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_____________________________________________________________________________________________ Pennsylvania Department of Health 2012-2013 Annual C.U.R.E. Report Magee Womens Research Institute and Foundation 2011 Formula Grant Page 1 Magee Womens Research Institute and Foundation Annual Progress Report: 2011 Formula Grant Reporting Period July 1, 2012 December 31, 2012 Formula Grant Overview Magee Womens Research Institute and Foundation received $971,921 in formula funds for the grant award period January 1, 2012 through December 31, 2012. Accomplishments for the reporting period are described below. Research Project 1: Project Title and Purpose In Vivo Analysis of Human C19MC MicroRNAs in a Transgenic Mouse Model The C19MC locus on chromosome 19 harbors the largest cluster of microRNAs (miRNAs) in humans. Interestingly, these miRNAs are primate-specific and uniquely expressed in placental trophoblasts, although they were recently found expressed infrequently in several forms of cancers. In trophoblasts the C19MC miRNAs constitute the most abundant family of miRNAs and hold a considerable regulatory potential. In this project, we seek to develop a transgenic mouse model that will allow us to investigate the function of this unique family of miRNAs. Duration of Project 1/1/2012 12/31/2012 Project Overview Our goal is to better understand placental biology and the causes of placental insufficiency leading to gestational diseases. MicroRNAs have emerged as critical regulators of virtually every biological process and their altered expression is increasingly found to be associated with pathological states. Recently, it was found that the placenta is an almost exclusive source of a large family of miRNAs originating from a unique cluster located on chromosome 19 (C19MC). While the expression of these miRNAs is normally restricted to placental trophoblasts, their aberrant expression in other cell types is often associated with malignant conditions. However, the relevant biological function of these miRNAs in the placenta remains poorly understood. In this project, we aim to develop a transgenic mouse line using a BAC plasmid containing the entire C19MC locus and flanking sequences. This “humanized” mouse model may enable us to examine the collective function of the C19MC miRNAs. In the first aim of this project we perform a phenotypic and physiologic characterization of the new transgenic mouse strain. In addition, we plan to carefully examine the expression of the transgenic miRNAs first in mouse placentas, and also in other organs. In the second aim we focus on the molecular pathways that

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Page 1: Magee Womens Research Institute and Foundation Annual ... Research... · Magee Womens Research Institute and Foundation – 2011 Formula Grant – Page 1 ... July 1, 2012 – December

_____________________________________________________________________________________________

Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report

Magee Womens Research Institute and Foundation – 2011 Formula Grant – Page 1

Magee Womens Research Institute and Foundation

Annual Progress Report: 2011 Formula Grant

Reporting Period

July 1, 2012 – December 31, 2012

Formula Grant Overview

Magee Womens Research Institute and Foundation received $971,921 in formula funds for the

grant award period January 1, 2012 through December 31, 2012. Accomplishments for the

reporting period are described below.

Research Project 1: Project Title and Purpose

In Vivo Analysis of Human C19MC MicroRNAs in a Transgenic Mouse Model – The C19MC

locus on chromosome 19 harbors the largest cluster of microRNAs (miRNAs) in humans.

Interestingly, these miRNAs are primate-specific and uniquely expressed in placental

trophoblasts, although they were recently found expressed infrequently in several forms of

cancers. In trophoblasts the C19MC miRNAs constitute the most abundant family of miRNAs

and hold a considerable regulatory potential. In this project, we seek to develop a transgenic

mouse model that will allow us to investigate the function of this unique family of miRNAs.

Duration of Project

1/1/2012 – 12/31/2012

Project Overview

Our goal is to better understand placental biology and the causes of placental insufficiency

leading to gestational diseases. MicroRNAs have emerged as critical regulators of virtually every

biological process and their altered expression is increasingly found to be associated with

pathological states. Recently, it was found that the placenta is an almost exclusive source of a

large family of miRNAs originating from a unique cluster located on chromosome 19 (C19MC).

While the expression of these miRNAs is normally restricted to placental trophoblasts, their

aberrant expression in other cell types is often associated with malignant conditions. However,

the relevant biological function of these miRNAs in the placenta remains poorly understood.

In this project, we aim to develop a transgenic mouse line using a BAC plasmid containing the

entire C19MC locus and flanking sequences. This “humanized” mouse model may enable us to

examine the collective function of the C19MC miRNAs. In the first aim of this project we

perform a phenotypic and physiologic characterization of the new transgenic mouse strain. In

addition, we plan to carefully examine the expression of the transgenic miRNAs first in mouse

placentas, and also in other organs. In the second aim we focus on the molecular pathways that

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may be targeted by these miRNAs. Using microarray analysis and computational methods we

compare gene expression in tissue from transgenic animals with their wild type counterparts and

identify potential targets of the C19MC miRNAs.

Principal Investigator

Jean-Francois Mouillet, PhD

Instructor

Magee-Womens Research Institute and Foundation

204 Craft Avenue, Room A634

Pittsburgh, PA 15213

Other Participating Researchers

Tianjiao Chu, PhD; Yoel Sadovsky, MD – employed by Magee Womens Research Institute and

Foundation

Expected Research Outcomes and Benefits

Placental insufficiency is one of the main complications of pregnancy, and may affect as many as

3-10% of all pregnancies. It is associated with compromised fetal growth and development and

poor pregnancy outcome. In addition, strong epidemiological evidence indicates that surviving

growth-restricted infants are at an increased risk of coronary heart disease, hypertension, stroke

and type-2 diabetes during adulthood. Despite the improvement in clinical imaging for diagnosis

of fetal growth restriction, little progress has been made in deciphering the causes of fetal growth

restriction (FGR) and the development of sensitive molecular probes designed to investigate fetal

growth restriction or assist in early diagnosis.

The discovery of miRNAs and the realization that miRNAs are abundant in the human placenta

offer new perspectives on placental development and pathophysiology. Intriguingly, the placenta

is the main source of a set of miRNAs deriving from a large miRNA cluster located on

chromosome 19 (C19MC). These C19-linked miRNAs are uniquely found in humans and non-

human primates, where they are primarily expressed in placental trophoblasts. Normally

restricted to the placenta, aberrant expression of miRNAs from this locus has been observed in

several cancers. Recent studies also revealed that undifferentiated embryonic stem cells express

some C19MC miRNAs, although their expression is lost when cells differentiate. The role of

these miRNAs and their impact on cellular function is unknown. The analysis of C19MC

miRNA function will illuminate molecular pathways governing trophoblast differentiation and

function. Furthermore, conclusions from our studies may uncover important mechanisms that

contribute to placental dysfunction.

Summary of Research Completed

This report covers the last six months of the project in which we generated a transgenic mouse

model expressing the human chromosome 19-microRNA cluster (C19MC). The experiments

performed during this period indicate that the C19MC transgene is expressed in the mouse with a

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pattern very similar to that seen in humans, with high expression mostly restricted to the

placenta. We also show that, as in humans, C19MC miRNAs produced in the placenta are

released in the maternal circulation. However, in spite of high basal placental levels of C19MC

miRNAs, transgenic placentas and fetuses do not show any phenotypic alterations.

Aim 1: Analyze the ectopic expression of the human C19MC miRNAs in mouse organs and

characterize their phenotype.

We continued efforts to characterize the expression pattern of the transgene and made detailed

analysis of the C19MC expression in the four lines of transgenic mice that were established.

Primary expression of the C19MC miRNAs in the placenta was confirmed while their detection

in other tissues was shown to be minimal, with the exception of testes (Fig.1A). RT-qPCR

analysis of a larger set of samples revealed a higher level of expression in transgenic testes than

was previously observed. We also analyzed mouse blood samples from pregnant and non-

pregnant transgenic mice in order to determine whether C19MC are miRNAs released in the

maternal circulation as it was observed in human pregnancies. Total RNA were extracted from

200 ul of mouse plasma using a NucleoSpin miRNA plasma kit from Macherey-Nagel.

Typically, one-fifth of the isolated RNA was used for detection using the miScript PCR System

from Qiagen. Our results clearly indicate that transgenic C19MC miRNAs are detected in the

mouse blood (Fig.1B). We can also confirm their placental origin because plasma samples from

non-pregnant transgenic females were negative while samples from pregnant wild-type females

were positive when carrying transgenic embryos. Finally, examination of C19MC miRNA levels

in the internal organs of wild-type females carrying transgenic embryos indicate that they do not

accumulate in tissues such as liver or kidneys despite their substantial levels in the systemic

circulation.

C19MC expression was also assessed in tissues from transgenic embryos. Overall, the C19MC

miRNAs were low in embryonic organs. We detected, however, some expression in the brain

(Fig 2A). Interestingly, we also detected the presence of the transgenic miRNAs in the plasma of

embryos (Fig. 2B). At this stage of the study, though, we have not clarified their origin (placenta

or internal organs).

During the course of these studies, we noticed a difference in the level of C19MC expression

depending on the sex of the parent from which the transgene was inherited. (Our transgenic mice

are maintained as hemizygotes and only one of the parents contribute to passing the transgene to

their offspring.) Interestingly, embryos that received the transgene from the male expressed a

significantly higher level of C19MC miRNAs compared to the embryos that inherited the

transgene from the female (Fig. 3). A recently published study has shown that the C19MC

cluster was imprinted in humans and was expressed exclusively from the male allele. Although

this restriction is not absolute in our mice, it nonetheless suggests that some level of imprinting

was retained in transgenic animals. In humans, a differentially methylated region has been

identified approximately 17kb upstream of the first miRNA gene of the cluster. This domain,

with a maternal-specific methylation imprint in the human placenta, is present in the DNA

sequences included in the BAC vector and may be responsible for the parent-of-origin-dependent

expression of the transgene in mice. To further address this question, we will investigate the

methylation status of this region in transgenic mice using bisulfite sequencing.

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We next tried to determine the precise distribution of the C19MC miRNAs in transgenic

placentas using in situ hybridization (ISH) on frozen sections using a digoxigenin (DIG)-labeled

locked nucleic acid (LNA) probe. Dissected mouse placentas were pre-fixed in 4%

paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight, then successively, in

10% sucrose in PBS for 30 minutes, 20% sucrose in PBS, and finally, mounted in Tissue-Tek

OCT (Sakura Finetek). Frozen sections (10 μm) were treated with 20 mg/ml proteinase K

(Roche), then fixed in 4% PFA and acetylated for 10 minutes with a mixture of 0.25% acetic

anhydride and 0.1 M triethanolamine. The postfixed tissues were subsequently incubated

overnight with DIG-labeled LNA probes from Exiqon according to the manufacturer’s

instructions. After stringent washes, the slides were incubated with alkaline phosphatase

conjugated with an anti-DIG antibody (Roche) and detected using BM Purple Substrate (Roche).

Finally, the slides were mounted in dextropropoxyphene (Fluka). Although we have successfully

used this approach in the past to detect several mouse placental miRNAs, we were not able to

detect a specific signal for any of the C19MC tested. We made several attempts to improve the

detection (1) by using double-DIG-labeled LNA probes to increase the strength of the signal and

(2) by employing different hybridization temperature, in addition to using several tissue fixation

procedures. Four C19MC-specific LNA probes were tested, but none of them produced a clear

and specific signal. These probes were also used on human placental sections, but again, no

specific signal was confirmed. The reason for this failure is not known although an earlier study

revealed that only 48 miRNA ISH probes were confirmed out of a total of 130.

To detect and localize the transgenic C19MC miRNAs, we are currently working on an

alternative technique using Fluorescent ISH (RNA-FISH). This recently described method to

allow visualization of C19MC expression in trophoblast cell lines uses a short cyanin 3 (Cy3)-

labeled DNA oligonucleotide that hybridizes to repetitive sequences within the C19MC primary

transcript. This method was first applied to primary human trophoblasts that were fixed in 4%

PFA and then permeabilized overnight in 70% ethanol at 4ºC. After fixation, cells were

hybridized overnight at 37ºC with the Cy3-labeled probe in a 15% formamide/2X SSPE buffer.

The slides were then washed and mounted on Mowiol-DAPI. A strong, unique nuclear signal

was detected in almost every cell, revealing transcription at the C19MC locus (Fig. 4). We are

planning to adapt this technique to work on placental sections in order to detect and identify the

cells that express the C19MC miRNAs.

At the phenotypic level, we initially noted a slight increase in the weight of transgenic placentas

compared to their wild-type counterparts. However, this trend was not confirmed upon analysis

of more samples, and apparently, there is no difference, at the level of the placenta or the

embryo, between transgenic and wild-type (Fig. 5). As part of our general phenotype

characterization of the transgenic mice, the histology of every organ collected from adult mice

(6-8 weeks old) was evaluated in tissue sections. We also obtained 10-µm sections from paraffin-

embedded embryos for detailed examination of the histology of transgenic embryos collected at

E18.5 dpc. However, to date we have not observed any phenotypic impact of the expression of

the C19MC miRNAs in the mouse. Also, because ectopic expression of these miRNAs has been

found associated with several types of cancers in humans, we looked carefully for the appearance

of tumors in aging transgenic mice. Several mice were kept over a period of one year, then

sacrificed for histological examination. No pathologies or anomalies were observed in these

mice.

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Aim 2: Examine gene expression profiles in transgenic organs and their wild-type counterparts

using microarrays and determine the cellular pathways impacted by these miRNAs.

Transgenic mice express high levels of C19MC miRNAs in the placenta in a pattern similar to

that observed in human placental trophoblasts, where they could potentially alter the expression

of a number of target genes. To investigate the molecular effect of these transgenic miRNAs in

mouse trophoblasts, we performed a microarray analysis of gene expression in mouse transgenic

placentas and wild-type placentas. Placentas from transgenic embryos and from their wild-type

counterparts were collected from the different transgenic lines, and total RNA was purified using

a miRNeasy Mini Kit from Qiagen, labeled using the Agilent Low RNA Input Linear

Amplification Kit in the presence of Cy3-CTP and hybridized to Agilent SurePrint G3 Mouse

GE 8x60K microarray. After hybridization and washing, the microarrays were processed with an

Agilent scanner. A total of 24 arrays, corresponding to 12 samples from transgenic placentas and

12 samples from wild-type placentas, were hybridized.

Microarray data were first Log2-transformed, then normalized using the cyclic loess

normalization method. The normalized data were then analyzed using two-way ANOVA with

two factors: the Treatment factor, representing whether the placenta is transgenic or wild-type;

and the Strain factor, representing the four transgenic lines. Moderated F-test, as implemented in

the R package limma, was performed for the Treatment factor to identify the genes that are

differentially expressed between the transgenic placenta and wild-type placenta. The q-value

method was used to adjust the p values of the moderated F-test to control for the false discovery

rate. Because no genes were found to have significant adjusted p values at the 0.05 level, we

retained the genes satisfying one of the following criteria: (1) the Log2 fold change between the

wild type and the transgenic placenta is at least 1 and the p-value of the moderated F-test is no

greater than 0.001, or (2) the Log2 fold change between the wild type and the transgenic placenta

is at least 2 and the p value of the moderated F-test is no greater than 0.01, or (3) the adjusted p

value of the moderated F-test is no greater than 0.1. This analysis did not allow us to clearly

identify a set of differentially expressed genes.

In addition, a subsequent hierarchical cluster analysis using Ward’s method revealed that the data

from the 24 microarrays clustered by transgenic line and not the transgenic status (Fig. 6).

Therefore, expression of the C19MC miRNAs in mouse placenta does not seem to have any

impact on gene expression.

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Fig. 1: Expression of transgenic C19MC miRNAs in mouse adult tissues. Total RNA samples were analyzed by real-time PCR of miR-517a using the miScript PCR System (Qiagen). (A) Relative expression level of miR-517a in tissues from adult transgenic mice. Bars represent the Log2 fold change in miR-517a levels in various tissues, taking the liver as the reference. (B) Relative miR-517a levels in plasma samples from pregnant transgenic (Tg) and wild-type (WT) females. Bars represent the Log2 fold change of miR-517a level in samples from transgenic mice, taking wild-type plasma samples as reference.

Fig. 2: Expression of transgenic C19MC miRNAs in mouse embryo tissues. Total RNA samples were analyzed by real-time PCR of miR-517a using the miScript PCR System (Qiagen). (A) Relative expression level of miR-517a in tissues from adult transgenic mice. Bars represent the Log2 fold change in miR-517a levels in various tissues, taking the liver as the reference. (B) Relative miR-517a levels in plasma samples from transgenic (Tg) and wild-type (WT) embryos. Bars represent the Log2 fold change of miR-517a level in samples from transgenic mice, taking wild-type plasma samples as reference.

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Fig. 3: Parent-of-origin effect on C19MC transgene expression. Relative miR-517a expression in the four transgenic lines was analyzed by real-time PCR in placentas from offspring which had inherited the transgenic locus paternally (orange bars) or maternally (gray bars). Expression in the offspring with maternal transmission was set at one for comparison. TG-F/WT-M: Transgenic female crossed with wild-type male. WT-F/TG-M: Wild-type female crossed with transgenic male.

Fig. 4: RNA-FISH analysis of expression at

the C19MC locus in primary human

trophoblast cells. A Cy3-labeled DNA

oligonucleotide probe (red) detecting

sequences from the primary C19MC transcript

was used. Nuclei were counterstained with

DAPI (blue). Note that most cells exhibit a

single RNA-FISH signal indicating

monoallelic expression of C19MC in human

trophoblasts.

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Fig. 5: Embryo weight and placenta weight in C19MC-transgenic mice (TG) and wild-type mice (WT). Embryos and placentas were collected at 18.5 days post coitum.

Fig. 6: Hierarchical cluster analysis of the 24 arrays using Ward’s method based on Euclidean distance. The distance between two arrays is defined as 1-the Pearson’s correlation between the expression levels of the genes with above-the-median expression level in the two arrays.

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Research Project 2: Project Title and Purpose

Glycocalyx Syndecan-1 and Preeclampsia Pathogenesis – The purpose of the project is to learn

more about what causes the human pregnancy-specific hypertensive disease preeclampsia. Our

preliminary data suggest that women with preeclampsia have reduced expression of the heparan

sulfate proteoglycan syndecan-1 (SDC-1; CD138) in the placenta, correlating with reduced

concentrations of soluble SDC-1 in the maternal circulation. SDC-1 and the related protein

glypican-1 (GPC-1) may play pivotal roles in regulation of cell interactions and function. The

project will 1) test if changes in maternal plasma soluble SDC-1 or GPC-1 herald the

development of preeclampsia, and 2) study the expression and biologic function of these factors

in normal and abnormal placenta.

Duration of Project

1/1/2012 – 12/31/2012

Project Overview

Preliminary data suggest that, compared to women with uncomplicated pregnancies (controls),

women with preeclampsia or fetal intrauterine growth restriction (IUGR) are distinguished by

reduced expression of the heparan sulfate proteoglycan (HSPG) syndecan-1 (SDC-1) on the

apical surface of placental syncytiotrophoblast, along with lower levels of soluble (shed) SDC-1

in maternal plasma. The expression of SDC-1 (present only on syncytiotrophoblast) suggests a

role for SDC-1 in communication between fetal villi and maternal blood that fails in

preeclampsia.

The objectives of this project are thus to 1) understand how maternal plasma concentrations of

soluble SDC-1 and the related HSPG glypican-1 (GPC1) relate to placental expression of SDC-1

and GPC1 in normal and preeclamptic pregnancies; 2) to examine whether reduced plasma SDC-

1 precedes the clinical onset of preeclampsia; and 3) to begin to explore functional consequences

of altered SDC-1 expression in placental trophoblasts.

The Aims of this project are to show that:

1) Maternal plasma concentrations of soluble SDC-1 and GPC1 are reduced both early in

pregnancy (before clinical disease) and during preeclampsia compared to women whose

pregnancies remain uncomplicated, and the reduced plasma soluble SDC-1 and GPC1 is

explained by reduced expression of these glycoproteins on syncytiotrophoblast of the placenta.

2) Local retention/binding of the placentally secreted soluble receptor, soluble vascular

endothelial growth factor (VEGF) receptor-1 (sFlt1, an anti-angiogenic decoy receptor

implicated in the pathogenesis of preeclampsia) is regulated by heparan sulfate components of

cell surface SDC-1 on trophoblast cells.

3) SDC-1 mediates binding and internalization of very low-density lipoprotein (VLDL) in human

trophoblasts suggesting an important role for SDC-1 in placental lipid trafficking with potential

implications for fetal growth.

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Principal Investigator

Carl A. Hubel, PhD

Associate Professor

Magee Womens Research Institute

204 Craft Avenue

Pittsburgh, PA 15213

Other Participating Researchers

Robert W. Powers, PhD; Arundhathi Jeyabalan, MD; Robin E. Gandley, PhD – employed by

Magee Womens Research Institute

Expected Research Outcomes and Benefits

The etiology and pathogenesis of the multi-system pregnancy disorder preeclampsia remain

poorly understood. Our preliminary data suggest that women with preeclampsia are

distinguished by abnormally low concentrations of the soluble (circulating) HSPG synedecan-1

(SDC-1) in maternal plasma compared to both controls with uncomplicated (normotensive)

pregnancies, and that low soluble SDC-1 is paralleled by reduced expression of SDC-1 on the

apical surface of placental villous syncytiotrophoblast in the same patients. Therefore, we wish

to further explore this association and probe the potential role of SDC-1 in the development of,

and prediction of, preeclampsia. If the hypotheses of this pilot project are supported, it will

springboard an NIH proposal to conduct detailed, mechanistic studies on the relationship

between the soluble and membrane-bound versions of SDC-1, and related HSPG’s including

glypican-1 (GPC1), and their relationship to placental function and vascular function during

pregnancy in the human, and in knockout mouse and cell culture models of preeclampsia.

Although this is not a clinical trial and not expected to have immediate ‘bench to bedside’

translation of research findings to patient care, this project will significantly advance our

understanding of the pathogenesis of preeclampsia. Placenta-derived factors in maternal blood--

if altered early in pregnancy-- could be incorporated into algorithms allowing for improved

predictive tests for preeclampsia, and may lead to therapeutic avenues. This biomedical research

project features collaboration, and is intended for new discovery, leveraging new grants, and

potential for addressing health disparities.

Summary of Research Completed

Specific Aim 1:

Cross-sectional patient data: During the first reporting period we showed that third trimester

(time of disease) plasma soluble SDC-1 levels were 2.5-fold lower in women with preeclampsia

compared to controls (P<0.01). During the July-December 2012 (current) reporting period we

examined correlations with clinical variables. It was noted that the mean baby birth weight

percentile in the preeclampsia group was 20 25 (SD). Seven of the 17 preeclampsia cases

delivered babies that were small for gestational age, defined as birth weight percentile < 10. The

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mean birth weight centile of this sub-group (2.3 1.7), was significantly different (P<0.01)

compared to birth weight centile of the 10 preeclampsia cases with more appropriately grown for

gestation age (AGA) babies (34.3 3.2). As shown in Figure 1, the low plasma SDC-1 values in

the preeclampsia group were predominantly clustered in the preeclampsia sub-group with SGA

infants [SDC-1 median (interquartile range): 221 (149-268)]. These values were significantly

different from the controls (p<0.01) whereas values in the preeclampsia sub-group with AGA

infants [SDC-1 median (interquartile range): 635 (255 – 779)] did not differ from the controls.

These data indicate an association of low maternal plasma sSDC-1 with the restricted infant

growth that often accompanies preeclampsia.

We compared plasma sSDC-1 levels between gestational hypertension (without proteinuria)

(n=8), normotensive SGA (n=6), and control (n=6) groups. Plasma sSDC-1 in the gestational-

age matched samples of these 3 patient groups not differ (Kruskal-Wallis test) (Table 1). These

data suggest that low plasma sSDC-1 is not merely a consequence of hypertension and is more

strongly related to preeclampsia with SGA than SGA alone.

Longitudinal normal pregnancy data: During the first reporting period Soluble SDC-1

concentrations were measured by ELISA in longitudinal (pregnancy and postpartum) plasma

samples obtained from 7 women with uncomplicated pregnancy. One additional patient with

samples extending to 4-9 weeks postpartum was added (n=8). These data are shown in Figure 2.

Mid-pregnancy evaluations of plasma sSDC-1 and triglycerides:

Maternal plasma sSDC-1 concentrations were measured in gestational age-matched samples

collected at mid-pregnancy from 9 women who later developed preeclampsia, 9 women who

later developed gestational hypertension (without proteinuria), and from 19 normotensive

controls with uncomplicated outcome. These patients comprised a nested-case subset of

longitudinal patients recruited by the PEPP study. Plasma sSDC-1 was measured by ELISA in

duplicate (Eli-pair kit from Diaclone/Cell Sciences Inc.). Plasma total triglycerides were

measured by colorimetric assay, with quality controls provided by the vendor (Pointe Scientific,

Inc.). The clinical characteristics of these patients, who are different individuals from the other

patient sets, are summarized in Table 2. Similar to the cross-sectional study subjects, longitudinal

subjects who developed preeclampsia delivered earlier (p<0.05), had babies with lower birth

weight percentiles (p<0.05), and by definition significantly higher blood pressures at admission

to labor and delivery (p<0.05). Women with gestational hypertension also had elevated blood

pressures and babies with lower birth weight percentiles (p<0.05), but did not deliver earlier,

compared to controls. As shown in Figure 3 and Table 2, Plasma sSDC-1 concentrations were

significantly reduced (36%, p<0.05) at 20 weeks of gestation (more than 10 weeks prior to

clinically evident disease) in women who later developed preeclampsia, but not gestational

hypertension, compared to controls. Median plasma triglycerides were higher at 20 weeks

gestation in patients who later developed preeclampsia (mg/dl: preeclampsia 127, gestational

hypertension 104, control 92), but not significantly so (P=0.10).

Syncytiotrophoblast Immunohistochemical Staining and Scoring: Extending the data in the first

reporting period, we excluded one prospective SGA patient who had a baby birth weight

percentile >10 and one uncomplicated pregnancy patient with birth weight percentile <10. Two

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SGA patients were added to give n=12 normal (uncomplicated) pregnancy controls, n=10

preeclamptics, n= 6 SGA. mRNA profiling was completed on these 2 patients and

imunostaining for syndecan-1 and glypican-1 was completed on all of the SGA patients. We

have developed a macro for more quantitative cell surface analysis using NIH Image J software.

Specific Aim 2:

In the Strategic Research Plan we presented data that blood concentrations of the anti-angiogenic

soluble decoy receptor for VEGF, sFlt1 increase ~250-fold shortly after intravenous heparin

administration to non-pregnant women of child-bearing age, equivalent to 3rd

trimester normal

pregnancy levels, and of sufficient quantity to reduce concentrations of free VEGF to near zero.

Measurements of heparin-induced release of sFlt1 from whole blood in vitro were performed

during this reporting period. We added heparin to whole blood samples from pregnant and non-

pregnant women to test the hypothesis that blood cells or other plasma components do not

contribute appreciably to releasable stores of sFlt-1. We obtained blood samples from four

women prior to delivery (mean ± standard deviation: 38.4 ± 2.1 weeks gestation), and seven

women between 6 and 21 months post-partum (13 ± 5 months). Blood was collected in

vacutainers containing EDTA. Pre-delivery samples were treated under six conditions as shown

in Figure 4: (1) Plasma was isolated within 2 hours of venipuncture; (2a-c) Whole blood was

incubated for 24 hours at 37°C before plasma was isolated and frozen with no additives (2a), or

after adding 20 U/ml of unfractionated (2b) or low molecular weight (2c) heparin to the plasma;

(3a,b) Whole blood was incubated with 20 U/ml of unfractionated (3a) or low molecular weight

(3b) heparin for 24 hours at 37°C before the plasma was isolated. Plasma for all conditions was

frozen at -80°C for later analysis. Post-partum samples were treated under four conditions (1, 2a,

2b, 3a), excluding the two conditions involving treatment with low molecular weight heparin (2c,

3b). Plasma sFlt1 was measured by ELISA kit (R&D Systems). Effects of treatments were

determined by Wilcoxon signed rank test. Results are shown in Figure 4. In pre-delivery

samples, plasma sFlt1 was slightly lower in samples processed within 2 hours than in samples in

which whole blood was incubated for 24 hours, or in which whole blood was incubated for 24

hours and LMWH was then added to plasma. Incubating whole blood or plasma with LMH or

UFH did not further increase sFlt1; in fact, sFlt1 was significantly lower in all conditions in

which UFH or LMWH was added to plasma than in samples incubated for 24 hours without

heparin (p<0.05). In post-partum samples, plasma sFlt1 was slightly higher in all conditions in

which whole blood was incubated for 24 hours compared to samples processed immediately

(P<0.05). However, adding UFH to whole blood or plasma had no additional effect on plasma

sFlt1. These data show negligible heparin-induced release of sFlt1 from blood cells or other

plasma components, supporting the premise that tissue cells in contact with maternal blood

(endothelium and syncytiotrophoblast) are the major reservoir of heparin-releasable sFlt1.

Specific Aim 3:

Analysis of the relationship between maternal and umbilical cord (fetal) blood Lipid levels:

The relationship between maternal plasma and umbilical cord (fetal) plasma concentrations of

total triglycerides and cholesterol was assessed in 11 preeclampsia cases and 23 uncomplicated

pregnancy controls. Plasma total triglycerides and cholesterol were measured using reagents by

colorimetric manual method (Pointe Scientific). Clinical characteristics and lipid data are given

in Table 3. Maternal lipid levels were higher than cord lipid levels. Triglyceride concentrations

in maternal plasma were significantly higher in preeclamptics compared to controls whereas

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cholesterol concentrations did not differ. In contrast, cholesterol concentrations in fetal (cord)

plasma were significantly higher in preeclamptics whereas cord triglyceride concentrations did

not differ between groups. This is plausibly consistent with the hypothesis that trans-placental

transfer of triglyceride-rich lipoproteins (VLDL and IDL) from maternal to fetal circulations is

diminished in preeclampsia. It might also be consistent with reports that glycocalyx disruption is

associated with impaired LDL retention by the glycocalyx and transcellular leakage of

cholesterol (LDL) into the intima (van den Berg, et al. Pflugers Arch 2009;457:1199-1206).

Figure 1. Box-plot of 3rd

trimester plasma soluble syndecan-1 (sSDC-1) concentrations (y-axis)

in gestational age-matched women with uncomplicated pregnancies (controls) compared to

preeclampsia without small for gestational age (SGA) and preeclampsia with SGA (SGA defined

as infant birth weight percentile less than 10; all were less than 5th

centile). The top, bottom, and

solid line through the interior of the box correspond to the 75th

percentile, 25th

percentile, and

50th

percentile (median), respectively. The whiskers (t bars) on the bottom and top denote the

10th

percentile and 90th

percentile, respectively. Solid lines through boxes denote median values.

Significant difference is denoted by the horizontal line above the boxes. The sSDC-1 value in

the preeclampsia+SGA subgroup (mean gestation age 30 weeks) approximates the value seen

during 7-11 weeks of normal pregnancy.

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Figure 3. Box-plot of sSDC-1 concentrations in gestational age-matched mid-pregnancy (20

week) plasma samples (y-axis) from women with uncomplicated pregnancy, preeclampsia, or

gestational hypertension (Gest HTN). The top, bottom, and solid line through the interior of the

box correspond to the 75th

percentile, 25th

percentile, and 50th

percentile (median), respectively.

The whiskers (t bars) on the bottom and top denote the 10th

percentile and 90th

percentile,

respectively. Solid lines through the boxes denote median values. Significant difference between

preeclampsia and control groups (p<0.05) is indicated by the vertical line.

Figure 2. Plasma SDC-

1 concentrations

increase with advancing

gestation and decline

postpartum. Data are

from a longitudinal

patient with

uncomplicated first

pregnancy.

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Figure 4. Pre-delivery samples were treated under six conditions: (1): Plasma was isolated within

2 hours of venipuncture; (2a-c): Whole blood was incubated for 24 hours at 37°C before plasma

was isolated and frozen with no additives (2a), or after adding 20 U/ml of unfractionated (2b) or

low molecular weight (2c) heparin to the plasma; (3a,b): Whole blood was incubated with 20

U/ml of unfractionated (3a) or low molecular weight (3b) heparin for 24 hours at 37°C before the

plasma was isolated. Plasma for all conditions was frozen at -80°C for later analysis. Post-

partum samples were treated under four conditions (1, 2a, 2b, 3a), excluding the two conditions

involving treatment with low molecular weight heparin (2c, 3b). In pre-delivery samples, plasma

sFlt1 was slightly but significantly lower in samples processed within 2 hours than in samples in

which whole blood was incubated for 24 hours, or in which whole blood was incubated for 24

hours and LMWH was then added to plasma. Incubating whole blood or plasma with LMH or

UFH did not further increase sFlt1; sFlt1 was significantly lower in all conditions in which UFH

or LMWH was added to plasma than in samples incubated for 24 hours without heparin

(p<0.05). In post-partum samples, plasma sFlt1 was slightly higher in all conditions in which

whole blood was incubated for 24 hours compared to samples processed immediately (P<0.05).

However, adding UFH to whole blood or plasma had no additional effect on sFlt1.

Table 1. Plasma Soluble Syndecan-1 Data; Cross-sectional Control, Gestational Hypertensive,

and Small for Gestational Age (SGA) Pregnancy Groups

Normotensive

Controls

(n=6)

Gestational

Hypertension

(n=8)

SGA

(n=6)

P value

Soluble syndecan-1

ng/mL

(median and interquartile range)

1358

(1098-1546)

923

(766-1101)

1177

(708-1908)

NS

NS, not significant

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Table 2. Clinical and Mid-pregnancy Soluble Syndecan-1 Data; Control, Gestational

Hypertensive, and Preeclampsia Pregnancy Groups

Normotensive

Controls

(n=19)

Gestational

Hypertension

(n=9)

Preeclampsia

(n=9)

P-

value

Age (years)

BMI pre-pregnancy (kg/m2)

25 4

29.5 3.6

24 6

28.6 5.7

26 8

31.1 7.1

NS

NS

Gestational weeks at venipuncture

Gestational weeks at delivery

20.4 1.9

39.3 2.7

20.3 2.3

39.9 1.2

20.0 1.4

#36.0 1.3

NS

<0.05

Early gestational BP (<20wks.)

Systolic (mm Hg)

Diastolic (mm Hg)

Pre-delivery BP:

Systolic (mm Hg)

Diastolic (mm Hg)

113 7

68 6

125 8

72 7

122 10

73 7

*147 8

*88 6

116 6

72 4

*154 17

*99 7

NS

NS

<0.05

<0.05

Birth weight percentile 71 18 *38 15 *30 29 <0.05

Cigarette smokers (%) 0% 0% 0% NS

Race (% black) 47% 44% 56% NS

Soluble syndecan-1

ng/mL

(median and interquartile range)

272

(245-595)

242

(148-369)

*174

(136-232)

<0.05

Continuous variables are given as mean (SD) or median (interquartile range; IQR);

NS, not significant; BMI, body mass index;

Birth weight percentiles were calculated using local (Western PA) race- and gender-specific

growth data adjusted for gestational age at delivery;

P values: #Indicated group versus the other two groups; *Indicated group versus Control.

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Table 3. Cross-Sectional Pregnancy Groups, Aim 3 Analysis of Cord and Maternal Lipids

Normotensive

Controls

(n=23)

Preeclamptics

(n=11)

P value

Age (years)

BMI pre-pregnancy (kg/m2)

26 6

24.9 4.8

25 5

28.3 5.5

NS

NS

Gestational weeks at venipuncture

Gestational weeks at delivery

35.3 4.6

39.6 1.9

35.0 3.4

35.2 3.4

NS

<0.05

Early gestational BP (<20wks.)

Systolic (mm Hg)

Diastolic (mm Hg)

Pre-delivery BP:

Systolic (mm Hg)

Diastolic (mm Hg)

112 8

70 6

120 8

68 11

119 6

74 3

154 10

97 8

NS

NS

<0.01

<0.01

Birth weight percentile 48 28 26 22 <0.01

Cigarette smokers (%) 17% 9% NS

Race (% black) 22% 36% NS

Maternal plasma triglyceride (mg/dL) 173 (132-193) *250(149-371) <0.04

Cord plasma triglyceride (mg/dL) 22.1 (18-44) 31.9 (26-80) NS

Maternal plasma cholesterol (mg/dL) 238 37 231 59 NS

Cord plasma cholesterol (mg/dL) 62 15 *73 12 <0.04

Continuous variables are given as mean (SD) or median (interquartile range).

NS, not significant; BMI, body mass index

Research Project 3: Project Title and Purpose

Targeting Women’s Cancer Cells with Novel Cell Cycle Inhibitors Blocking Centrosome

Clustering – Centrosome aberrations cause cancers and birth defects by inducing chromosome

errors leading to aneuploidies after mitosis. Genomic instabilities correlate with the degree of

centrosomal abnormalities. At mitosis, extra centrosomes increase spindle multipolarity, a

hallmark of many cancers. Recently, some cancers cluster supernumerary centrosomes at a

bipolar spindle, avoiding multipolarity and activation of the internal cell death program while

preserving reasonable genomic stability after cell division. Here, dynamic confocal imaging

with living markers for centrioles and microtubules is used to investigate cell cycle inhibitors’

impact on centrosome clustering and multipolarity at mitosis in normal somatic and cancerous

cells.

Duration of Project

1/1/2012 – 12/31/2012

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Project Overview

Specific Aims:

AIM 1. Is centrosome clustering dependent on the expression patterns of molecular motors

(dynein, HSET, dynactin) and NuMA at the spindle poles in cancerous and noncancerous cancer

cells?

AIM 2. How does the cdk1 inhibitor RO3306 affect cell cycle progression and centrosome

duplication in cancerous versus non-cancerous cells and expression of microtubule minus end

molecular motors?

AIM 3. Using dynamic confocal imaging with a living marker for centrioles or microtubules,

does Cdk1 inhibitor RO3306 prevent centrosome clustering and/or increase spindle multipolarity

at metaphase in cancer cells following drug rescue?

AIM 4. Does the PARP inhibitor PJ-34 block cell cycle progression and centrosome clustering

at the metaphase spindle poles with or without RO3306 exposure in cancer versus noncancerous

cells?

Methods:

Methods employed in this study include: (i) transduction of cancerous and noncancerous cell

lines with GFP NuMA, GFP-centrin or mCherry Red tubulin plasmids to dynamically follow

endogenous NuMA, centrin, or microtubule proteins in live cells by either conventional or laser

scanning confocal microscopy. We will also employ the G1S cell cycle plasmid to accurate

determine the phase of the cell cycle in cancerous and noncancerous cells by live cell imaging;

(ii) indirect immunofluorescence using monospecific affinity purified antibodies to NuMA,

centrosomes, molecular motors (dynein, dynactin, kinesins), and microtubules; (iii) siRNA

probes to knockdown endogenous NuMA protein; (iv) Western blotting to confirm NuMA

knockdown experiments; (v) cytoskeletal and cell cycle inhibitor drugs to disrupt endogenous

proteins in order to explore their effects on centrosome clustering during metaphase spindle

assembly.

Principal Investigator

Calvin Simerly, PhD

Research Associate Professor

Magee Womens Research Institute

204 Craft Avenue, B603

Pittsburgh, PA 15213

Other Participating Researchers

Gerald Schatten, PhD – employed by Magee Womens Research Institute and Foundation

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Expected Research Outcomes and Benefits

Understanding the dynamics and molecular composition of the cell’s spindle poles and the

centrosomes in normal and cancerous cells affords new targets for designing chemotherapeutic

strategies in oncology. This may well translate into innovative cancer treatments, but is also of

keen importance for understanding the basic biology of every cell during division.

Understanding the molecular mechanism of increasing multipolarity in cancer cells without

subsequent effects on normal somatic cells would permit new strategies for the treatment of

malignancies. It may also afford a much richer understanding of the link between spindle

multipolarity, aneuploidy and malignancy transformation in humans and perhaps give insights

into innovative approaches for personalized treatment of cancers.

Summary of Research Completed

AIM 1. Is centrosome clustering dependent on the expression patterns of molecular motors

(dynein, HSET, dynactin) and NuMA at the spindle poles in cancerous and noncancerous cells?

For this reporting period, we explored whether the knockdown of endogenous nuclear mitotic

apparatus protein (NuMA) with a shRNA NuMA probe (Santa Cruz Biotechnology, Inc, Santa

Cruz, CA) would increase spindle multipolarity and centrosome dispersion at the spindle poles in

cancer cells. NCI H292 lung carcinoma and MCF7 breast cancer cells were plated in a T-25 flask

for 24 hours to approximately 50% confluency. The cell medium was replaced with 5 ml of a 5

g ml-1

Polybrene before adding 20 l shRNA NuMA lentiviral particles to flasks for overnight

incubation at 37°C in 5% CO2. The following day, cells were rinsed three times in normal

culture media and incubated overnight. Cells expressing shRNA were passaged 1:3 to 1:5 for 48

hrs before selection of stably transduced cells using 10 g ml-1

puromycin dihydrocholoride over

a 3-4 day period. As shown in Figure 1, Western blots of protein extracts of control versus

transduced shRNA NuMA cells showed measurable changes in endogenous NuMA in both NCI

H292 cells (-31%) and MCF7 cells (-42%). Attempts at increasing shRNA NuMA transduction

were found to be detrimental to cell survivability. As shown in Fig. 1, immunocytochemistry

(ICC) analysis did not show complete disruption of NuMA from spindle pole microtubules or the

cell cortex. Spindles were largely bipolar (Figs. 1A and 2), with normal centriolar organization

and aligned chromosomes. We also investigated whether combined shRNA NuMA knockdown

with the Cdk-1 inhibitor RO3306 (RO3306i), in conjunction with the multidrug resistance

compound verapamil, would impact spindle multipolarity and centrosome organization. Control

NCI H292 cells treated with15M RO3306/10 M verapamil for 72 hrs demonstrated an

increased percentage of cells expressing abnormal spindle poles, centriole amplification, and

misaligned chromosomes (Figs. 1B and 2). NuMA was observed in most spindle pole

microtubules, with some reduction observed at extra supernumerary spindle poles (Fig. 1B).

Likewise, shRNA NuMA NCI H292 cells treated with 15M RO3306/10 M verapamil for 72

hrs showed similar increases in multipolar spindles, centriole hyperamplification, abnormal

NuMA at some poles, and misaligned chromosomes (Fig. 1C and Fig 2). Taken together, Cdk-1

inhibition by RO3306/verapamil initiates a significant increase in spindle multipolarity, centriole

amplification, and DNA misalignment in cancer cells. Knockdown of endogenous NuMA by the

lentiviral shRNA NuMA at amounts sufficient to ensure cell survival was probably not sufficient

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to drop NuMA below the critical thresholds necessary to impact microtubule minus end

organization in spindle poles.

AIM 2. How does the Cdk1 inhibitor RO3306 affect cell cycle progression and centrosome

duplication in cancerous versus noncancerous cells and expression of microtubule minus end

molecular motors? In this reporting period, we investigated whether Cdk-1 inhibition blocked

cell cycle progression in cancer cells by examining bromodeoxyuridine (BrDU) incorporation

after RO3306i for 48 or 72 hrs. MCF7 cells were plated at 4 x 104 cells per well for 24 hrs before

adding 10 M RO3306/10 M verapamil. About 16 hrs prior to fixation, 20 M BrDU labeling

reagent was added to the cells. After fixation according to the manufacturer’s instructions, BrDU

was detected with a mouse anti-BrDU antibody and the number of BrDU-positive cells counted

in 5-8 fields. We determined that RO3306i reduced the number of MCF7 cells progressing

through S-phase of the cell cycle by 75% at 48 hrs and 70% by 72 hrs post-inhibitor treatment

compared to control cells exposed to 10 M verapamil alone (data not presented). This

surprising finding suggests that RO3306i might be impacting DNA replication onset, with cells

arresting earlier in the cell cycle than the predicted G2/S phase as reported for Chinese hamster

ovary cells.

AIM 3. Using dynamic confocal imaging with a living marker for centrioles or microtubules,

does Cdk1 inhibitor RO3306 prevent centrosome clustering and/or increase spindle

multipolarity at metaphase in cancer cells following drug rescue? In this reporting period, we

captured an additional 74 time-lapse video microscopy (TLVM) images on NCI H292 and

MCF7 cancer cells stably transduced with the pEGFP-CETN2 (GFP-centrin) construct prepared

previously (Table 1). Control WI-38 lung fibroblast cells were inefficient at transduction with the

GFP-centrin construct, with only 1 of 4 attempts successful at four times the concentration of

lentiviral particles which produced success in the cancer lines. However, the WI-38 cells

exposed to the highest concentration of GFP-centrin construct did not propagate effectively in

vitro, and mitotic cells were rarely observed in culture. As figure 3 demonstrates, exposure of

successfully transduced GFP-centrin WI-38 cells to 12 M RO3306/10 M verapamil did show

hyperamplification of centrioles in a few interphase cells, although most displayed normal

centriole pairs after duplication. For GFP-centrin-tagged control NCI H292 lung carcinoma cells,

we imaged 9 interphase and 10 mitotic cells (Table 1). Interphase cells imaged by TLVM rarely

entered mitosis, probably owing to photodamage by lengthy fluorescent exposure despite using

fully attenuated light. Nevertheless, all 10 control mitotic NCI H292 cells completed normal cell

division within 1-2 hrs postimaging (Fig. 4A-F). An additional 18 GFP-centrin-tagged NCI H292

cells were imaged following 15 M RO3306/10 M verapamil for 72 hrs (Table 1). Of six

mitotic, only 50% completed cell division (Table 1). Indeed, TLVM imaging showed amplified

centrioles in most mitotic spindles which did not cluster at a bipolar spindle or complete

cytokinesis (Fig. 4 G-L). For GFP-centrin-tagged MCF7 breast cancer cells, we imaged eight

control cells during mitosis, with only 63% completing normal cytokinesis (Table 1). After GFP-

centrin MCF7 cells were exposed to 20 M RO3306/10 M verapamil for 48 hrs, many cells

showed hyperamplification of centrioles at interphase (Fig. 5). At mitosis, most cells assembled

mitotic spindles with multiple centrioles that did not cluster at the poles (Fig. 5B-F). Some GFP-

centrin centrioles were observed to migrate away from the spindle poles into the cytoplasm, (Fig.

5C-D) with the eventual collapse of the bipolar spindle without cytokinesis ensuing (Fig. 5F).

Only 17% of GFP-centrin-tagged MCF7 cells treated with RO3306i completed cell division

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(Table 1), and most divided 1-to-3 or 1-to-4 (not shown). Collectively, these data suggest that

cancer cells with hyperamplified centrioles rarely cluster them at bipolar spindles to avoid

activating programmed cell death. Indeed, it is interesting to speculate that the rare cells that do

cluster extra centrioles to avoid apoptosis are specialized cells, perhaps even cancer stem cells or

their early progenitors, and that this ability might convey unique advantages to these cells for

propagating tumors.

AIM 4. Does the PARP inhibitor PJ-34 block cell cycle progression and centrosome clustering

at the metaphase spindle poles with or without RO3306 exposure in cancer versus noncancerous

cells? For this reporting period, we re-tested the effects of the poly (ADP-ribose) polymerase

(PARP) inhibitor N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethyl-acetamide (PJ-34) on

bipolar spindle assembly and centrosome amplification in our cancerous cell lines (Fig. 6). By

raising the concentration of PJ-34 to 20 M and co-incubating cells with 1 M Tariquidar, a

potent third-generation p-glycoprotein inhibitor effective at blocking multidrug resistance in

cancer cells, we demonstrated a dramatic effect on the mitotic index rate in NCI H292 and

MCF7 cancer cells. Indeed, we showed a 136-fold decrease in the number of total mitotic cells in

NCI H292 treated with PJ-34/Tariquidar compared to Tariquidar-only control cells and a similar

86-fold reduction of mitotic MCF7 cells treated with PJ-34/Tariquidar compared to paired

control cells. ICC analysis of control mitotic cancer cell lines demonstrated abundant multipolar

spindles with amplified centrioles and poorly aligned chromosomes (Fig. 6A and C). Conversely,

cancer cells treated with 20 M PJ-34/1 M Tariquidar for 72 hrs showed only bipolar spindles

with normal centriole numbers and position at the poles (Fig. 6B and D), although some cells

displayed misaligned chromosomes. Taken together, it appears that PJ-34 does not prevent cell

cycle progression in cancer cell lines but greatly diminishes the incidence of mitotic cells and,

particularly, the presence of multipolar spindles classically identified in cancer cells. We

speculate that PJ-34 may actually increase multipolar spindles early by interfering with centriole

clustering at the spindle poles and with the consequence of increasing programmed cell death

that eliminates abnormal cancer cells carrying amplified centrioles. By 72 hrs post drug

treatment, mitotic cell numbers are greatly diminished, and only bipolar spindles with correctly

positioned centrioles at their poles are able to avoid activation of programmed cell death and

apoptosis. Inducing amplification of centrioles with the Cdk-1 inhibitor RO3306, followed by

treatment with PJ-34, is predicted to vastly diminish abnormal mitotic cancer cells in vitro and

may represent a novel cancer therapy for future testing.

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Figure 1. Knockdown of Endogenous NuMA with shRNA in NCI H292 and MCF7 Cancer

Cells. Left: Western blot of MCF7 (Lanes 1 and 2) and NCI H292 (Lanes 3 and 4) protein

extracts showing 30-42% reduction of endogenous NuMA protein after shRNA transduction.

Right: Representative ICC figures of NCI H292 lung fibroblast cells after shRNA NuMA

transduction and exposure to 15 M RO3306/ 10 M verapamil for 72 hrs. A: control mitotic

NCI H292 cells (A: blue, DNA) after shRNA NuMA knockdown showing typical bipolar spindle

assembly (A: red, microtubules) and endogenous NuMA at the spindle poles (A: green, arrows;

double arrows: NuMA at cell cortex). B: Tetrapolar NCI H292 cells after RO3306i for 72 hrs but

without shRNA NuMA knockdown. NuMA (B: green, arrows) is strongly localized to 3 of 4

spindle poles (B: red, microtubules; blue, DNA). C: mitotic NCI H292 cell after shRNA NuMA

knockdown and treatment with RO3306i. This cell has 5 NuMA-localized spindle poles despite

NuMA knockdown (C: green, arrows; red, microtubules) and misaligned DNA (C: blue). All

insets: centrin (green), microtubules (red) and DNA (blue). Bars= 5m.

Figure 2. Graphic analysis of NCI

H292 cells following shRNA

NuMA knockdown and Cdk-1

inhibition by RO3306. Blue bars:

analysis of spindle pole numbers

in control NCI H292 cells treated

with shRNA NuMA. No increase

in multipolar spindle assembly

was evident. Green bars: NCI

H292 cells, treated with 15 M

RO3306/10 M verapamil for

72 hrs, demonstrated a significant

increase in multipolar spindle

assembly. Red bars: NCI H292

cells, after shRNA NuMA and 15

M RO3306/10 M verapamil for

72 hrs, showed increased

multipolar mitotic spindles but no

significant difference from cells

treated with RO3306i alone.

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Figure 3. Interphase GFP-centrin-tagged

WI-38 lung fibroblasts treated 48 hrs with 12

M RO3306/10 M verapamil. A: cell with 8

GFP-centrin-expressing centrioles (green,

arrowheads; inset: centriole details) after

RO3306i inhibition. Centrioles separated

unequally along the nuclear surface (Nu). B:

duplicated GFP-centrin-expressing centrioles

(green, arrowhead; inset: centriole details)

adjacent to nuclear surface (Nu) after

RO3306i. Bar=5 m.

Figure 4. Selected TLVM images of mitotic progression in GFP-centrin-tagged NCI H292 lung

carcinoma cells after 10 M verapamil (Controls) or 15 M RO3306i for 72 hrs. A-F: Control

cells. Middle cell in panels A-F depicts normal cell division beginning at anaphase (A: 0 min;

arrowheads: GFP-centrin-tagged centrioles) and ending with normal cell division after 44

minutes (F: arrowheads; GFP-centrin-tagged centrioles). Lower right cell in panels A-F shows

progression from late interphase (A: 0 min; arrowheads; centrioles) through normal cell division

(F: inset; arrowheads, centrioles) by 50 min. G-L: tetrapolar cell with four GFP-centrin-tagged

centrioles (arrowheads) following RO3306i. Imaging 24 hrs post drug recovery. Neither

centriole clustering nor cell division was observed after 146 min (L). Bars=5 m.

Figure 5. TLVM of GFP-centrin-tagged MCF7 Breast Cancer cells

following 20 M RO3306/10 M verapamil for 48 hrs. Imaging at 24 hrs

post RO3306i recovery. A: MCF7 interphase cell depicting centriole

hyperamplification (A: green, arrowheads; inset: centriole details; Nu:

nucleus) after RO3306i. B-F: Panels from a TLVM movie of a mitotic cell

with four distinct centrioles (B: green, arrowheads). By 56 min post

imaging, the metaphase spindle collapses without cytokinesis onset (C-F:

green, arrowheads, centrioles). Three of four centrioles remain with the

collapsing spindle, and one centriole displaces towards the cytoplasm.

Bar=5 m.

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Table 1. Summary of TLVM Experiments on GFP-Centrin-Expressing Cancer Cells

Cancer cell

type

Number of

TLVM trials

(total cells)

Cancer cell

treatment1

Number of cells imaged at: Number of

developing from

interphase → mitosis

(%)

Number of

mitotics which

divided

(%) Interphase Mitosis

NCI H292 5

(37)

Control 9 10 1/9

(11) 10/10 (100)

RO3306i 12 6 1/12

(8)

3/6

(50)

MCF7 9

(42)

Control 1 8 0/1

(0)

5/8

(63)

RO3306i 15 18 1/15

(7)

3/18

(17) 1Control: 10 M verapamil or Ttariquidar anti-MDR compound only; RO3306i: inhibitor of Cdk-1 (10-20 M).

Figure 6. The PARP inhibitor PJ-34 did not block cell cycle progression but greatly reduced the

mitotic index in NCI H292 and MCF7 cancer cells. Left graph: total counts of mitotic cells

observed in control (1 M Tariquidar) and 20 M PJ-34/1 M Tariquidar-treated cancer cells.

Total mitotic cell counts dropped 136-fold in NCI H292 lung carcinoma cells and 86-fold in

MCF7 breast cancer cells. Right image: representative images from control and PJ-34-treated

cancer cells. Control metaphase NCI H292 (A: blue, DNA) and MCF7 cells (C: blue, DNA)

displayed many tetrapolar spindles and amplified centrosomes (A, C: red, microtubules; green;

centrosomes, arrowheads). Conversely, exposure to PJ-34 for 72 hrs in NCI H292 (B: blue,

DNA) and MCF7 cells (D: blue, DNA) greatly reduced, but did not eliminate, mitotic cells,

although no multipolar spindles or amplified centrosome numbers were observed in PJ-34-

exposed cells (B and D: red, microtubules; green, centrosomes, arrowheads). Bars=5 m.

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Research Project 4: Project Title and Purpose

Regulation of Spermatogenesis by Classical and Non-classical Testosterone Signaling –

Testosterone (T) is essential for male fertility. However, there is a lack of information regarding

the mechanisms by which T acts to support spermatogenesis and male fertility. Our studies will

identify the molecular and cellular mechanisms by which T supports critical spermatogenesis

processes including maintaining the blood-testis barrier (BTB), preventing the premature release

of developing germ cells and stimulating the release of mature spermatozoa. These studies will

provide 1) information needed to treat specific male infertility conditions and 2) long-needed

new targets for male contraceptive research.

Duration of Project

1/1/2012 – 12/31/2012

Project Overview

The major objective of this project is to identify the molecular mechanisms by which

testosterone (T) supports male fertility. T acts via two signaling pathways in somatic Sertoli cells

to support the survival and development of male germ cells in the testes. In the classical

pathway, T acts through the androgen receptor (AR) to regulate the expression of genes required

to support spermatogenesis. In the non-classical pathway, T acts through AR to stimulate the

phosphorylation and activation of a series of kinases that support germ cell survival and release.

Rat ex vivo and in vivo testis models will be used to determine the molecular mechanisms by

which testosterone supports spermatogenesis and fertility. In Aim 1, seminiferous tubules

isolated from rat testes will be incubated with inhibitors of the non-classical pathway to

determine whether the non-classical pathway is required for the release of mature spermatozoa.

In Aim 2, small fragments of testis tissue (4 mm3) will be placed into culture and incubated with

inhibitors of classical and non-classical T signaling. We will determine the extent to which each

pathway is required to activate kinases in Sertoli cells that are required for mainitaining

sprematogenesis. We also will identify Sertoli cell genes that are regulated by the classical

pathway and whether either pathway is required to form specialized tight junctions between

Sertoli cells that form the blood-testis barrier (BTB). In Aim 3, adenovirus constructs expressing

inhibitors of classical or non-classical T signaling will be injected into the lumen of seminiferous

tubules in the testes of rats. We will identify genes that are regulated by the classcial and non-

classical pathways. We will determine whether the number of tight junctions and the integrity of

the BTB is altered by blocking either pathway. The survival and attachment of germ cells to

Sertoli cells as well as the corect timing of sperm release will be assessed after blocking each

pathway.

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Principal Investigator

William H. Walker, PhD

Associate Professor

Magee Womens Research Institute and Foundation

204 Craft Avenue, B305

Pittsburgh, PA, 15213

Other Participating Researchers

None

Expected Research Outcomes and Benefits

This project will benefit the 10-15% of all couples that are unable to have children due to

infertility. In half of all infertile couples, there is a male defect. The results of the proposed

studies will identify new mechanisms and factors through which testosterone acts to support

spermatogenesis and male fertility. As a result, this work will provide new diagnostic targets that

will identify the causes of defects in male spermatogenesis. These findings will also provide the

fundamental knowledge required to design new personalized therapies for male infertility. It is

anticipated that after identification of the testosterone-regulated factors that are required for

fertility, genome surveys will find that the factors are defective in infertile men. Directed

research to repair or replace the defective factors may then be performed to cure the infertility.

These studies will also identify new factors that are required to maintain spermatogenesis. These

factors will be useful targets to design better strategies for male contraceptives. Because we will

identify factors regulated by T, the only hormone that is essential for spermatogenesis, we expect

to find central, critical targets for contraception research. The factors regulated by T control the

initial development of germ cells prior to the production of sperm, thus fertility can be blocked at

the beginning point in the developmental pathway before sperm are produced. It is anticipated

that the newly identified fertility determining factors can be inhibited with drugs targeted to post

translational modifications (phosphorylation of proteins) without the clinical challenges of

altering hormone levels.

Summary of Research Completed

The major objective of this proposal was to identify the molecular mechanisms by which

testosterone (T) supports male fertility. We focused on the processes that are required for

spermatogenesis and fertility that are regulated by classical and non-classical T signaling. We

hypothesized that each pathway regulates specific factors that are essential for maintaining

spermatogenesis. We proposed that these studies would provide (1) information needed to treat

specific male infertility conditions and (2) long-needed new targets for male contraceptive

research.

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Aim 1: Determine whether inhibiting non-classical T signaling blocks sperm release.

Progress: Eighty percent of the goals for Aim 1 were completed during the first 6 months. No

further studies were performed during the last 6 months.

Aim 2: Determine whether inhibiting classical or non-classical T signaling blocks

spermatogenesis in testis explants.

Progress: We built upon the success of our studies of inhibitors in testis explants to expand our

studies to determine whether activators of classical and non-classical signaling could alter T

signaling and spermatogenesis in testis explants. For these studies, explants were cultured from

AR-defective testicular feminized (tfm) rats in which T-mediated gene expression is inhibited

and spermatogenesis is blocked during meiosis. The explants were infected on the first day of

culture with adenovirus constructs expressing wild type AR or mutant ARs that selectively

activate either only the classical pathway or only the non-classical pathway. Adenovirus

constructs do not efficiently infect germ cells but they do effectively infect Sertoli cells. RNA

was extracted from the explants 7 days after adenovirus infection, and reverse transcription-

polymerase chain reaction (RT-PCR) assays were performed to determine the expression levels

of the housekeeping gene GAPDH and Rhox5, a homeobox gene that is highly inducible by T

via the classical signaling pathway. Rhox5 expression was below the level of detection in the

absence of exogenous AR expression. In contrast, Rhox5 was stimulated after infection by

adenovirus constructs expressing wild type AR or the classical pathway activator AdAR372-

385 (Fig. 1). As expected, an adenovirus expressing an activator of the non-classical pathway,

AdARC562, did not stimulate Rhox5 expression. Together, these data confirm that our AR

mutants can be used to selectively activate pathway-specific T signaling in testis tissue.

Further studies were performed using explants isolated from wild-type rats and adenovirus

constructs expressing selective inhibitors of the classical or non-classical pathways. In these

studies, Rhox5 expression was observed in uninfected explants and explants infected with a

control adenovirus expressing enhanced green fluorescent protein (EGFP) (Fig. 1). Infection

with adenoviruses expressing either classical or non-classical inhibitors resulted in decreased

Rhox5 expression and both inhibitors together synergized to decrease Rhox5 expression further.

These data again indicate that T signaling in explants can be regulated by inhibitors of the

classical and non-classical pathways. Although inhibition of classical pathway–regulated Rhox5

gene expression with the classical pathway inhibitor was expected, the decreased expression of

Rhox5 in the presence of the non-classical inhibitor was not anticipated. The non-classical

inhibitor did not alter the expression of the GAPDH control gene, suggesting that the inhibitor

does not have a general poisoning effect on gene expression or Sertoli cells’ health. It is possible

that inhibition of non-classical T signaling may alter regulatory pathways in Sertoli cells such

that T-mediated gene expression is indirectly inhibited.

The findings that activators and inhibitors of T signaling can regulate gene expression in explants

raised the possibility that pathway-specific activators could be used to alter Sertoli cell functions

in the explants that regulate germ cell development. To test this hypothesis, the expression of

genes expressed specifically in spermatogonia germ cells was assayed in testis explants isolated

from tfm rats in which spermatogenesis is halted. T and AR are known to not directly regulate

expression of genes in germ cells.

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Expression of the spermatogonia-specific genes PLZF and c-kit in the cultured explants was

assayed by quantitative PCR (qPCR). Infection of the explants with an adenovirus expressing

wild-type AR resulted in a fivefold increase in expression of the PLZF gene (Fig. 2). Activation

of only the classical pathway was as effective as wild-type AR in increasing PLZF expression,

whereas the non-classical pathway activator ARC562G increased PLZF expression in

spermatogonia twofold. Expression of the classical and non-classical pathways together further

increased PLZF expression, but the increase was not statistically significant. The c-kit gene was

stimulated to a similar extent after infection of Sertoli cells with wild-type AR, the classical

pathway activator, the non-classical pathway activator, or the combination of the classical and

non-classical pathway-selective AR mutants. These data indicate that activation of the classical

pathway in Sertoli cells is more effective than the non-classical pathway in supporting the

expression of PLZF, which is a marker for renewing spermatogonial stem cells. Activation of

either the classical or the non-classical pathway in Sertoli cells increases c-kit expression, which

is a marker for the differentiation and expansion of differentiated spermatogonia. Together, these

results support the idea that the classical and non-classical pathways both contribute to providing

the factors that are required to reinitiate the progression of spermatogenesis in the tfm explants.

These data provide important support for the funding of future studies in which transgenic mouse

models expressing only the classical or only the non-classical pathway will be created to identify

the spermatogenesis processes that are regulated by each pathway.

Aim 3: Determine whether the classical and/or non-classical pathway is required to maintain

spermatogenesis in vivo.

Progress: Non-classical T signaling increases ERK phosphorylation in Sertoli cells in vivo.

To test whether non-classical T signaling occurs in Sertoli cells in vivo, we developed a rat

model in which T levels could be rapidly manipulated. Adult rats were injected intraperitoneally

with the gonadotropin-releasing hormone antagonist Cetrorelix (50 g/100 l) to transiently

decrease gonadotropin (luteinizing hormone and follicle-stimulating hormone) secretion and

T levels. After 7 hours, testicular T levels fell to 16% of control levels, in agreement with

previous studies (Fig. 3A). Subsequent injections of T propionate (TP, 5 mg, ip) plus T enanthate

(TE, 10 mg, im) first elevated testicular T levels after 30 min, but T increased to 137% of control

levels after 1 hour. These initial studies determined that intratesticular T levels could be rapidly

decreased and increased in rats.

To determine whether intratesticular T levels correlated with ERkinase phosphorylation, a key

component in the non-classical T signaling pathway, phosphorylated ERK (p-ERK) levels were

assayed in T-sensitive stage VII seminiferous tubule cross sections. p-ERK immunostaining

decreased to 34% of normal levels 7 hours after Cetrorelix injection but, then, was restored to

103% of control levels 1 hour after injection of TP and TE (Fig. 3B). Inspection of stage VII

cross sections revealed that, in control and Cetrorelix + T-treated rats, p-ERK immunostaining

was present in the Sertoli cell cytoplasm associated with the blood-testis barrier region and

Sertoli–elongated spermatid adhesion sites (Fig. 3C). In contrast, p-ERK staining was reduced at

both sites after Cetrorelix treatment alone.

The Cetrorelix model data show that testicular T levels can be rapidly decreased and then

restored to normal levels within 1 hour. Importantly, ERK kinase phosphorylation is rapidly

increased within 1 hour of administration of TP and TE to restore T levels. Because at least 30

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minutes is required to elevate T levels in the testis after TP and TE injections (data not shown),

and T-stimulated transcription requires 30 to 45 minutes to be productive, with protein

production reported to occur even later, the increase in p-ERK levels observed within 1 hour is

not consistent with a classical pathway model. The rapid increase in T-mediated p-ERK levels

supports the hypothesis that the non-classical pathway activates kinase signaling in Sertoli cells

in vivo.

Spermatogenesis is disrupted after inhibition of either classical or non-classical signaling. To

determine whether inhibition of the classical or non-classical pathways disrupts spermatogenesis,

adenovirus constructs expressing pathway-selective inhibitors were injected into the testes of

14-day-old mice via the rete testis. We and other groups have shown that adenovirus constructs

at this concentration (50 l of 1 x 1010

particles/ml), injected by this protocol, infect only Sertoli

cells and do not cause an immune response. Testes were isolated 4 days after injection and fixed

in Bouin’s solution and embedded in paraffin. Staining of testis sections with periodic acid

Schiff-hematoxylin revealed that injection of an adenovirus expressing EGFP (AdEGFP) did not

affect testis morphology or spermatogenesis (Fig. 4A). In contrast, after injection of AdS1, an

adenovirus expressing the S1 inhibitor of the non-classical pathway, spermatogenesis was

disrupted, with spermatocytes (the most advanced germ cell at this age) being absent in at least

50% of tubules (Fig. 4B).

Further studies of adult mice injected with adenoviruses via the rete testis revealed that the

adenovirus-mediated expression of the EGFP control protein had no effect (Fig. 4C), but the S1

non-classical inhibitor (AdS1 caused vacuoles in the seminiferous epithelium due to the loss of

spermatocytes (Fig. 4D). AdH-K-AR122, which inhibits classical signaling, caused immature

germ cells to be released prematurely into the lumen of seminiferous tubules (Fig. 4E). The

combined inhibition of both pathways (AdS1 and AdH-K-AR122) resulted in an additive effect

including the loss of spermatocytes and the detachment of immature germ cells (Fig. 4F). These

data indicate that both T signaling pathways are required to maintain spermatogenesis and that

alteration of either signaling pathway contributes to a defective spermatogenesis phenotype.

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Fig. 1. Rhox 5 is expressed in testis explants

and is regulated by classical and non-classical

signaling. (A) Testis explants from 35 day-old

wild type rats were exposed for 2 h to media

alone (no virus), adenovirus constructs

expressing control (-galactosidase), non-

classical inhibitor S1, classical inhibitor H-K-

AR122, or classical + non-classical inhibitors or

control inhibitor S1 scrambled. (B) Testis

explants from 35 day-old AR-defective (tfm)

rats were exposed for 2 h to media alone (no

virus), adenovirus constructs expressing AR that

activates only classical, non-classical or

classical + non classical pathways, or wild type

AR.

Fig. 2. Pathway-selective AR mutants

increase spermatogonia-specific gene

expression in testis explants from tfm rats.

Top: The initial steps of spermatogenesis are

provided, with the expression patterns of the

PLZF and c-kit genes. Bottom: The results of

qPCR assays of testis explants from tfm rats.

The relative gene expression is provided for

the PLZF and c-kit genes after infection of

the explants with adenovirus construct

expressing EGFP (control), wild-type AR

(wt-AR), non-classical pathway activator

(ARC562G), classical pathway activator

(AR∆372-385), or both activators (ARC562G

+ AR∆372-385) (n=3).

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Fig. 3. T stimulates rapid ERK phosphorylation

in Sertoli cells in vivo: The Cetrorelix model for

deprivation and restoration of T. (A) T levels in

control testes and after 7 h of Cetrorelix

treatment or 7 h Cetrorelix followed by 1 h T

(n=3). (B) p-ERK immunostaining was

quantified as relative integrated optical density

(IOD) in stage VII tubules without treatment and

7 h after Cetrorelix treatment and Cetrorelix (7h)

+ T for 1 (n=5). (C) Representative

immunofluorescence microscopy images of p-

ERK (red) staining in stage VII tubules in

control and after Cetrorelix (7 h) or Cetrorelix +

T (1 h) treatments. Arrows indicate p-ERK

staining in the blood-testis barrier region, and

arrowheads denote p-ERK staining associated

with elongated spermatids. Nuclei are stained

blue with DAPI.

Fig. 4. Peptides inhibiting classical or non-

classical signaling disrupt spermatogenesis.

A+B: Hematoxylin staining of 18 day-old

mouse testis 4 days after injection of (A)

control AdEGFP (B) bon-classical pathway

inhibitor AdS1. C-F: Hematoxylin staining of

adult mouse testis 4 days after injection of

(C) AdEGFP (D) AdS1. (E) classical pathway

inhibitor Ad-H-K-AR122 and F) AdS1 + Ad-

H-K-AR122. Asterisks (*) indicate tubules

with disrupted spermatogenesis in panel B.

Short arrows depict vacuoles lacking germ

cells. Long arrows depict prematurely

detached germ cells in the lumen. Circles and

ovals indicate regions lacking germ cells.