displayed classic morphological signs of molar villi, including
vessel swelling, circumferential hyperplasia and cistern formation. Molar placentae typically
present with few or no villous blood vessels, and those present are usually collapsed and empty
(Kim et al., 2006). The two cases presented here also had no obvious vessel architecture
indicative of progressed vessel deterioration. In addition it is characteristic for molar placentae to
have apoptotic debris in the stromal area, as seen in the two cases presented. Pathological
description of these two cases suggests that second trimester molar twin cases are grossly
equivalent to first trimester moles that develop as a singleton pregnancy.
Importantly, these rare, molar-twin cases provide a unique model to study the molecular
pathophysiology of molar development, as each molar case developed adjacent to a genetically
normal twin placenta, used as a baseline control in the analysis of the data. Our findings are
therefore highly reflective of the innate genetic and molecular differences between molar tissue
development and placental development, represented by the co-existing twin placenta. However,
due to the complications associated with the molar twin pregnancies, the molecular findings were
also compared to normal age-matched twin controls. Interestingly the first case of molar twins
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displayed low levels of all the molecules examined, with the exception of TUNEL reactivity.
This finding suggests that the pregnancy in case 1 was severely compromised, possibly due to
the bacterial infection. Nevertheless, when the molar tissue in case 1 was compared to its co-twin
placenta, the trends were found to be similar to that of case 2. In case 2 the genetically normal
co-placenta displayed slight elevations in the apoptotic markers cleaved Parp1 and cleaved
caspase 8, as well as elevated levels of cyclin E1 and p27 in comparison to the control twin sets.
The increase in apoptotic markers likely reflects the clinical finding that the co-twin in case 2
was IUGR and presented with preeclampsia, two pathologies associated with increased placental
apoptosis. Likewise the increase in cyclin E1 and p27 may also reflect the incidence of early
onset preeclampsia, as seen in chapter 3.
Importantly, molar tissue provides a unique model to study the mechanisms and molecules that
regulate both cell proliferation and cell death. We have previously reported on Mtd, a pro-
apoptotic member of the Bcl-2 family, to be highly expressed in the early stages of normal
placental development and in severe early onset preeclampsia, where it was found to regulate
apoptotic cell death as well as cell proliferation (Soleymanlou et al., 2005b;Ray et al., 2009).
The high level of Mtd expression found in molar tissue in the current report was therefore not
surprising, as molar pregnancy represents an exemplary pathological state of excessive
trophoblast proliferation and placental apoptosis.
Previous reports have described apoptosis occurring in both the cytotrophoblast layer and in the
villous stroma in first trimester molar pregnancy (Halperin et al., 2000;Kim et al., 2006).
Similarly, in this study Mtd was found to co-localize to TUNEL positive cells in both the
cytotrophoblast and stromal region supporting the idea that Mtd plays a role in apoptosis in this
pathology. In the stroma of molar tissue, cell death has been associated with the destruction of
the primitive vascular network, resulting in the fluid accumulation and villous swelling (Kim et
al., 2006), as well as the classic acellular stromal characteristic. Whereas apoptosis in the
trophoblast layers is associated with the exacerbated proliferation, similar to that seen in
preeclampsia. It is therefore possible that Mtd may contribute to both the acellular and avascular
stromal feature of molar development, as well as the excessive trophoblastic shedding and
subsequently high incidence of preeclampsia in this pathology.
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In addition, a number of studies have linked the level of apoptosis in molar pregnancy with the
potential for malignant transformation, where high apoptotic rates are suggested to be indicative
of a favorable prognosis (Qiao et al., 1998;Wong et al., 1999;Chiu et al., 2001). These findings
have been based primarily on TUNEL reactivity and the detection of caspase-cleaved
cytokeratin, although additional studies have suggested that the expression of Bcl-2 family
members may also provide further insight as to the aggressiveness of this pathology. It has been
theorized that the Bcl-2/Bax ratio may be an important indicator of overall cell death in the mole
(Qiao et al., 1998). However, a more recent study reported that Mcl-1 expression displayed a
more significant correlation to the prognosis of gestational trophoblastic disease (GTD)
compared to either Bcl-2 or Bax (Qiao et al., 1998;Fong et al., 2005). High levels of Mcl-1 were
found to be indicative of a poor prognosis, likely due to the anti-apoptotic effect of the protein.
Importantly Mcl-1 is the primary binding partner of Mtd. The interaction of Mcl-1 with Mtd
prevents the apoptotic effect of Mtd and has been shown to lead to the decreased incidence of
cell death (Soleymanlou et al., 2007). Since molar tissue expresses high levels of Mtd, the
Mtd/Mcl-1 rheostat may be of significance to molar development and progression. Interestingly,
in the two cases presented we found elevated Mtd expression and no difference in Mcl-1 levels
compared to control; an Mtd/Mcl-1 ratio that in theory would be favorable for high levels of cell
death and thus molar regression. This is supported by the high incidence of apoptosis seen in our
cases. A follow up study of these patients would be required to confirm that molar regression
resulted in these cases following molar evacuation, however in case 2 a full hysterectomy was
performed preventing further follow up analysis.
In general, apoptosis can result from activation of the extrinsic or intrinsic apoptotic pathways
involving the caspase cascade. Activation of caspase initiators (casp-2, 8, 9,10) leads to cleavage
and activation of the executioner caspases such as caspase 3 and 7 directly or through
mitochondrial depolarization (intrinsic pathway, involving members of the Bcl-2 family).
Activation of downstream executioner caspases results in cellular changes, such as cytoskeletal
rearrangement, due to cleavage of a variety of cellular proteins including Parp1. Interestingly,
although the clinical cases presented demonstrated an increased Mtd/Mcl-1 ratio and increased
TUNEL positivity, the majority of the apoptotic molecules examined (cleaved caspase-8, cleaved
caspase-3, cleaved Parp1) displayed lower or equivalent levels of expression compared to
controls. This suggests that the majority of the molecules tested may not be significant
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contributors to apoptosis in the mole. Similarly Wong 1999 and Qiau 1998 found that Bax had
no correlation with the apoptotic index in molar tissue and Fong et at 2006 found no difference
in expression of either caspase-8 or caspase-10 between molar tissue and placentae from normal
pregnancy. It is therefore possible that apoptosis in molar tissue is carried out through related
caspases such as initiator caspases 2 or 9 and executioner caspase 7, not examined in this study.
Further examination of these molecules in association with Bcl-2 family members such as Mtd
and Mcl-1in molar tissue is therefore warranted.
p53 is another pro-apoptotic molecule involved in both cell death and cell proliferation and,
similar to Mtd, it is elevated in the trophoblast cells of molar tissue (Qiao et al., 1998;Halperin et
al., 2000). Interestingly, mutation of the p53 gene has been associated with its abundance in a
number of cancers, however, in molar tissue it is the wild type form of p53 that is overexpressed
(Cheung et al., 1994;Halperin et al., 2000). Importantly, p53 has been shown to induce Mtd
expression, and it may therefore contribute to the elevated levels of Mtd seen in this pathology
(Yakovlev et al., 2004). In addition, studies have shown that p53 may be important in the
neoplastic nature of the mole (Qiao et al., 1998). It would therefore be interesting to compare the
level of Mtd in molar samples that regress to those that persist, to examine whether Mtd has a
similar effect. This area of study warrants further investigation.
In the current report Mtd co-localized to both proliferative (Ki67 and cyclin E1 positive) as well
as apoptotic (TUNEL positive) cells in the two molar cases, suggesting that in molar tissue Mtd
functions in promoting cell cycle progression in addition to cell death, similar to its function in
normal placentation and in preeclampsia. Furthermore, in the first data chapter we found Mtd to
influence the cell cycle through its positive impact on cyclin E1 (Ray et al., 2009). Our finding
that Mtd co-localized with Ki67 and cyclin E1 in trophoblast cells in molar tissue, therefore
suggests that Mtd may contribute to the hyperproliferative nature of the molar pathology by
promoting the expression of cyclin E1. This was supported by the finding that the two molar
cases displayed increased levels of cyclin E1 and Ki67 compared to the co-existing twin or
control twin sets. Similarly, Ki67 and cyclin E1 have also been found to be increased in first
trimester molar placentae compared to normal placental development of the same gestation
(Fukunaga, 2004). Olvera et al 2001 had comparable findings for cyclin E1 and Ki67 and in
addition reported increased levels of Cdk2 and E2F1 in the trophoblastic layers of molar tissue
(Olvera et al., 2001). Activation of Cdk2 by cyclin E1 leads to the release of E2F1 from its
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inhibitor, Rb, and the subsequent transcription of E2F1 regulated genes. Interestingly, E2F1
regulates transcription of both cyclin E1 and Mtd, and this may explain the high levels of these
molecules observed in our second trimester molar cases.
In addition to Rb, the Cyclin E-CDK2 complex also phosphorylates the CDK inhibitor p27 at the
Thr187 site leading to its destruction in the nucleus (Vlach et al., 1997). This may explain the
low levels of p27 observed in the molar tissue. Furthermore, the low level of p27 maintained in
the trophoblast cells is likely insufficient to fully inhibit cell cycle progression. Interestingly, the
low level of p27 seen in the molar pathology is in contrast to the elevated levels of p27 seen in
severe early onset preeclampsia in the previous chapter. This observation indicates that although
the two placental pathologies share similar characteristics, there are likely key differences in
upstream events that underlie the two pathologies. Nevertheless, in both preeclamptic and molar
placentation the inhibitory effect of p27 appears to be hampered thus, contributing to the
proliferative nature the disorders.
Although low levels of p27 were observed in the two molar cases presented, we did see an
increase in expression of the related CDK inhibitor p21 in the trophoblastic layers. p21 can
hinder cell cycle progression by inhibiting the function of the cyclin E1-CDK2 complex, or it can
function in the assembly of cyclin D-CDK complexes and aid in promoting cell cycle
progression (Labaer et al., 1997;Cheng et al., 1999). Interestingly, in the control samples p21
was occasionally co-localized with cyclin D3. It is therefore possible that p21 functions in the
assembly of cyclin D3-CDK complexes in normal placental development. In contrast, no cyclin
D1 or cyclin D3 was observed in the trophoblastic layers in the molar tissue. This suggests that
the high levels of p21 may function to inhibit, rather than promote, cell cycle progression in the
trophoblast cells of molar tissue. Furthermore, p21 expression was primarily restricted to the
more distal trophoblast cells in the molar pathology, consistent with a role in differentiation or
quiescence. Future studies are warranted to determine if p21 functions in the differentiation of
the trophoblast cells in molar tissue. If so it could serve as a good prognostic indicator against
malignant transformation in molar cases. However, Cheung et al 1998 have previously reported
that there was no significant difference in the expression of p21 between cases that persisted and
those that regressed (Cheung et al., 1998).
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In the current study, we found that the molar tissue expressed very low levels of cyclin D1 and
cyclin D3. In addition, the small amount of protein expressed in the mole was restricted
primarily to the stromal tissue, whereas in normal placental tissue both cyclin D1 and cyclin D3
were expressed by the trophoblastic layers. Expression of nuclear cyclin D1 in the stromal cells
of the molar tissue is similar to the pattern of expression seen for cyclin D1 in early first
trimester (data not shown) suggesting that the stromal tissue in molar placentae may remain in an
undifferentiated state. Cytoplasmic cyclin D1, such as that seen in the syncytium of the control
sections, has been linked to cell differentiation of murine embryonic stem cells (Bryja et al.,
2008). Therefore the lack of cyclin D1 in the trophoblast of the mole may also reflect a poorly
differentiated state of the molar tissue. Alternatively the low level of cyclin D1 expression may
be due to increased expression of E2F-1 which has been shown to inhibit activation of the cyclin
D1 promoter (Watanabe et al., 1998).
Interestingly, reports on murine models suggest that D type cyclins are not crucial for placental
development, as cyclin D triple knockout mice were found to have no defects in placentation
(Kozar et al., 2004). In addition, certain cell types, including some embryonic tissues, have been
shown to proliferate efficiently in the absence of D type cyclins (Kozar et al., 2004). D type
cyclins are normally controlled by mitogenic stimuli and function to link cues from the extra
cellular environment with control over cell proliferation. The fact that trophoblast cells in the
mole proliferate independent of cyclin D expression suggests that molar tissue may be unaffected
by external cues. In addition these finding also suggest that molar tissue could be used to study
the difference in cyclin D dependent and cyclin D independent cell cycle machinery.
Many studies have focused on uncovering traits that will distinguish whether a molar case will
become persistent or if it will regress. Although the presence, or absence, of cell cycle regulating
molecules have been associated with oncogenic transformation, (Sgambato et al.,
2000;Slingerland et al., 2000;Kozar et al., 2004) studies have yet to find a cell cycle regulating
molecule that has prognostic value in the mole. There is however evidence that the level of
apoptosis may be a reliable diagnostic tool. It is therefore important to learn more about the
proliferative and apoptotic activity in the pathophysiology of molar pregnancy so that diagnostic
methods and clinical treatments can be identified.
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6 Summary and Future Directions
Proper formation of the placenta is essential for fetal development and a successful healthy
pregnancy. In humans, this process involves a balance in cellular proliferation, differentiation
and death of the trophoblast lineage, the cells forming the placenta. Consequently, abnormality in
the regulation of trophoblast cell fate may lead to inadequate placental function and subsequent
pregnancy related complications.
Preeclampsia is a life threatening disorder of pregnancy, common in the obstetric field. It has
been well established that the cause of preeclampsia stems from the placenta, however to date,
no reliable prognostic or diagnostic tools for preeclampsia exist. Moreover, the current standard
of treatment for preeclampsia remains delivery and removal of the placenta. Prior research has
established that the maternal symptoms of preeclampsia arise from inflammation and endothelial
dysfunction, occurring from a reaction of the maternal endothelium to placental-derived factors.
In cases of severe early onset preeclampsia this is believed to occur due to excessive deportation
of placental debris into the maternal circulation. In contrast, late onset preeclampsia appears to
result from a sensitivity of the maternal endothelium to normal levels of placental debris, and is
thus a maternally derived disorder. In either case, women with preeclampsia may require prompt
delivery of the fetus, causing potential developmental complications to the newborn.
Complete molar pregnancy is yet another placental disorder. In these cases, placental tissue
develops in the absence of a fetus, and presents a high risk of developing preeclampsia and
choriocarcinoma requiring chemotherapy. Although separate pathologies, both preeclampsia and
molar pregnancy share similar characteristics including an immature proliferative trophoblast
phenotype accompanied by excessive cell death and increased trophoblast turnover. Deciphering
the mechanisms that regulate cell proliferation and cell death in the placenta will therefore allow
us to gain insight into the pathogenesis of both these pregnancy-related disorders.
The objective of the current dissertation was to further our knowledge on the systems governing
the balance between cell death and cell proliferation in the placenta and to identify alterations in
the expression of cell fate regulatory molecules that may contribute to placental pathologies.
Work presented in this thesis provides novel molecular insights into the regulation of trophoblast
cell proliferation by Mtd and cell cycle regulating molecules in normal placental development as
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well as in preeclampsia and molar pregnancy. Importantly the data presented in this dissertation
also describe molecular differences between clinically related but distinct placental pathologies,
further advancing the potential for future diagnostic, prognostic and treatment strategies.
The first data chapter of the dissertation (Chapter 3) examined the role of Mtd, a pro-apoptotic
molecule, in normal placental development. This line of investigation identified a novel role for
Mtd in the regulation of trophoblast cell cycle in placentation. The dual roles of Mtd were
associated with changes in its subcellular localization, with Mtd localizing to the mitochondria
during apoptosis, and to the nucleus in proliferative cells. Although the current dissertation
revealed insight as to how cell death and proliferation in the trophoblast may be interconnected
by Mtd, we recognize that these processes are extremely complex and involve a multitude of
players. Recently a number of classical apoptotic and cell cycle regulating molecules have been
identified as having multiple roles related to cell fate. Caspases (cysteine-aspartate proteases) are
among this group. Although caspases are classically known for their prominent role in apoptosis,
recent studies have revealed a variety of non-apoptotic functions of these cysteine proteases,
including roles in cell differentiation, cell motility, and importantly cell cycle regulation
(Feinstein-Rotkopf and Arama, 2009). In future studies, it would be of value to determine if
caspases, in conjunction with Mtd, are involved in the regulation of trophoblast cell cycle in the
human placenta.
Studies presented in this dissertation also revealed that the Mtd-L isoform had a positive effect
on cyclin E1 expression and promoted G1-S phase transition. However the means by which Mtd
impacts cyclin E1 expression remains unknown. As such, it would be of significance to
determine the mechanism linking Mtd to cyclin E1 expression. The Caniggia lab has previously
shown that active caspase-3 is a downstream effecter of Mtd function during apoptosis
(Soleymanlou et al., 2005b). Importantly, a number of molecules involved in cell cycle
regulation, including cell cycle inhibitors p21 and p27, are reported to be cleaved by caspases
(Eymin et al., 1999;Woo et al., 2003). To date, caspase-3 has been found to promote cell cycle
progression in lymphoid cells, forebrain cells and keratinocytes. In lymphoid cells this has been
attributed to caspase cleavage of cell cycle inhibitor p27. In contrast, caspase-3 has been found to
inhibit proliferation of B cells through a mechanism involving cleavage of p21 (Waga et al.,
1994;Woo et al., 2003). The data presented in this dissertation suggests that p27 is a prominent
antagonist to cyclin E1 in the placenta. Therefore it would be of interest to determine if caspase
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mediated cleavage and subsequent inactivation of p27 in trophoblast cells may be the link
between Mtd and cyclin E1 expression. A putative model is depicted in Figure 6.1.
In the second and third data chapters (Chapters 4 and 5), an association between Mtd and
increased levels of cyclin E1 was discovered in cytotrophoblast cells from placentae of early
onset severe preeclampsia and molar pregnancy. These findings demonstrate that Mtd plays an
intricate role in maintaining the natural homeostasis in trophoblast cell fate, and it suggests that
the increased expression of Mtd previously seen in preeclampsia (Soleymanlou 2005) likely
contributes to both the hyperproliferative and apoptotic nature of the placenta typical of this
disorder.
Studies presented in Chapter 4 of the dissertation also addressed the cause of the
hyperproliferative state of trophoblast cells in preeclampsia at the level of the cell cycle. First, in
order to understand the potential consequences of elevated cyclin E1 expression in placental
disease, its role and regulation in normal placentation were assessed. This series of studies
indicated that cyclin E1 was maximally expressed in the early stages of placentation where its
expression was inversely correlated with that of the CDK inhibitor p27. We also determined that
a low oxygen environment in conjunction with high levels of TGF3, such as that previously
found in early first trimester and in the preeclamptic pathology, can lead to increased cyclin E1
and p27 protein expression. In light of this data we hypothesized that placental pathologies, such
as preeclampsia, would display increased Mtd and cyclin E1 and would exhibit low levels of p27
resembling the molecular profile of an immature early first trimester placenta. In contrast to our
hypothesis, we discovered an increased level of p27 expression in severe early onset
preeclampsia. However, p27 in this pathology was localized primarily to the cytoplasm.
Moreover, this led to the identification of elevated levels of p27 phosphorylation at the Ser10 site
in severe early onset preeclampsia. We speculated that this post-translational modification led to
the observed increase in cytoplasmic localization of p27, preventing its inhibition on cyclin E1-
mediated CDK2 activation. We further hypothesized that this may contribute to the increased
trophoblast turnover and syncytial shedding seen in preeclampsia. We did not however examine
the mechanism by which p27 was phosphorylated in the preeclamptic pathology. Excessive
trophoblast deportation is one of the primary factors leading to endothelial dysfunction in severe
early onset preeclampsia. Thus, identifying the upstream factors leading to the increased
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Figure 6-1 Putative model of the mechanism linking Mtd to cyclin E1 expression
Mtd initiates mitochondrial depolarization leading to caspase-3 cleavage and activation. Activated caspase-3 cleaves
the CDK inhibitors p21 and p27 alleviating this inhibition from the cyclin E-CDK2 complex. The activated cyclin E
CDK2 complex then phosphorylates Rb which in turn dissociates from the E2F transcription factor. Once free, E2F
activates the transcription of multiple genes, including Mtd and cyclin E1, thereby creating a positive feedback loop.
140
trophoblast turnover may aid to the discovery of potential treatments to help alleviate the clinical
symptoms of preeclampsia and thus the overall severity of the disease.
Ser10 phosphorylation of p27 has been reported to occur at the G1 phase downstream of KIS,
PKB/Akt, and ERK signaling (Besson et al., 2008) (Figure 6.2). Phosphorylation at this site
during the G1 phase promotes nuclear export by providing a binding site for CRM1/Exportin.
Alternatively, in G0, Ser10 phosphorylation results from activation of Mirk/Dyrk kinase
therefore conferring p27 stability in the nucleus (Deng et al., 2004). Since our observations in
preeclampsia are consistent with an increase in G1 phase activity, it would be relevant to
examine the pathways that have been reported to phosphorylate p27 during the G1 phase.
hKIS (human kinase interacting stathmin) was discovered in 2002 to be the major kinase that
phosphorylates Ser10 in p27 (Boehm et al., 2002). In addition, it was shown to promote cell
cycle progression in mouse embryonic fibroblast cells as well as human leukemia cells
(Nakamura et al., 2008) by stabilizing p27 in the cytoplasm during the G1 phase. Interestingly,
Boehm et al also found that hKIS was highly expressed in the placenta; supporting our
hypothesis that p27 may be phosphorylated by hKIS during placentation. To our knowledge
there are no reports on hKIS activity in the placenta or on the expression of hKIS in
preeclampsia. It would therefore be informative to determine if hKIS phosphorylates p27 in
trophoblast cells and to examine whether hKIS levels are altered in severe early onset
preeclampsia.
A second pathway described to phosphorylate p27 Ser10 in the G1 phase is the PKB/Akt
pathway (Fujita et al., 2002;Besson et al., 2004). However, it is unlikely that the increased p27
phosphorylation seen in our preeclamptic samples results from Akt phosphorylation, as the Akt
pathway is reported as being either unaltered or diminished in this pathology (Orcy et al.,
2008;Chiang et al., 2009). Alternatively, p27 could be phosphorylated at Ser10 by ERK
(extracellular signal-regulated kinase) downstream of the MAPK (mitogen activated protein
kinase) pathway (Ishida et al., 2000). This is supported by Shin et al who have reported ERK to
be increasingly active in severe preeclampsia (Shin et al., 2009).
Our studies also revealed that severe early onset preeclampsia is associated with cytoplasmic
localization of p27, and we hypothesize that p27 is stabilized in the cytoplasm in this pathology.
Stabilization of p27 in the cytoplasm has been reported to occur through its interaction with
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Figure 6-2 Phosphorylation of p27 at the ser10 site.
Ser10 phosphorylation of p27 occurs in the G0 phase by the Mirk/Dyrk kinase and in the G1 phase downstream of
KIS, PKB/Akt, and ERK signaling. Phosphorylation at this site during the G1 phase promotes nuclear export by
providing a binding site for CRM1/Exportin. In G0, ser10 phosphorylation confers p27 stability in the nucleus by
binding to Mirk/Dyrk.
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cyclin D-CDK complexes or by further phosphorylation at its Thr-157 or Thr-198 sites, leading
to its interaction with 14-3-3 (Besson et al., 2008;Vervoorts et al., 2008) (Figure 6.3).
Determining the mechanism that facilitates p27 stability in the cytoplasm in preeclampsia may
be of interest, as therapeutic treatment targeted at disrupting the stabilizing factor may allow p27
to reenter the nucleus and prevent trophoblast hyperproliferation.
The comparison of cyclin E1 and p27 expression, between preeclampsia-related pathologies
including late onset preeclampsia and IUGR was another important study presented in the
dissertation. Here we discovered that the high levels of cyclin E1 and cytoplasmic p27 were
unique to the severe early onset form of preeclampsia, whereas neither feature was associated
with either late onset preeclampsia or IUGR. These findings indicate that cyclin E1, in addition
to altered p27 regulation, aids in the hyperproliferative state of severe early onset PE and that
this may be potentiated by the low pO2 and elevated levels of TGF in the preeclamptic
environment. These molecular data also support the theory that severe early onset preeclampsia
is a distinct pathology from late onset preeclampsia. A further understanding of the differences
between placental-related pathologies will provide a basis with which to improve diagnostic
methods in the future and aid in tailoring more direct and effective approaches to treatment.
In the final chapter two unique cases of second trimester twin pregnancies, including a complete
mole and co-existing twin, were examined. Through these studies Mtd was identified as being
highly expressed in the molar pathology, localizing to both apoptotic cells present in the
trophoblast and stromal regions, as well as to the hyperproliferative areas of trophoblast cells. In
addition, Mtd expression was abundant in trophoblast cells positive for cyclin E1, similar to what
we have shown in severe early onset preeclamptic cases. In contrast to the elevated levels of p27
seen in severe early onset preeclampsia, molar tissue displayed decreased levels of p27. We
therefore propose that the high abundance of Mtd and cyclin E1 in conjunction with the low level
of p27, may contribute to the hyperproliferative nature of the disorder. Low levels of p27 are
commonly associated with various cancers, resulting primarily from increased proteolytic
degradation rather than allelic loss (Sherr et al., 2004). Hence, it would be informative to
determine if the low level of p27 associated with molar development results from altered p27
transcription or from increased proteolytic degradation of the protein.
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Figure 6-3 Phosphorylation of p27 and the effect on protein localization and stability
The cyclin E1-CDK2 complex phosphorylates p27 at the Thr187 residue revealing a binding site for Skp2-SCF.
Ser10 phosphorylation of p27 in the G0 phase by the Mirk/Dyrk kinase confers p27 stability in the nucleus by
binding to Mirk/Dyrk. Phosphorylation at ser 10 by KIS, PKB/Akt, or ERK during the G1 phase promotes nuclear
export by providing a binding site for CRM1/Exportin. P27 is stabilized in the cytoplasm by interaction with cyclin
D-CDK complexes or by further phosphorylation at Thr157 or Thr198 leading to its interaction with 14-3-3.
Interaction of p27 with RhoA is involved in cell migration.
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Both severe early onset preeclampsia and molar pregnancy are hyperproliferative placental
pathologies that display elevated levels of trophoblast apoptosis. Importantly, in this dissertation
we discovered that placentae from both disorders displayed similarities in their molecular profile,
including increased Mtd and cyclin E1 expression. However, our data also highlighted the
differences that occur in cell cycle regulation between the pathologies, namely the differences in
p27 regulation. A putative model comparing Mtd, cyclin E1 and p27 expression in molar
pregnancy and severe early onset preeclampsia is summarized in (Figure 6-4). This underscores
the importance of investigating cell cycle regulation in independent placental pathologies, as
potential treatments strategies may have different effects in each case.
Current clinical practice following evacuation of complete molar tissue is to monitor the human
chorionic gonadotropin (HCG) levels in the women in order to determine if their molar
pathology fully regressed. Remaining HCG levels indicate persisting tissue, and only then can
the physician plan additional treatment, often including chemotherapy. This current method of
practice is time consuming for both the patient and the physician and most importantly can result
in a lengthy period of uncertainty and anguish for the already devastated woman. It is therefore
imperative that research continues toward discovery of prognostic indicators of molar pregnancy
outcomes. Previous studies suggest that cell death regulating molecules may be the link to the
behavior of molar tissue. Both TUNEL positivity and Mcl-1 have been hypothesized to be
potential indicators of molar regression and persistence (respectively) (Qiao et al., 1998;Wong et
al., 1999;Chiu et al., 2001). Importantly, Mtd is a pro-apoptotic molecule that interacts with Mcl-
1, and since we found Mtd to be increased in molar pathology, it would be worth studying the
relationship between Mtd and molar progression in cases that regress compared to those that
persist.
Further investigation into the cell cycle regulation of molar tissue uncovered multiple levels of
disregulation at the G1 phase. These included decreased levels of cyclin D1 and cyclin D3 as
well as high levels of the CDK inhibitor p21. These findings suggested that cyclin E1 is
sufficient to push the cell cycle through the G1 phase in trophoblast cells, and highlights a
potential role for p21 in molar development. It will be important in later studies to decipher the
level of interaction and interplay between the D type cyclins, cyclin E1, p21 and p27 in normal
placental development as well as in preeclampsia and molar pregnancy.
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Figure 6-4: Model of Mtd, cyclin E1 and p27 expression in placentae from severe early onset preeclampsia,
and complete molar pregnancy
Both severe early onset preeclampsia and complete molar pregnancy display increased levels of Mtd expression.
This is associated with increased apoptosis and cell cycle progression. Increased proliferation is likely related to the
elevated levels of the cell cycle activator cyclin E1 (E1), seen in both disorders. In molar pregnancy, the CDK
inhibitor, p27, is decreased. This likely aids in the activating effect of cyclin E1. In contrast, the levels of p27 are
elevated in severe early onset preeclampsia; however, p27 is phosphorylated at the Ser10 site and translocated to the
cytoplasm where it likely does not inhibit cell cycle progression.
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Together the data presented in this dissertation support our hypothesis that Mtd is an important
regulator of cell death and proliferation in the placenta. Furthermore, this work lends to the
knowledge of how cell cycle regulation may contribute to placentation and suggests that the
balance between Mtd, cyclin E1 and p27 are key to determining the fate of cell cycle progression
in the trophoblast. In conclusion, the work presented in this dissertation has uncovered novel
insights into the regulation of trophoblast apoptosis and cell cycle regulation. Not only is Mtd a
pro-apoptotic regulating molecule, but in the trophoblast it performs a second role in cell cycle
regulation. Importantly, the impact of Mtd on cyclin E1 to promote G1-S transition is a novel
and significant molecular mechanism found to regulate trophoblast cell proliferation in normal
and pathological placentation. Equally important is our identification of molecular differences
that may help to differentiate placental pathologies including early and late onset preeclampsia,
IUGR and molar pregnancy. Cell death and cell cycle regulation are however extremely complex
processes involving a multitude of players. Therefore, although we have uncovered a portion of
the puzzle there are many aspects involved in these events that still need to be addressed further.
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7 Future Directions – Experimental Design and
Preliminary Data
7.1 Determine if caspase-3 is the connecting link between Mtd and
cyclin E1 through the cleavage of p21 and p27.
The direct mechanism linking Mtd to cyclin E1 expression remains unknown. We hypothesize
that caspase activation, downstream of Mtd, leads to cleavage of p27 and allows for cell cycle
progression. Analysis of the human placental lysates used in our studies revealed the presence of
potential p21 and p27 cleavage products, in addition to the full length protein. Through Western
blot analysis one potential p21 and two potential p27 cleaved products that were identified, were
were differentially expressed across gestation. These bands were consistent with the predicted
size of cleaved p21 and p27, generated by caspases (Figure 7.1a). Moreover, the pattern of the
p27 (c-terminal: 12kd and N-terminal: 15kd, 23kd) bands correlated with the presence of cleaved
caspase-3 over placental development. Examination of cleaved caspase-3 by fluorescence
immunohistochemisty revealed cleaved caspase-3 to be expressed at low levels in the nuclei of
proliferating cytotrophoblast cells in early gestation supporting our hypothesis that caspase
activity may be associated with trophoblast proliferation (Figure 7.1a,b). In future studies it will
be important to verify that theses fragments are truly products of caspase cleavage. This may be
done by assessing the protein cleavage of p21 and p27 from cultured first trimester explants
treated with or without caspase inhibitors. Preliminary studies conducted so far have revealed
that inhibition of caspase-3/7 prevented the cleavage of p27, and this was associated with a
decrease in cyclin D1 expression (Figure 7.1c). The effect of p21/p27 cleavage on cyclin E1
needs to be completed to determine whether caspase activation can potentially link Mtd to cyclin
E1 expression. In addition, it would be informative to assess the rate of proliferation in human
placental explants following treatment with caspase inhibitors. This would help to determine if
caspase activation and p27 cleavage functionally results in the transition from G1-S phase in the
cell cycle.
Future studies comparing the proliferative capabilities of wild type (WT) murine trophoblast
stem cells (TS) and caspase-3 -/- TS cells are also underway to further verify caspase-3
involvement in trophoblast proliferation. This model will ensure effective and specific
knockdown of caspase-3 (Woo et al., 1999). It will be important to first verify caspase-3 specific
148
Figure 7-1 Caspase-3 cleavage of CDK inhibitors in the placenta
a: Protein expression of cleaved caspase-3, p21 (total and cleaved) and p27 (total and cleaved), in total placental
lysates across gestation. Actin was used as an internal control. blue box depicts cleaved caspase-3 and cleaved p27
in early first trimester b: Spatial localization of cleaved caspase-3 (red-top panels) and immunohistochemical dual
staining of cleaved caspase-3 (red) with Ki67 (green) (bottom panels) in early first trimester (5weeks) floating villi.
Nuclei are visualized by DAPI labeled chromatin (blue). Middle and right panels: high magnification of the boxed
area. Circle: cytotrophoblast cell expressing both cleaved caspase-3 and Ki67 (CT: cytotrophoblast; S: stroma; ST:
syncytiotrophoblast). c: Western blot of first trimester placental explants treated with caspase-3/7 specific inhibitor
DEVD, and blotted for total p27 (top panel) and cyclin D1 (bottom panel). Star denotes decreased levels of cleaved
p27 and cyclin D1 in explants treated with DEVD.
149
cleavage of p21 and p27 by comparing their cleavage profiles between caspase3 null and wild
type TS cells. The effect of caspase 3 knockdown on cyclin expression and proliferation could
then be compared between wild type and caspase-3 null TS under proliferating (stem cell
conditions) and differentiating (withdrawal of FGF4) conditions (Tanaka et al., 1998). This could
be done by assessing the expression of cyclin mRNA and protein levels as well as by BrdU
incorporation and FACS cell cycle analysis. To date three TS cell derivations have been
conducted, resulting in 5 wild type cell lines and 3 caspase-3 knock out lines (Figure 7.2).
In light of the data presented in the current dissertation, we hypothesize that targeting p27
cleavage in particular will have a significant impact on cell cycle progression. This could be
investigated further by studying the proliferative capacity of trophoblast cell lines transfected
with p27 constructs with targeted mutations to their caspase-3 cleavage site (ex. D136PSD139S
in p27) (Eymin et al., 1999). This would specifically prevent the cleavage of p27. By comparing
the affect of cleavable and uncleavable p27 on the cell cycle, the significance of p27 cleavage
could be assessed. These studies could be accomplished through BrdU and FACS analysis.
Staining BrdU labeled cells with TO-PRO-3 (T3605, molecular probes) and propidium iodide
(molecular probes) would enable the quantification of both BrdU-labeled and non-labeled cells
so that the percentage of cells actively cycling could be quantified. FACS analysis would also
provide information as to the number of cells in G0, G1,S,G2 and M phase of the cell cycle so
that it could be determined whether cells are arrested or spending less time in a particular phase
of the cell cycle when p27 cleavage is prevented.
7.2 Determine the upstream pathway in preeclampsia leading to the
phosphorylation of p27at Ser10 and its translocation to the
cytoplasm
Determining the mechanism in severe early onset preeclampsia leading to p27 Ser10
phosphorylation and nuclear export will be important to understanding the upstream events
contributing to the disease. Immunoprecipitation studies of p27 with ERK and hKIS in
preeclamptic samples would be useful to determine which kinase interacts with p27 in the
preeclamptic pathology. Additionally, hKIS overexpression studies could be conducted in
trophoblast cell lines to determine if KIS activity leads to p27 phosphorylation and nuclear
export. ERK mediated phosphorylation of p27 could also be verified in trophoblast cell lines by
150
Figure 7-2 Caspase-3 null TS cell derivation
Wild type and caspase-3 deficient TS cells were generated from blastocysts of heterozygous mice carrying a
disrupted Caspase-3 gene. Blastocysts were collected from caspae-3 mutated heterozygous crosses and cultured as
previously described (Tanaka et al., 1998). Cells from each colony were genotyped to identify knockout and Wild
Type (WT) groups.
151
inhibiting MEK activation using the MEK inhibitor PD98059 or U0126 MEK1/2 inhibitor (Cell
Signaling). These studies could be conducted in either BeWo or JEG choriocarcinoma cell lines,
as both cell lines have been shown to have functional ERK pathways (Oufkir et al., 2010). In
addition, estrogens have been shown to cause nuclear export of Ser10 phosphorylated p27 in
early to mid-G1 phase through a mechanism involving the ERK signaling pathway (Foster et al.,
2003). Estrogen levels in preeclampsia should be assessed to determine if estrogen may play a
role upstream in this pathway.
It would also be important to determine if blocking p27 Ser10 phosphorylation in trophoblast
cells would prevent cyclin E1-CDK2 activity and inhibit the positive feedback loop generated by
the excessive cyclin E1 levels in preeclampsia. This could be accomplished by preventing the
kinase activity that targets p27 Ser10 (identified by the experiments outlined above) and
examining cell cycle progression by FACS analysis. Alternatively, this could be accomplished
by knocking down endogenous p27 in trophoblast cell lines and transfecting a p27 construct with
the serine 10 site mutated to an alanine (Ser10Ala). The effect on cyclin E1 mediated CDK2
activity could be assessed by histone phosphorylation assays following cyclin E
immunoprecipitation. In addition, co-immunoprecipitation studies of p27 with cyclin D, CDK4/6
or 14-3-3 may help identify the mode of p27 stabilization.
7.3 Determine the mechanism leading to low p27 expression in molar
tissue and determine if it contributes to increased Mtd and cyclin
E1 expression in the pathology
We hypothesize that the low level of p27 in molar pregnancy may contribute to the
hyperproliferative nature of the disorder. Strategies targeted at elevating p27 in molar cases, or
averting its degradation, may aid in preventing the hyperproliferative component of molar
pathogenesis. Degradation of p27 in the nucleus follows p27 phosphorylation at Thr187 by
CDK2, and involves ubiquitination by the skp2-CKS1 ubiquitin ligase complex (Vlach et al.,
1997;Montagnoli et al., 1999) (Figure 6.5). It would therefore be of interest to examine the
Thr187 phosphorylated and ubiquitinated state of p27 in molar tissue, and to test whether forced
expression of p27 would decrease cyclin E1 and Mtd expression. This could be tested by
blocking p27 ubiquitination using antisense oligos towards skp2 in molar placental explants, and
assessing for CDK2 activation by H1 phosphorylation, as well as cyclin E1 and Mtd expression.
152
REFERENCES
1. (2002). ACOG practice bulletin. Diagnosis and management of preeclampsia and
eclampsia. Number 33, January 2002. American College of Obstetricians and
Gynecologists. Int. J. Gynaecol. Obstet., 77, 67-75.
2. Abrahams,V.M., Kim,Y.M., Straszewski,S.L., Romero,R., and Mor,G. (2004).
Macrophages and apoptotic cell clearance during pregnancy. Am. J. Reprod. Immunol.,
51, 275-282.
3. Adams,P.D. (2001). Regulation of the retinoblastoma tumor suppressor protein by
cyclin/cdks. Biochim. Biophys. Acta, 1471, M123-M133.
4. Alam,A., Cohen,L.Y., Aouad,S., and Sekaly,R.P. (1999). Early activation of caspases
during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic
cells. J. Exp. Med., 190, 1879-1890.
5. Allaire,A.D., Ballenger,K.A., Wells,S.R., McMahon,M.J., and Lessey,B.A. (2000).
Placental apoptosis in preeclampsia. Obstet. Gynecol., 96, 271-276.
6. Aly,A.S., Khandelwal,M., Zhao,J., Mehmet,A.H., Sammel,M.D., and Parry,S. (2004).
Neutrophils are stimulated by syncytiotrophoblast microvillous membranes to generate
superoxide radicals in women with preeclampsia. Am. J. Obstet. Gynecol., 190, 252-258.
7. Antonsson,B., Montessuit,S., Lauper,S., Eskes,R., and Martinou,J.C. (2000). Bax
oligomerization is required for channel-forming activity in liposomes and to trigger
cytochrome c release from mitochondria. Biochem. J., 345 Pt 2, 271-278.
8. Antonsson,B., Montessuit,S., Sanchez,B., and Martinou,J.C. (2001). Bax is present as a
high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic
cells. J. Biol. Chem., 276, 11615-11623.
9. Arkwright,P.D., Rademacher,T.W., Dwek,R.A., and Redman,C.W. (1993). Pre-eclampsia
is associated with an increase in trophoblast glycogen content and glycogen synthase
activity, similar to that found in hydatidiform moles. J. Clin. Invest, 91, 2744-2753.
10. Arnholdt,H., Meisel,F., Fandrey,K., and Lohrs,U. (1991). Proliferation of villous
trophoblast of the human placenta in normal and abnormal pregnancies. Virchows Arch.
B Cell Pathol. Incl. Mol. Pathol., 60, 365-372.
11. Asada,M., Yamada,T., Ichijo,H., Delia,D., Miyazono,K., Fukumuro,K., and Mizutani,S.
(1999). Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic
differentiation. EMBO J., 18, 1223-1234.
12. Ashton,S.V., Whitley,G.S., Dash,P.R., Wareing,M., Crocker,I.P., Baker,P.N., and
Cartwright,J.E. (2005). Uterine spiral artery remodeling involves endothelial apoptosis
153
induced by extravillous trophoblasts through Fas/FasL interactions. Arterioscler. Thromb.
Vasc. Biol., 25, 102-108.
13. Axt-Fliedner,R., Friedrich,M., Kordina,A., Wasemann,C., Mink,D., Reitnauer,K., and
Schmidt,W. (2001). The immunolocalization of Bcl-2 in human term placenta. Clin. Exp.
Obstet. Gynecol., 28, 144-147.
14. Baczyk,D., Drewlo,S., Proctor,L., Dunk,C., Lye,S., and Kingdom,J. (2009). Glial cell
missing-1 transcription factor is required for the differentiation of the human trophoblast.
Cell Death. Differ., 16, 719-727.
15. Bailly,E., Pines,J., Hunter,T., and Bornens,M. (1992). Cytoplasmic accumulation of
cyclin B1 in human cells: association with a detergent-resistant compartment and with the
centrosome. J. Cell Sci., 101 ( Pt 3), 529-545.
16. Bamberger,A., Sudahl,S., Bamberger,C.M., Schulte,H.M., and Loning,T. (1999).
Expression patterns of the cell-cycle inhibitor p27 and the cell-cycle promoter cyclin E in
the human placenta throughout gestation: implications for the control of proliferation.
Placenta, 20, 401-406.
17. Bamberger,A.M., Aupers,S., Milde-Langosch,K., and Loning,T. (2003). Expression
pattern of the cell cycle promoter cyclin e in benign extravillous trophoblast and
gestational trophoblastic lesions: correlation with expression of Ki-67. Int. J. Gynecol.
Pathol., 22, 156-161.
18. Barker,D.J. (1998). In utero programming of chronic disease. Clin. Sci. (Lond), 95, 115-
128.
19. Barker,D.J. (2003). The developmental origins of adult disease. Eur. J. Epidemiol., 18,
733-736.
20. Barlow,P.W. and Sherman,M.I. (1972). The biochemistry of differentiation of mouse
trophoblast: studies on polyploidy. J. Embryol. Exp. Morphol., 27, 447-465.
21. Bartholomeusz,G., Wu,Y., Ali,S.M., Xia,W., Kwong,K.Y., Hortobagyi,G., and
Hung,M.C. (2006). Nuclear translocation of the pro-apoptotic Bcl-2 family member Bok
induces apoptosis. Mol. Carcinog., 45, 73-83.
22. Besson,A., Dowdy,S.F., and Roberts,J.M. (2008). CDK inhibitors: cell cycle regulators
and beyond. Dev. Cell, 14, 159-169.
23. Besson,A., Gurian-West,M., Chen,X., Kelly-Spratt,K.S., Kemp,C.J., and Roberts,J.M.
(2006). A pathway in quiescent cells that controls p27Kip1 stability, subcellular
localization, and tumor suppression. Genes Dev., 20, 47-64.
24. Besson,A., Gurian-West,M., Schmidt,A., Hall,A., and Roberts,J.M. (2004). p27Kip1
modulates cell migration through the regulation of RhoA activation. Genes Dev., 18, 862-
876.
154
25. Black,S., Kadyrov,M., Kaufmann,P., Ugele,B., Emans,N., and Huppertz,B. (2004).
Syncytial fusion of human trophoblast depends on caspase 8. Cell Death. Differ., 11, 90-
98.
26. Boehm,M., Yoshimoto,T., Crook,M.F., Nallamshetty,S., True,A., Nabel,G.J., and
Nabel,E.G. (2002). A growth factor-dependent nuclear kinase phosphorylates p27(Kip1)
and regulates cell cycle progression. EMBO J., 21, 3390-3401.
27. Bonnefoy-Berard,N., Aouacheria,A., Verschelde,C., Quemeneur,L., Marcais,A., and
Marvel,J. (2004). Control of proliferation by Bcl-2 family members. Biochim. Biophys.
Acta, 1644, 159-168.
28. Boyd,J.D. and Hamilton,W.J. (1970). The Human Placenta. W.Heffer & Sons:
Cambridge, MA. pp 365.
29. Brady,H.J., Gil-Gomez,G., Kirberg,J., and Berns,A.J. (1996). Bax alpha perturbs T cell
development and affects cell cycle entry of T cells. EMBO J., 15, 6991-7001.
30. Brown,L.M., Lacey,H.A., Baker,P.N., and Crocker,I.P. (2005). E-cadherin in the
assessment of aberrant placental cytotrophoblast turnover in pregnancies complicated by
pre-eclampsia. Histochem. Cell Biol., 124, 499-506.
31. Brunelle,J.K. and Chandel,N.S. (2002). Oxygen deprivation induced cell death: an
update. Apoptosis., 7, 475-482.
32. Bryja,V., Pachernik,J., Vondracek,J., Soucek,K., Cajanek,L., Horvath,V., Holubcova,Z.,
Dvorak,P., and Hampl,A. (2008). Lineage specific composition of cyclin D-
CDK4/CDK6-p27 complexes reveals distinct functions of CDK4, CDK6 and individual
D-type cyclins in differentiating cells of embryonic origin. Cell Prolif., 41, 875-893.
33. Burton,G.J. (2009). Oxygen, the Janus gas; its effects on human placental development
and function. J. Anat., 215, 27-35.
34. Burton,G.J. and Jauniaux,E. (2004). Placental oxidative stress: from miscarriage to
preeclampsia. J. Soc. Gynecol. Investig., 11, 342-352.
35. Burton,G.J., Jauniaux,E., and Watson,A.L. (1999). Maternal arterial connections to the
placental intervillous space during the first trimester of human pregnancy: the Boyd
collection revisited. Am. J. Obstet. Gynecol., 181, 718-724.
36. Caniggia,I., Grisaru-Gravnosky,S., Kuliszewsky,M., Post,M., and Lye,S.J. (1999).
Inhibition of TGF-beta 3 restores the invasive capability of extravillous trophoblasts in
preeclamptic pregnancies. J. Clin. Invest, 103, 1641-1650.
37. Caniggia,I., Mostachfi,H., Winter,J., Gassmann,M., Lye,S.J., Kuliszewski,M., and
Post,M. (2000). Hypoxia-inducible factor-1 mediates the biological effects of oxygen on
human trophoblast differentiation through TGFbeta(3). J. Clin. Invest, 105, 577-587.
155
38. Caniggia,I., Taylor,C.V., Ritchie,J.W., Lye,S.J., and Letarte,M. (1997). Endoglin
regulates trophoblast differentiation along the invasive pathway in human placental
villous explants. Endocrinology, 138, 4977-4988.
39. Caniggia,I. and Winter,J.L. (2002). Adriana and Luisa Castellucci Award lecture 2001.
Hypoxia inducible factor-1: oxygen regulation of trophoblast differentiation in normal
and pre-eclamptic pregnancies--a review. Placenta, 23 Suppl A, S47-S57.
40. Cartwright,J.E., Kenny,L.C., Dash,P.R., Crocker,I.P., Aplin,J.D., Baker,P.N., and
Whitley,G.S. (2002). Trophoblast invasion of spiral arteries: a novel in vitro model.
Placenta, 23, 232-235.
41. Cartwright,J.E. and Wareing,M. (2006). An in vitro model of trophoblast invasion of
spiral arteries. Methods Mol. Med., 122, 59-74.
42. Cetin,I. and Alvino,G. (2009). Intrauterine growth restriction: implications for placental
metabolism and transport. A review. Placenta, 30 Suppl A, S77-S82.
43. Cetin,I., Foidart,J.M., Miozzo,M., Raun,T., Jansson,T., Tsatsaris,V., Reik,W., Cross,J.,
Hauguel-de-Mouzon,S., Illsley,N., Kingdom,J., and Huppertz,B. (2004). Fetal growth
restriction: a workshop report. Placenta, 25, 753-757.
44. Chen,C.P., Bajoria,R., and Aplin,J.D. (2002). Decreased vascularization and cell
proliferation in placentas of intrauterine growth-restricted fetuses with abnormal
umbilical artery flow velocity waveforms. Am. J. Obstet. Gynecol., 187, 764-769.
45. Cheng,M., Olivier,P., Diehl,J.A., Fero,M., Roussel,M.F., Roberts,J.M., and Sherr,C.J.
(1999). The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin
D-dependent kinases in murine fibroblasts. EMBO J., 18, 1571-1583.
46. Cheung,A.N., Shen,D.H., Khoo,U.S., Wong,L.C., and Ngan,H.Y. (1998).
p21WAF1/CIP1 expression in gestational trophoblastic disease: correlation with
clinicopathological parameters, and Ki67 and p53 gene expression. J. Clin. Pathol., 51,
159-162.
47. Cheung,A.N., Srivastava,G., Chung,L.P., Ngan,H.Y., Man,T.K., Liu,Y.T., Chen,W.Z.,
Collins,R.J., Wong,L.C., and Ma,H.K. (1994). Expression of the p53 gene in
trophoblastic cells in hydatidiform moles and normal human placentas. J. Reprod. Med.,
39, 223-227.
48. Chiang,M.H., Liang,F.Y., Chen,C.P., Chang,C.W., Cheong,M.L., Wang,L.J., Liang,C.Y.,
Lin,F.Y., Chou,C.C., and Chen,H. (2009). Mechanism of hypoxia-induced GCM1
degradation: implications for the pathogenesis of preeclampsia. J. Biol. Chem., 284,
17411-17419.
49. Child,E.S. and Mann,D.J. (2006). The intricacies of p21 phosphorylation: protein/protein
interactions, subcellular localization and stability. Cell Cycle, 5, 1313-1319.
156
50. Chiu,P.M., Ngan,Y.S., Khoo,U.S., and Cheung,A.N. (2001). Apoptotic activity in
gestational trophoblastic disease correlates with clinical outcome: assessment by the
caspase-related M30 CytoDeath antibody. Histopathology, 38, 243-249.
51. Connor,M.K., Kotchetkov,R., Cariou,S., Resch,A., Lupetti,R., Beniston,R.G.,
Melchior,F., Hengst,L., and Slingerland,J.M. (2003). CRM1/Ran-mediated nuclear
export of p27(Kip1) involves a nuclear export signal and links p27 export and
proteolysis. Mol. Biol. Cell, 14, 201-213.
52. Coverley,D., Laman,H., and Laskey,R.A. (2002). Distinct roles for cyclins E and A
during DNA replication complex assembly and activation. Nat. Cell Biol., 4, 523-528.
53. Crocker,I. (2007). Gabor Than Award Lecture 2006: pre-eclampsia and villous
trophoblast turnover: perspectives and possibilities. Placenta, 28 Suppl A, S4-13.
54. Cross,J.C., Werb,Z., and Fisher,S.J. (1994). Implantation and the placenta: key pieces of
the development puzzle. Science, 266, 1508-1518.
55. Damsky,C.H., Librach,C., Lim,K.H., Fitzgerald,M.L., McMaster,M.T., Janatpour,M.,
Zhou,Y., Logan,S.K., and Fisher,S.J. (1994). Integrin switching regulates normal
trophoblast invasion. Development, 120, 3657-3666.
56. Danihel,L., Gomolcak,P., Korbel,M., Pruzinec,J., Vojtassak,J., Janik,P., and Babal,P.
(2002). Expression of proliferation and apoptotic markers in human placenta during
pregnancy. Acta Histochem., 104, 335-338.
57. De Falco,M., De Luca,L., Acanfora,F., Cavallotti,I., Cottone,G., Laforgia,V., De Luca,B.,
Baldi,A., and De Luca,A. (2001). Alteration of the Bcl-2:Bax ratio in the placenta as
pregnancy proceeds. Histochem. J., 33, 421-425.
58. De Falco,M., Fedele,V., Cobellis,L., Mastrogiacomo,A., Giraldi,D., Leone,S., De
Luca,L., Laforgia,V., and De Luca,A. (2004). Pattern of expression of cyclin D1/CDK4
complex in human placenta during gestation. Cell Tissue Res., 317, 187-194.
59. Degterev,A., Boyce,M., and Yuan,J. (2003). A decade of caspases. Oncogene, 22, 8543-
8567.
60. Dejean,L.M., Martinez-Caballero,S., Guo,L., Hughes,C., Teijido,O., Ducret,T., Ichas,F.,
Korsmeyer,S.J., Antonsson,B., Jonas,E.A., and Kinnally,K.W. (2005). Oligomeric Bax is
a component of the putative cytochrome c release channel MAC, mitochondrial
apoptosis-induced channel. Mol. Biol. Cell, 16, 2424-2432.
61. DeLoia,J.A., Burlingame,J.M., and Krasnow,J.S. (1997). Differential expression of G1
cyclins during human placentogenesis. Placenta, 18, 9-16.
62. Deng,X., Mercer,S.E., Shah,S., Ewton,D.Z., and Friedman,E. (2004). The cyclin-
dependent kinase inhibitor p27Kip1 is stabilized in G(0) by Mirk/dyrk1B kinase. J. Biol.
Chem., 279, 22498-22504.
157
63. DiFederico,E., Genbacev,O., and Fisher,S.J. (1999). Preeclampsia is associated with
widespread apoptosis of placental cytotrophoblasts within the uterine wall. Am. J.
Pathol., 155, 293-301.
64. Du,C., Fang,M., Li,Y., Li,L., and Wang,X. (2000). Smac, a mitochondrial protein that
promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell,
102, 33-42.
65. Dulic,V., Lees,E., and Reed,S.I. (1992). Association of human cyclin E with a periodic
G1-S phase protein kinase. Science, 257, 1958-1961.
66. Dunk,C., Petkovic,L., Baczyk,D., Rossant,J., Winterhager,E., and Lye,S. (2003). A novel
in vitro model of trophoblast-mediated decidual blood vessel remodeling. Lab Invest, 83,
1821-1828.
67. Dyson,N. (1998). The regulation of E2F by pRB-family proteins. Genes Dev., 12, 2245-
2262.
68. Endl,E. and Gerdes,J. (2000). The Ki-67 protein: fascinating forms and an unknown
function. Exp. Cell Res., 257, 231-237.
69. Everett,R.B. and MacDonald,P.C. (1979). Endocrinology of the placenta. Annu. Rev.
Med., 30, 473-488.
70. Eymin,B., Sordet,O., Droin,N., Munsch,B., Haugg,M., Van de,C.M., Vandenabeele,P.,
and Solary,E. (1999). Caspase-induced proteolysis of the cyclin-dependent kinase
inhibitor p27Kip1 mediates its anti-apoptotic activity. Oncogene, 18, 4839-4847.
71. Feinstein-Rotkopf,Y. and Arama,E. (2009). Can't live without them, can live with them:
roles of caspases during vital cellular processes. Apoptosis., 14, 980-995.
72. Ferrazzi,E., Bozzo,M., Rigano,S., Bellotti,M., Morabito,A., Pardi,G., Battaglia,F.C., and
Galan,H.L. (2002). Temporal sequence of abnormal Doppler changes in the peripheral
and central circulatory systems of the severely growth-restricted fetus. Ultrasound
Obstet. Gynecol., 19, 140-146.
73. Finucane,D.M., Bossy-Wetzel,E., Waterhouse,N.J., Cotter,T.G., and Green,D.R. (1999).
Bax-induced caspase activation and apoptosis via cytochrome c release from
mitochondria is inhibitable by Bcl-xL. J. Biol. Chem., 274, 2225-2233.
74. Fisher,R.A., Nucci,M.R., Thaker,H.M., Weremowicz,S., Genest,D.R., and
Castrillon,D.H. (2004). Complete hydatidiform mole retaining a chromosome 11 of
maternal origin: molecular genetic analysis of a case. Mod. Pathol., 17, 1155-1160.
75. Fisher,S.J., Cui,T.Y., Zhang,L., Hartman,L., Grahl,K., Zhang,G.Y., Tarpey,J., and
Damsky,C.H. (1989). Adhesive and degradative properties of human placental
cytotrophoblast cells in vitro. J. Cell Biol., 109, 891-902.
158
76. Fong,P.Y., Xue,W.C., Ngan,H.Y., Chan,K.Y., Khoo,U.S., Tsao,S.W., Chiu,P.M.,
Man,L.S., and Cheung,A.N. (2005). Mcl-1 expression in gestational trophoblastic disease
correlates with clinical outcome: a differential expression study. Cancer, 103, 268-276.
77. Fong,P.Y., Xue,W.C., Ngan,H.Y., Chiu,P.M., Chan,K.Y., Tsao,S.W., and Cheung,A.N.
(2006). Caspase activity is downregulated in choriocarcinoma: a cDNA array differential
expression study. J. Clin. Pathol., 59, 179-183.
78. Foster,J.S., Fernando,R.I., Ishida,N., Nakayama,K.I., and Wimalasena,J. (2003).
Estrogens down-regulate p27Kip1 in breast cancer cells through Skp2 and through
nuclear export mediated by the ERK pathway. J. Biol. Chem., 278, 41355-41366.
79. Fuentes-Prior,P. and Salvesen,G.S. (2004). The protein structures that shape caspase
activity, specificity, activation and inhibition. Biochem. J., 384, 201-232.
80. Fujise,K., Zhang,D., Liu,J., and Yeh,E.T. (2000). Regulation of apoptosis and cell cycle
progression by MCL1. Differential role of proliferating cell nuclear antigen. J. Biol.
Chem., 275, 39458-39465.
81. Fujita,N., Sato,S., Katayama,K., and Tsuruo,T. (2002). Akt-dependent phosphorylation of
p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization. J. Biol. Chem., 277,
28706-28713.
82. Fukunaga,M. (2004). Immunohistochemical characterization of cyclin E and p27KIP1
expression in early hydatidiform moles. Int. J. Gynecol. Pathol., 23, 259-264.
83. Galan,A., O'Connor,J.E., Valbuena,D., Herrer,R., Remohi,J., Pampfer,S., Pellicer,A., and
Simon,C. (2000). The human blastocyst regulates endometrial epithelial apoptosis in
embryonic adhesion. Biol. Reprod., 63, 430-439.
84. Gao,S., Fu,W., Durrenberger,M., De Geyter,C., and Zhang,H. (2005). Membrane
translocation and oligomerization of hBok are triggered in response to apoptotic stimuli
and Bnip3. Cell Mol. Life Sci., 62, 1015-1024.
85. Genbacev,O., DiFederico,E., McMaster,M., and Fisher,S.J. (1999). Invasive
cytotrophoblast apoptosis in pre-eclampsia. Hum. Reprod., 14 Suppl 2, 59-66.
86. Genbacev,O., Joslin,R., Damsky,C.H., Polliotti,B.M., and Fisher,S.J. (1996). Hypoxia
alters early gestation human cytotrophoblast differentiation/invasion in vitro and models
the placental defects that occur in preeclampsia. J. Clin. Invest, 97, 540-550.
87. Genbacev,O., McMaster,M.T., and Fisher,S.J. (2000). A repertoire of cell cycle
regulators whose expression is coordinated with human cytotrophoblast differentiation.
Am. J. Pathol., 157, 1337-1351.
88. Genbacev,O., Zhou,Y., Ludlow,J.W., and Fisher,S.J. (1997). Regulation of human
placental development by oxygen tension. Science, 277, 1669-1672.
159
89. Geng,Y., Eaton,E.N., Picon,M., Roberts,J.M., Lundberg,A.S., Gifford,A., Sardet,C., and
Weinberg,R.A. (1996). Regulation of cyclin E transcription by E2Fs and retinoblastoma
protein. Oncogene, 12, 1173-1180.
90. Geng,Y., Yu,Q., Sicinska,E., Das,M., Schneider,J.E., Bhattacharya,S., Rideout,W.M.,
Bronson,R.T., Gardner,H., and Sicinski,P. (2003). Cyclin E ablation in the mouse. Cell,
114, 431-443.
91. Geng,Y., Yu,Q., Whoriskey,W., Dick,F., Tsai,K.Y., Ford,H.L., Biswas,D.K.,
Pardee,A.B., Amati,B., Jacks,T., Richardson,A., Dyson,N., and Sicinski,P. (2001).
Expression of cyclins E1 and E2 during mouse development and in neoplasia. Proc. Natl.
Acad. Sci. U. S. A, 98, 13138-13143.
92. Gerretsen,G., Huisjes,H.J., and Elema,J.D. (1981). Morphological changes of the spiral
arteries in the placental bed in relation to pre-eclampsia and fetal growth retardation. Br.
J. Obstet. Gynaecol., 88, 876-881.
93. Graham,C.H. and Lala,P.K. (1991). Mechanism of control of trophoblast invasion in situ.
J. Cell Physiol, 148, 228-234.
94. Graham,C.H. and Lala,P.K. (1992). Mechanisms of placental invasion of the uterus and
their control. Biochem. Cell Biol., 70, 867-874.
95. Green,D.R. and Reed,J.C. (1998). Mitochondria and apoptosis. Science, 281, 1309-1312.
96. Gross,A., McDonnell,J.M., and Korsmeyer,S.J. (1999). BCL-2 family members and the
mitochondria in apoptosis. Genes Dev., 13, 1899-1911.
97. Gurel,D., Ozer,E., Altunyurt,S., Guclu,S., and Demir,N. (2003). Expression of IGR-IR
and VEGF and trophoblastic proliferative activity in placentas from pregnancies
complicated by IUGR. Pathol. Res. Pract., 199, 803-809.
98. Habli,M., Levine,R.J., Qian,C., and Sibai,B. (2007). Neonatal outcomes in pregnancies
with preeclampsia or gestational hypertension and in normotensive pregnancies that
delivered at 35, 36, or 37 weeks of gestation. Am. J. Obstet. Gynecol., 197, 406-407.
99. Halperin,R., Peller,S., Sandbank,J., Bukovsky,I., and Schneider,D. (2000). Expression of
the p53 gene and apoptosis in gestational trophoblastic disease. Placenta, 21, 58-62.
100. Hansen,A.R., Collins,M.H., Genest,D., Heller,D., Shen-Schwarz,S., Banagon,P.,
Allred,E.N., and Leviton,A. (2000). Very low birthweight placenta: clustering of
morphologic characteristics. Pediatr. Dev. Pathol., 3, 431-438.
101. Hauth,J.C., Ewell,M.G., Levine,R.J., Esterlitz,J.R., Sibai,B., Curet,L.B., Catalano,P.M.,
and Morris,C.D. (2000). Pregnancy outcomes in healthy nulliparas who developed
hypertension. Calcium for Preeclampsia Prevention Study Group. Obstet. Gynecol., 95,
24-28.
160
102. Heazell,A.E. and Crocker,I.P. (2008a). Live and let die - regulation of villous trophoblast
apoptosis in normal and abnormal pregnancies. Placenta, 29, 772-783.
103. Heazell,A.E., Lacey,H.A., Jones,C.J., Huppertz,B., Baker,P.N., and Crocker,I.P. (2008b).
Effects of oxygen on cell turnover and expression of regulators of apoptosis in human
placental trophoblast. Placenta, 29, 175-186.
104. Hsu,S.Y. and Hsueh,A.J. (2000). Tissue-specific Bcl-2 protein partners in apoptosis: An
ovarian paradigm. Physiol Rev., 80, 593-614.
105. Hsu,S.Y., Kaipia,A., McGee,E., Lomeli,M., and Hsueh,A.J. (1997). Bok is a pro-
apoptotic Bcl-2 protein with restricted expression in reproductive tissues and
heterodimerizes with selective anti-apoptotic Bcl-2 family members. Proc. Natl. Acad.
Sci. U. S. A, 94, 12401-12406.
106. Hu,R., Zhou,S., and Li,X. (2006). Altered Bcl-2 and Bax expression is associated with
cultured first trimester human cytotrophoblasts apoptosis induced by hypoxia. Life Sci.,
79, 351-355.
107. Hubel,C.A. (1999). Oxidative stress in the pathogenesis of preeclampsia. Proc. Soc. Exp.
Biol. Med., 222, 222-235.
108. Hung,T.H., Skepper,J.N., and Burton,G.J. (2001). In vitro ischemia-reperfusion injury in
term human placenta as a model for oxidative stress in pathological pregnancies. Am. J.
Pathol., 159, 1031-1043.
109. Hung,T.H., Skepper,J.N., Charnock-Jones,D.S., and Burton,G.J. (2002). Hypoxia-
reoxygenation: a potent inducer of apoptotic changes in the human placenta and possible
etiological factor in preeclampsia. Circ. Res., 90, 1274-1281.
110. Huppertz,B., Bartz,C., and Kokozidou,M. (2006). Trophoblast fusion: Fusogenic
proteins, syncytins and ADAMs, and other prerequisites for syncytial fusion. Micron..
111. Huppertz,B., Frank,H.G., Kingdom,J.C., Reister,F., and Kaufmann,P. (1998). Villous
cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta.
Histochem. Cell Biol., 110, 495-508.
112. Huppertz,B., Frank,H.G., Reister,F., Kingdom,J., Korr,H., and Kaufmann,P. (1999).
Apoptosis cascade progresses during turnover of human trophoblast: analysis of villous
cytotrophoblast and syncytial fragments in vitro. Lab Invest, 79, 1687-1702.
113. Huppertz,B., Kingdom,J., Caniggia,I., Desoye,G., Black,S., Korr,H., and Kaufmann,P.
(2003). Hypoxia favours necrotic versus apoptotic shedding of placental
syncytiotrophoblast into the maternal circulation. Placenta, 24, 181-190.
114. Huppertz,B. and Kingdom,J.C. (2004). Apoptosis in the trophoblast--role of apoptosis in
placental morphogenesis. J. Soc. Gynecol. Investig., 11, 353-362.
161
115. Ietta,F., Wu,Y., Romagnoli,R., Soleymanlou,N., Orsini,B., Zamudio,S., Paulesu,L., and
Caniggia,I. (2007). Oxygen regulation of macrophage migration inhibitory factor in
human placenta. Am. J. Physiol Endocrinol. Metab, 292, E272-E280.
116. Ietta,F., Wu,Y., Winter,J., Xu,J., Wang,J., Post,M., and Caniggia,I. (2006). Dynamic
HIF1A regulation during human placental development. Biol. Reprod., 75, 112-121.
117. Inohara,N., Ekhterae,D., Garcia,I., Carrio,R., Merino,J., Merry,A., Chen,S., and
Nunez,G. (1998). Mtd, a novel Bcl-2 family member activates apoptosis in the absence of
heterodimerization with Bcl-2 and Bcl-XL. J. Biol. Chem., 273, 8705-8710.
118. Irving,J.A. and Lala,P.K. (1995). Functional role of cell surface integrins on human
trophoblast cell migration: regulation by TGF-beta, IGF-II, and IGFBP-1. Exp. Cell Res.,
217, 419-427.
119. Ishida,N., Kitagawa,M., Hatakeyama,S., and Nakayama,K. (2000). Phosphorylation at
serine 10, a major phosphorylation site of p27(Kip1), increases its protein stability. J.
Biol. Chem., 275, 25146-25154.
120. Ishihara,N., Matsuo,H., Murakoshi,H., Laoag-Fernandez,J.B., Samoto,T., and Maruo,T.
(2002). Increased apoptosis in the syncytiotrophoblast in human term placentas
complicated by either preeclampsia or intrauterine growth retardation. Am. J. Obstet.
Gynecol., 186, 158-166.
121. Jackman,M., Kubota,Y., den Elzen,N., Hagting,A., and Pines,J. (2002). Cyclin A- and
cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm. Mol. Biol. Cell,
13, 1030-1045.
122. Jaffe,R., Jauniaux,E., and Hustin,J. (1997). Maternal circulation in the first-trimester
human placenta--myth or reality? Am. J. Obstet. Gynecol., 176, 695-705.
123. Jamil,S., Sobouti,R., Hojabrpour,P., Raj,M., Kast,J., and Duronio,V. (2005). A
proteolytic fragment of Mcl-1 exhibits nuclear localization and regulates cell growth by
interaction with Cdk1. Biochem. J., 387, 659-667.
124. Jauniaux,E., Watson,A.L., Hempstock,J., Bao,Y.P., Skepper,J.N., and Burton,G.J.
(2000). Onset of maternal arterial blood flow and placental oxidative stress. A possible
factor in human early pregnancy failure. Am. J. Pathol., 157, 2111-2122.
125. Jeschke,U., Schiessl,B., Mylonas,I., Kunze,S., Kuhn,C., Schulze,S., Friese,K., and
Mayr,D. (2006). Expression of the proliferation marker Ki-67 and of p53 tumor protein in
trophoblastic tissue of preeclamptic, HELLP, and intrauterine growth-restricted
pregnancies. Int. J. Gynecol. Pathol., 25, 354-360.
126. Johansen,M., Redman,C.W., Wilkins,T., and Sargent,I.L. (1999). Trophoblast
deportation in human pregnancy--its relevance for pre-eclampsia. Placenta, 20, 531-539.
162
127. Kajantie,E., Eriksson,J.G., Osmond,C., Thornburg,K., and Barker,D.J. (2009). Pre-
eclampsia is associated with increased risk of stroke in the adult offspring: the Helsinki
birth cohort study. Stroke, 40, 1176-1180.
128. Kale,A., Soylemez,F., and Ensari,A. (2001). Expressions of proliferation markers (Ki-67,
proliferating cell nuclear antigen, and silver-staining nucleolar organizer regions) and of
p53 tumor protein in gestational trophoblastic disease. Am. J. Obstet. Gynecol., 184, 567-
574.
129. Kaufmann,P. (1982). Development and differentiation of the human placental villous
tree. Bibl. Anat., 29-39.
130. Kim,K.R., Park,B.H., Hong,Y.O., Kwon,H.C., and Robboy,S.J. (2009). The villous
stromal constituents of complete hydatidiform mole differ histologically in very early
pregnancy from the normally developing placenta. Am. J. Surg. Pathol., 33, 176-185.
131. Kim,M.J., Kim,K.R., Ro,J.Y., Lage,J.M., and Lee,H.I. (2006). Diagnostic and
pathogenetic significance of increased stromal apoptosis and incomplete vasculogenesis
in complete hydatidiform moles in very early pregnancy periods. Am. J. Surg. Pathol.,
30, 362-369.
132. Kim,Y.T., Cho,N.H., Ko,J.H., Yang,W.I., Kim,J.W., Choi,E.K., and Lee,S.H. (2000).
Expression of cyclin E in placentas with hydropic change and gestational trophoblastic
diseases: implications for the malignant transformation of trophoblasts. Cancer, 89, 673-
679.
133. Kingdom,J.C. and Kaufmann,P. (1997). Oxygen and placental villous development:
origins of fetal hypoxia. Placenta, 18, 613-621.
134. Kipp,B.R., Ketterling,R.P., Oberg,T.N., Cousin,M.A., Plagge,A.M., Wiktor,A.E.,
Ihrke,J.M., Meyers,C.H., Morice,W.G., Halling,K.C., and Clayton,A.C. (2010).
Comparison of fluorescence in situ hybridization, p57 immunostaining, flow cytometry,
and digital image analysis for diagnosing molar and nonmolar products of conception.
Am. J. Clin. Pathol., 133, 196-204.
135. Knoblich,J.A., Sauer,K., Jones,L., Richardson,H., Saint,R., and Lehner,C.F. (1994).
Cyclin E controls S phase progression and its down-regulation during Drosophila
embryogenesis is required for the arrest of cell proliferation. Cell, 77, 107-120.
136. Knudson,C.M., Johnson,G.M., Lin,Y., and Korsmeyer,S.J. (2001). Bax accelerates
tumorigenesis in p53-deficient mice. Cancer Res., 61, 659-665.
137. Koff,A., Giordano,A., Desai,D., Yamashita,K., Harper,J.W., Elledge,S., Nishimoto,T.,
Morgan,D.O., Franza,B.R., and Roberts,J.M. (1992). Formation and activation of a cyclin
E-cdk2 complex during the G1 phase of the human cell cycle. Science, 257, 1689-1694.
138. Koga,K., Osuga,Y., Tajima,T., Hirota,Y., Igarashi,T., Fujii,T., Yano,T., and Taketani,Y.
(2009). Elevated serum soluble fms-like tyrosine kinase 1 (sFlt1) level in women with
hydatidiform mole. Fertil. Steril..
163
139. Korgun,E.T., Celik-Ozenci,C., Acar,N., Cayli,S., Desoye,G., and Demir,R. (2006).
Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle
inhibitors p21, p27 and p57 in human first trimester placenta and deciduas. Histochem.
Cell Biol..
140. Kozar,K., Ciemerych,M.A., Rebel,V.I., Shigematsu,H., Zagozdzon,A., Sicinska,E.,
Geng,Y., Yu,Q., Bhattacharya,S., Bronson,R.T., Akashi,K., and Sicinski,P. (2004).
Mouse development and cell proliferation in the absence of D-cyclins. Cell, 118, 477-
491.
141. Kurman,R.J. (1991a). Pathology of trophoblast. Monogr Pathol., 195-227.
142. Kurman,R.J. (1991b). The morphology, biology, and pathology of intermediate
trophoblast: a look back to the present. Hum. Pathol., 22, 847-855.
143. Labaer,J., Garrett,M.D., Stevenson,L.F., Slingerland,J.M., Sandhu,C., Chou,H.S.,
Fattaey,A., and Harlow,E. (1997). New functional activities for the p21 family of CDK
inhibitors. Genes Dev., 11, 847-862.
144. Lamkanfi,M., Festjens,N., Declercq,W., Berghe,T.V., and Vandenabeele,P. (2007).
Caspases in cell survival, proliferation and differentiation. Cell Death. Differ., 14, 44-55.
145. Launay,S., Hermine,O., Fontenay,M., Kroemer,G., Solary,E., and Garrido,C. (2005).
Vital functions for lethal caspases. Oncogene, 24, 5137-5148.
146. Lea,R.G., Riley,S.C., Antipatis,C., Hannah,L., Ashworth,C.J., Clark,D.A., and
Critchley,H.O. (1999). Cytokines and the regulation of apoptosis in reproductive tissues:
a review. Am. J. Reprod. Immunol., 42, 100-109.
147. Leung,D.N., Smith,S.C., To,K.F., Sahota,D.S., and Baker,P.N. (2001). Increased
placental apoptosis in pregnancies complicated by preeclampsia. Am. J. Obstet. Gynecol.,
184, 1249-1250.
148. Levine,R.J., Maynard,S.E., Qian,C., Lim,K.H., England,L.J., Yu,K.F., Schisterman,E.F.,
Thadhani,R., Sachs,B.P., Epstein,F.H., Sibai,B.M., Sukhatme,V.P., and Karumanchi,S.A.
(2004). Circulating angiogenic factors and the risk of preeclampsia. N. Engl. J. Med.,
350, 672-683.
149. Levy,R. and Nelson,D.M. (2000). To be, or not to be, that is the question. Apoptosis in
human trophoblast. Placenta, 21, 1-13.
150. Levy,R., Smith,S.D., Yusuf,K., Huettner,P.C., Kraus,F.T., Sadovsky,Y., and
Nelson,D.M. (2002). Trophoblast apoptosis from pregnancies complicated by fetal
growth restriction is associated with enhanced p53 expression. Am. J. Obstet. Gynecol.,
186, 1056-1061.
151. Lew,D.J., Dulic,V., and Reed,S.I. (1991). Isolation of three novel human cyclins by
rescue of G1 cyclin (Cln) function in yeast. Cell, 66, 1197-1206.
164
152. Li,H.W., Tsao,S.W., and Cheung,A.N. (2002). Current understandings of the molecular
genetics of gestational trophoblastic diseases. Placenta, 23, 20-31.
153. Liang,J., Shao,S.H., Xu,Z.X., Hennessy,B., Ding,Z., Larrea,M., Kondo,S., Dumont,D.J.,
Gutterman,J.U., Walker,C.L., Slingerland,J.M., and Mills,G.B. (2007). The energy
sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the
decision to enter autophagy or apoptosis. Nat. Cell Biol., 9, 218-224.
154. Liu,Y., Yeh,N., Zhu,X.H., Leversha,M., Cordon-Cardo,C., Ghossein,R., Singh,B.,
Holland,E., and Koff,A. (2007). Somatic cell type specific gene transfer reveals a tumor-
promoting function for p21(Waf1/Cip1). EMBO J., 26, 4683-4693.
155. Livak,K.J. and Schmittgen,T.D. (2001). Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25, 402-408.
156. Lundberg,A.S. and Weinberg,R.A. (1998). Functional inactivation of the retinoblastoma
protein requires sequential modification by at least two distinct cyclin-cdk complexes.
Mol. Cell Biol., 18, 753-761.
157. MacAuley,A., Cross,J.C., and Werb,Z. (1998). Reprogramming the cell cycle for
endoreduplication in rodent trophoblast cells. Mol. Biol. Cell, 9, 795-807.
158. MacPhee,D.J., Mostachfi,H., Han,R., Lye,S.J., Post,M., and Caniggia,I. (2001). Focal
adhesion kinase is a key mediator of human trophoblast development. Lab Invest, 81,
1469-1483.
159. Maddika,S., Ande,S.R., Panigrahi,S., Paranjothy,T., Weglarczyk,K., Zuse,A.,
Eshraghi,M., Manda,K.D., Wiechec,E., and Los,M. (2007). Cell survival, cell death and
cell cycle pathways are interconnected: implications for cancer therapy. Drug Resist.
Updat., 10, 13-29.
160. Massague,J. (1998). TGF-beta signal transduction. Annu. Rev. Biochem., 67, 753-791.
161. Massardier,J., Golfier,F., Journet,D., Frappart,L., Zalaquett,M., Schott,A.M.,
Lenoir,V.T., Dupuis,O., Hajri,T., and Raudrant,D. (2009). Twin pregnancy with complete
hydatidiform mole and coexistent fetus: obstetrical and oncological outcomes in a series
of 14 cases. Eur. J. Obstet. Gynecol. Reprod. Biol., 143, 84-87.
162. Matsui,H., Iitsuka,Y., Yamazawa,K., Tanaka,N., Mitsuhashi,A., Seki,K., and Sekiya,S.
(2003). Criteria for initiating chemotherapy in patients after evacuation of hydatidiform
mole. Tumour. Biol., 24, 140-146.
163. Maulik,D. (2006). Management of fetal growth restriction: an evidence-based approach.
Clin. Obstet. Gynecol., 49, 320-334.
164. Mazur,M.T. and Kurman,R.J. (1994). Gestational trophoblastic disease and related
lesions. Blaustein's pathology of the female genital tract, . New York: Springer-Verlag,
pp. 1049-1093.
165
165. McAllister,S.S., Becker-Hapak,M., Pintucci,G., Pagano,M., and Dowdy,S.F. (2003).
Novel p27(kip1) C-terminal scatter domain mediates Rac-dependent cell migration
independent of cell cycle arrest functions. Mol. Cell Biol., 23, 216-228.
166. McConnell,T.G., Murphy,K.M., Hafez,M., Vang,R., and Ronnett,B.M. (2009a).
Diagnosis and subclassification of hydatidiform moles using p57 immunohistochemistry
and molecular genotyping: validation and prospective analysis in routine and consultation
practice settings with development of an algorithmic approach. Am. J. Surg. Pathol., 33,
805-817.
167. McConnell,T.G., Norris-Kirby,A., Hagenkord,J.M., Ronnett,B.M., and Murphy,K.M.
(2009b). Complete hydatidiform mole with retained maternal chromosomes 6 and 11.
Am. J. Surg. Pathol., 33, 1409-1415.
168. McKenzie,P.P., Foster,J.S., House,S., Bukovsky,A., Caudle,M.R., and Wimalasena,J.
(1998). Expression of G1 cyclins and cyclin-dependent kinase-2 activity during terminal
differentiation of cultured human trophoblast. Biol. Reprod., 58, 1283-1289.
169. Miyashita,T., Krajewski,S., Krajewska,M., Wang,H.G., Lin,H.K., Liebermann,D.A.,
Hoffman,B., and Reed,J.C. (1994). Tumor suppressor p53 is a regulator of bcl-2 and bax
gene expression in vitro and in vivo. Oncogene, 9, 1799-1805.
170. Montagnoli,A., Fiore,F., Eytan,E., Carrano,A.C., Draetta,G.F., Hershko,A., and
Pagano,M. (1999). Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation
and trimeric complex formation. Genes Dev., 13, 1181-1189.
171. Moore and Persaud (1998). The Developing Human: Clinically Oriented Embryology.
172. Morgan,D.O. (1997). Cyclin-dependent kinases: engines, clocks, and microprocessors.
Annu. Rev. Cell Dev. Biol., 13, 261-291.
173. Moroy,T. and Geisen,C. (2004). Cyclin E. Int. J. Biochem. Cell Biol., 36, 1424-1439.
174. Morrish,D.W., Dakour,J., and Li,H. (1998). Functional regulation of human trophoblast
differentiation. J. Reprod. Immunol., 39, 179-195.
175. Myatt,L. (2006). Placental adaptive responses and fetal programming. J. Physiol, 572,
25-30.
176. Nakamura,S., Okinaka,K., Hirano,I., Ono,T., Sugimoto,Y., Shigeno,K., Fujisawa,S.,
Shinjo,K., and Ohnishi,K. (2008). KIS induces proliferation and the cell cycle
progression through the phosphorylation of p27Kip1 in leukemia cells. Leuk. Res., 32,
1358-1365.
177. Nevins,J.R. (1998). Toward an understanding of the functional complexity of the E2F
and retinoblastoma families. Cell Growth Differ., 9, 585-593.
178. Nevo,O., Many,A., Xu,J., Kingdom,J., Piccoli,E., Zamudio,S., Post,M., Bocking,A.,
Todros,T., and Caniggia,I. (2008). Placental expression of soluble fms-like tyrosine
166
kinase 1 is increased in singletons and twin pregnancies with intrauterine growth
restriction. J. Clin. Endocrinol. Metab, 93, 285-292.
179. Nevo,O., Soleymanlou,N., Wu,Y., Xu,J., Kingdom,J., Many,A., Zamudio,S., and
Caniggia,I. (2006). Increased expression of sFlt-1 in in vivo and in vitro models of
human placental hypoxia is mediated by HIF-1. Am. J. Physiol Regul. Integr. Comp
Physiol, 291, R1085-R1093.
180. Olvera,M., Harris,S., Amezcua,C.A., McCourty,A., Rezk,S., Koo,C., Felix,J.C., and
Brynes,R.K. (2001). Immunohistochemical expression of cell cycle proteins E2F-1, Cdk-
2, Cyclin E, p27(kip1), and Ki-67 in normal placenta and gestational trophoblastic
disease. Mod. Pathol., 14, 1036-1042.
181. Orcy,R.B., Schroeder,S., Martins-Costa,S.H., Ramos,J.G., Schechinger,W., Klein,H.,
Brum,I.S., von Eye,C.H., and Capp,E. (2008). Signalization of Akt/PKB in the placenta,
skeletal muscle and adipose tissue of preeclampsia patients. Gynecol. Obstet. Invest, 66,
231-236.
182. Oufkir,T., Arseneault,M., Sanderson,J.T., and Vaillancourt,C. (2010). The 5-HT(2A)
serotonin receptor enhances cell viability, affects cell cycle progression and activates
MEK-ERK1/2 and JAK2-STAT3 signalling pathways in human choriocarcinoma cell
lines. Placenta.
183. Palmer,J.R. (1994). Advances in the epidemiology of gestational trophoblastic disease. J.
Reprod. Med., 39, 155-162.
184. Panichkul,P.C., Al Hussaini,T.K., Sierra,R., Kashork,C.D., Popek,E.J., Stockton,D.W.,
and Van,d.V., I (2005). Recurrent biparental hydatidiform mole: additional evidence for a
1.1-Mb locus in 19q13.4 and candidate gene analysis. J. Soc. Gynecol. Investig., 12, 376-
383.
185. Paradinas,F.J., Browne,P., Fisher,R.A., Foskett,M., Bagshawe,K.D., and Newlands,E.
(1996). A clinical, histopathological and flow cytometric study of 149 complete moles,
146 partial moles and 107 non-molar hydropic abortions. Histopathology, 28, 101-110.
186. Parisi,T., Beck,A.R., Rougier,N., McNeil,T., Lucian,L., Werb,Z., and Amati,B. (2003).
Cyclins E1 and E2 are required for endoreplication in placental trophoblast giant cells.
EMBO J., 22, 4794-4803.
187. Pollack,R.N. and Divon,M.Y. (1992). Intrauterine growth retardation: definition,
classification, and etiology. Clin. Obstet. Gynecol., 35, 99-107.
188. Polyak,K., Kato,J.Y., Solomon,M.J., Sherr,C.J., Massague,J., Roberts,J.M., and Koff,A.
(1994). p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and
contact inhibition to cell cycle arrest. Genes Dev., 8, 9-22.
189. Potgens,A.J., Schmitz,U., Bose,P., Versmold,A., Kaufmann,P., and Frank,H.G. (2002).
Mechanisms of syncytial fusion: a review. Placenta, 23 Suppl A, S107-S113.
167
190. Qiao,S., Nagasaka,T., Harada,T., and Nakashima,N. (1998). p53, Bax and Bcl-2
expression, and apoptosis in gestational trophoblast of complete hydatidiform mole.
Placenta, 19, 361-369.
191. Rajakumar,A. and Conrad,K.P. (2000). Expression, ontogeny, and regulation of hypoxia-
inducible transcription factors in the human placenta. Biol. Reprod., 63, 559-569.
192. Ratts,V.S., Tao,X.J., Webster,C.B., Swanson,P.E., Smith,S.D., Brownbill,P.,
Krajewski,S., Reed,J.C., Tilly,J.L., and Nelson,D.M. (2000). Expression of BCL-2, BAX
and BAK in the trophoblast layer of the term human placenta: a unique model of
apoptosis within a syncytium. Placenta, 21, 361-366.
193. Ray,J., Jurisicova,A., and Caniggia,I. (2008). IFPA Trophoblast Research Award
Lecture: The Dynamic Role of Bcl-2 Family Members in Trophoblast Cell Fate.
Placenta.
194. Ray,J.E., Garcia,J., Jurisicova,A., and Caniggia,I. (2009). Mtd/Bok takes a swing:
proapoptotic Mtd/Bok regulates trophoblast cell proliferation during human placental
development and in preeclampsia. Cell Death. Differ..
195. Redline,R.W. (2008). Placental pathology: a systematic approach with clinical
correlations. Placenta, 29 Suppl A, S86-S91.
196. Redline,R.W. and Patterson,P. (1995). Pre-eclampsia is associated with an excess of
proliferative immature intermediate trophoblast. Hum. Pathol., 26, 594-600.
197. Redman,C.W., Sacks,G.P., and Sargent,I.L. (1999). Preeclampsia: an excessive maternal
inflammatory response to pregnancy. Am. J. Obstet. Gynecol., 180, 499-506.
198. Redman,C.W. and Sargent,I.L. (2000). Placental debris, oxidative stress and pre-
eclampsia. Placenta, 21, 597-602.
199. Redman,C.W. and Sargent,I.L. (2003). Pre-eclampsia, the placenta and the maternal
systemic inflammatory response--a review. Placenta, 24 Suppl A, S21-S27.
200. Roberts,J.M. and Cooper,D.W. (2001). Pathogenesis and genetics of pre-eclampsia.
Lancet, 357, 53-56.
201. Rodesch,F., Simon,P., Donner,C., and Jauniaux,E. (1992). Oxygen measurements in
endometrial and trophoblastic tissues during early pregnancy. Obstet. Gynecol., 80, 283-
285.
202. Rodier,G., Montagnoli,A., Di Marcotullio,L., Coulombe,P., Draetta,G.F., Pagano,M., and
Meloche,S. (2001). p27 cytoplasmic localization is regulated by phosphorylation on
Ser10 and is not a prerequisite for its proteolysis. EMBO J., 20, 6672-6682.
203. Rodriguez,J.M., Glozak,M.A., Ma,Y., and Cress,W.D. (2006). Bok, Bcl-2-related
Ovarian Killer, Is Cell Cycle-regulated and Sensitizes to Stress-induced Apoptosis. J.
Biol. Chem., 281, 22729-22735.
168
204. Romero,R., Nien,J.K., Espinoza,J., Todem,D., Fu,W., Chung,H., Kusanovic,J.P.,
Gotsch,F., Erez,O., Mazaki-Tovi,S., Gomez,R., Edwin,S., Chaiworapongsa,T.,
Levine,R.J., and Karumanchi,S.A. (2008). A longitudinal study of angiogenic (placental
growth factor) and anti-angiogenic (soluble endoglin and soluble vascular endothelial
growth factor receptor-1) factors in normal pregnancy and patients destined to develop
preeclampsia and deliver a small for gestational age neonate. J. Matern. Fetal Neonatal
Med., 21, 9-23.
205. Russo,A.A., Jeffrey,P.D., Patten,A.K., Massague,J., and Pavletich,N.P. (1996). Crystal
structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2
complex. Nature, 382, 325-331.
206. Sargent,I.L., Germain,S.J., Sacks,G.P., Kumar,S., and Redman,C.W. (2003). Trophoblast
deportation and the maternal inflammatory response in pre-eclampsia. J. Reprod.
Immunol., 59, 153-160.
207. Sebire,N.J. (2010). Histopathological diagnosis of hydatidiform mole: contemporary
features and clinical implications. Fetal Pediatr. Pathol., 29, 1-16.
208. Seckl,M.J., Fisher,R.A., Salerno,G., Rees,H., Paradinas,F.J., Foskett,M., and
Newlands,E.S. (2000). Choriocarcinoma and partial hydatidiform moles. Lancet, 356, 36-
39.
209. Sekimoto,T., Fukumoto,M., and Yoneda,Y. (2004). 14-3-3 suppresses the nuclear
localization of threonine 157-phosphorylated p27(Kip1). EMBO J., 23, 1934-1942.
210. Sgambato,A., Cittadini,A., Faraglia,B., and Weinstein,I.B. (2000). Multiple functions of
p27(Kip1) and its alterations in tumor cells: a review. J. Cell Physiol, 183, 18-27.
211. Sherr,C.J. (1994). G1 phase progression: cycling on cue. Cell, 79, 551-555.
212. Sherr,C.J. (1995). D-type cyclins. Trends Biochem. Sci., 20, 187-190.
213. Sherr,C.J. (1996). Cancer cell cycles. Science, 274, 1672-1677.
214. Sherr,C.J. and Roberts,J.M. (1995). Inhibitors of mammalian G1 cyclin-dependent
kinases. Genes Dev., 9, 1149-1163.
215. Sherr,C.J. and Roberts,J.M. (1999). CDK inhibitors: positive and negative regulators of
G1-phase progression. Genes Dev., 13, 1501-1512.
216. Sherr,C.J. and Roberts,J.M. (2004). Living with or without cyclins and cyclin-dependent
kinases. Genes Dev., 18, 2699-2711.
217. Shih,I. and Kurman,R.J. (2002). Molecular basis of gestational trophoblastic diseases.
Curr. Mol. Med., 2, 1-12.
218. Shin,J.K., Jeong,Y.T., Jo,H.C., Kang,M.Y., Chang,I.S., Baek,J.C., Park,J.K., Lee,S.A.,
Lee,J.H., Choi,W.S., and Paik,W.Y. (2009). Increased interaction between heat shock
169
protein 27 and mitogen-activated protein kinase (p38 and extracellular signal-regulated
kinase) in pre-eclamptic placentas. J. Obstet. Gynaecol. Res., 35, 888-894.
219. Sibai,B., Dekker,G., and Kupferminc,M. (2005). Pre-eclampsia. Lancet, 365, 785-799.
220. Sibai,B.M. (2003). Diagnosis and management of gestational hypertension and
preeclampsia. Obstet. Gynecol., 102, 181-192.
221. Slim,R. and Mehio,A. (2007). The genetics of hydatidiform moles: new lights on an
ancient disease. Clin. Genet., 71, 25-34.
222. Slingerland,J. and Pagano,M. (2000). Regulation of the cdk inhibitor p27 and its
deregulation in cancer. J. Cell Physiol, 183, 10-17.
223. Smith,S.C., Baker,P.N., and Symonds,E.M. (1997). Placental apoptosis in normal human
pregnancy. Am. J. Obstet. Gynecol., 177, 57-65.
224. Smith,S.C., Leung,T.N., To,K.F., and Baker,P.N. (2000). Apoptosis is a rare event in
first-trimester placental tissue. Am. J. Obstet. Gynecol., 183, 697-699.
225. Smith,S.C., Price,E., Hewitt,M.J., Symonds,E.M., and Baker,P.N. (1998). Cellular
proliferation in the placenta in normal human pregnancy and pregnancy complicated by
intrauterine growth restriction. J. Soc. Gynecol. Investig., 5, 317-323.
226. Soleymanlou,N., Jurisica,I., Nevo,O., Ietta,F., Zhang,X., Zamudio,S., Post,M., and
Caniggia,I. (2005a). Molecular evidence of placental hypoxia in preeclampsia. J. Clin.
Endocrinol. Metab, 90, 4299-4308.
227. Soleymanlou,N., Jurisicova,A., Wu,Y., Chijiiwa,M., Ray,J.E., Detmar,J., Todros,T.,
Zamudio,S., Post,M., and Caniggia,I. (2007). Hypoxic Switch in Mitochondrial Myeloid
Cell Leukemia Factor-1/Mtd Apoptotic Rheostat Contributes to Human Trophoblast Cell
Death in Preeclampsia. Am. J. Pathol..
228. Soleymanlou,N., Wu,Y., Wang,J.X., Todros,T., Ietta,F., Jurisicova,A., Post,M., and
Caniggia,I. (2005b). A novel Mtd splice isoform is responsible for trophoblast cell death
in pre-eclampsia. Cell Death. Differ., 12, 441-452.
229. Soothill,P.W., Nicolaides,K.H., Rodeck,C.H., and Campbell,S. (1986). Effect of
gestational age on fetal and intervillous blood gas and acid-base values in human
pregnancy. Fetal Ther., 1, 168-175.
230. Soto-Wright,V., Bernstein,M., Goldstein,D.P., and Berkowitz,R.S. (1995). The changing
clinical presentation of complete molar pregnancy. Obstet. Gynecol., 86, 775-779.
231. St Jacques,S., Forte,M., Lye,S.J., and Letarte,M. (1994). Localization of endoglin, a
transforming growth factor-beta binding protein, and of CD44 and integrins in placenta
during the first trimester of pregnancy. Biol. Reprod., 51, 405-413.
170
232. Steigrad,S.J. (2003). Epidemiology of gestational trophoblastic diseases. Best. Pract. Res.
Clin. Obstet. Gynaecol., 17, 837-847.
233. Straszewski-Chavez,S.L., Abrahams,V.M., and Mor,G. (2005). The role of apoptosis in
the regulation of trophoblast survival and differentiation during pregnancy. Endocr. Rev.,
26, 877-897.
234. Susin,S.A., Zamzami,N., Castedo,M., Hirsch,T., Marchetti,P., Macho,A., Daugas,E.,
Geuskens,M., and Kroemer,G. (1996). Bcl-2 inhibits the mitochondrial release of an
apoptogenic protease. J. Exp. Med., 184, 1331-1341.
235. Suzuki,A., Tsutomi,Y., Akahane,K., Araki,T., and Miura,M. (1998). Resistance to Fas-
mediated apoptosis: activation of caspase 3 is regulated by cell cycle regulator p21WAF1
and IAP gene family ILP. Oncogene, 17, 931-939.
236. Szulman,A.E. and Surti,U. (1984). Complete and partial hydatidiform moles: cytogenetic
and morphological aspects. Adv. Exp. Med. Biol., 176, 135-146.
237. Tanaka,S., Kunath,T., Hadjantonakis,A.K., Nagy,A., and Rossant,J. (1998). Promotion of
trophoblast stem cell proliferation by FGF4. Science, 282, 2072-2075.
238. Tham,B.W., Everard,J.E., Tidy,J.A., Drew,D., and Hancock,B.W. (2003). Gestational
trophoblastic disease in the Asian population of Northern England and North Wales.
BJOG., 110, 555-559.
239. Timmer,J.C. and Salvesen,G.S. (2007). Caspase substrates. Cell Death. Differ., 14, 66-
72.
240. Vaisbuch,E., Ben Arie,A., Dgani,R., Perlman,S., Sokolovsky,N., and Hagay,Z. (2005).
Twin pregnancy consisting of a complete hydatidiform mole and co-existent fetus: report
of two cases and review of literature. Gynecol. Oncol., 98, 19-23.
241. Venkatesha,S., Toporsian,M., Lam,C., Hanai,J., Mammoto,T., Kim,Y.M., Bdolah,Y.,
Lim,K.H., Yuan,H.T., Libermann,T.A., Stillman,I.E., Roberts,D., D'Amore,P.A.,
Epstein,F.H., Sellke,F.W., Romero,R., Sukhatme,V.P., Letarte,M., and Karumanchi,S.A.
(2006). Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat. Med., 12,
642-649.
242. Vervoorts,J. and Luscher,B. (2008). Post-translational regulation of the tumor suppressor
p27(KIP1). Cell Mol. Life Sci., 65, 3255-3264.
243. Villar,J., Carroli,G., Wojdyla,D., Abalos,E., Giordano,D., Ba'aqeel,H., Farnot,U.,
Bergsjo,P., Bakketeig,L., Lumbiganon,P., Campodonico,L., Al Mazrou,Y.,
Lindheimer,M., and Kramer,M. (2006). Preeclampsia, gestational hypertension and
intrauterine growth restriction, related or independent conditions? Am. J. Obstet.
Gynecol., 194, 921-931.
244. Vlach,J., Hennecke,S., and Amati,B. (1997). Phosphorylation-dependent degradation of
the cyclin-dependent kinase inhibitor p27. EMBO J., 16, 5334-5344.
171
245. Waga,S., Hannon,G.J., Beach,D., and Stillman,B. (1994). The p21 inhibitor of cyclin-
dependent kinases controls DNA replication by interaction with PCNA. Nature, 369,
574-578.
246. Wake,N., Seki,T., Fujita,H., Okubo,H., Sakai,K., Okuyama,K., Hayashi,H., Shiina,Y.,
Sato,H., Kuroda,M., and . (1984). Malignant potential of homozygous and heterozygous
complete moles. Cancer Res., 44, 1226-1230.
247. Watanabe,G., Albanese,C., Lee,R.J., Reutens,A., Vairo,G., Henglein,B., and Pestell,R.G.
(1998). Inhibition of cyclin D1 kinase activity is associated with E2F-mediated inhibition
of cyclin D1 promoter activity through E2F and Sp1. Mol. Cell Biol., 18, 3212-3222.
248. Weier,J.F., Weier,H.U., Jung,C.J., Gormley,M., Zhou,Y., Chu,L.W., Genbacev,O.,
Wright,A.A., and Fisher,S.J. (2005). Human cytotrophoblasts acquire aneuploidies as
they differentiate to an invasive phenotype. Dev. Biol., 279, 420-432.
249. Wilson,B.J., Watson,M.S., Prescott,G.J., Sunderland,S., Campbell,D.M., Hannaford,P.,
and Smith,W.C. (2003). Hypertensive diseases of pregnancy and risk of hypertension and
stroke in later life: results from cohort study. BMJ, 326, 845.
250. Wolter,K.G., Hsu,Y.T., Smith,C.L., Nechushtan,A., Xi,X.G., and Youle,R.J. (1997).
Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol., 139,
1281-1292.
251. Wong,S.Y., Ngan,H.Y., Chan,C.C., and Cheung,A.N. (1999). Apoptosis in gestational
trophoblastic disease is correlated with clinical outcome and Bcl-2 expression but not
Bax expression. Mod. Pathol., 12, 1025-1033.
252. Woo,M., Hakem,A., Elia,A.J., Hakem,R., Duncan,G.S., Patterson,B.J., and Mak,T.W.
(1999). In vivo evidence that caspase-3 is required for Fas-mediated apoptosis of
hepatocytes. J. Immunol., 163, 4909-4916.
253. Woo,M., Hakem,R., Furlonger,C., Hakem,A., Duncan,G.S., Sasaki,T., Bouchard,D.,
Lu,L., Wu,G.E., Paige,C.J., and Mak,T.W. (2003). Caspase-3 regulates cell cycle in B
cells: a consequence of substrate specificity. Nat. Immunol., 4, 1016-1022.
254. Xue,W.C., Feng,H.C., Chan,K.Y., Chiu,P.M., Ngan,H.Y., Khoo,U.S., Tsao,S.W.,
Chan,K.W., and Cheung,A.N. (2005). Id helix-loop-helix proteins are differentially
expressed in gestational trophoblastic disease. Histopathology, 47, 303-309.
255. Yakovlev,A.G., Di Giovanni,S., Wang,G., Liu,W., Stoica,B., and Faden,A.I. (2004).
BOK and NOXA are essential mediators of p53-dependent apoptosis. J. Biol. Chem.,
279, 28367-28374.
256. Yang,J., Liu,X., Bhalla,K., Kim,C.N., Ibrado,A.M., Cai,J., Peng,T.I., Jones,D.P., and
Wang,X. (1997). Prevention of apoptosis by Bcl-2: release of cytochrome c from
mitochondria blocked. Science, 275, 1129-1132.
172
257. Yasuda,M., Umemura,S., Osamura,R.Y., Kenjo,T., and Tsutsumi,Y. (1995). Apoptotic
cells in the human endometrium and placental villi: pitfalls in applying the TUNEL
method. Arch. Histol. Cytol., 58, 185-190.
258. Yinon,Y., Nevo,O., Xu,J., Many,A., Rolfo,A., Todros,T., Post,M., and Caniggia,I.
(2008). Severe intrauterine growth restriction pregnancies have increased placental
endoglin levels: hypoxic regulation via transforming growth factor-beta 3. Am. J. Pathol.,
172, 77-85.
259. Zhong,X.Y., Laivuori,H., Livingston,J.C., Ylikorkala,O., Sibai,B.M., Holzgreve,W., and
Hahn,S. (2001). Elevation of both maternal and fetal extracellular circulating
deoxyribonucleic acid concentrations in the plasma of pregnant women with
preeclampsia. Am. J. Obstet. Gynecol., 184, 414-419.
260. Zhou,Y., Damsky,C.H., and Fisher,S.J. (1997). Preeclampsia is associated with failure of
human cytotrophoblasts to mimic a vascular adhesion phenotype. One cause of defective
endovascular invasion in this syndrome? J. Clin. Invest, 99, 2152-2164.
261. Zhou,Y., Genbacev,O., Damsky,C.H., and Fisher,S.J. (1998). Oxygen regulates human
cytotrophoblast differentiation and invasion: implications for endovascular invasion in
normal pregnancy and in pre-eclampsia. J. Reprod. Immunol., 39, 197-213.
262. Zinkel,S., Gross,A., and Yang,E. (2006). BCL2 family in DNA damage and cell cycle
control. Cell Death. Differ., 13, 1351-1359.
263. Zybina,T.G., Frank,H.G., Biesterfeld,S., and Kaufmann,P. (2004). Genome
multiplication of extravillous trophoblast cells in human placenta in the course of
differentiation and invasion into endometrium and myometrium. II. Mechanisms of
polyploidization. Tsitologiia, 46, 640-648.
264. Zybina,T.G., Kaufmann,P., Frank,H.G., Freed,J., Kadyrov,M., and Biesterfeld,S. (2002).
Genome multiplication of extravillous trophoblast cells in human placenta in the course
of differentiation and invasion into endometrium and myometrium. I. Dynamics of
polyploidization. Tsitologiia, 44, 1058-1067.
265. Zybina,T.G. and Zybina,E.V. (2005). Cell reproduction and genome multiplication in the
proliferative and invasive trophoblast cell populations of mammalian placenta. Cell Biol.
Int., 29, 1071-1083.