signalling pathways- with a focus on telomerase...

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Signalling pathways in Renal cell carcinoma with a focus on telomerase regulation

Raviprakash T. Sitaram

Department of Medical Biosciences, Pathology Umeå University Umeå 2010

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Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) Copyrights © Raviprakash T. Sitaram ISBN: 978-91-7459-111-8 ISSN: 0346-6612 Cover designs: Munender Vodnala E -version available at http://umu.diva-portal.org/ Printed by: Arkitektkopia, Umeå Umeå, Sweden 2010

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Dream is yours, make it to roll. Choice is yours, put into action. Be thou an artist, be thou a scientist, But true to thy heart and conscience. Be a human by first for just which tends to ultimate quest For divinity the truth.

To My family

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TABLE OF CONTENT

ABSTRACT .......................................................................................................... 6

LIST OF ABBREVIATIONS ............................................................................... 7

ORIGINAL ARTICLES ........................................................................................ 8

INTRODUCTION ................................................................................................ 9

Renal cell carcinoma ...................................................................................... 9

Epidemiology, etiology and risk factors .................................................... 9

Classification of RCC ...................................................................................... 10

1.ccRCC ................................................................................................................ 10

2.pRCC ................................................................................................................. 10

3. chRCC ............................................................................................................... 11

4.Collecting duct RCC .......................................................................................... 11

5.Unclassified RCC ............................................................................................... 11

Clinical features of RCC ................................................................................ 12

Symptoms ........................................................................................................ 12

Tumour grade ................................................................................................. 12

Tumour stage ................................................................................................. 13

Treatment ........................................................................................................ 14

Telomere biology .......................................................................................... 15

The end replication problem ...................................................................... 15

Telomere structure and associated proteins ......................................... 16 The telomere hypothesis of aging and immortalization .................... 17

The telomerase complex ............................................................................. 18

Regulation of hTERT ...................................................................................... 18

Alternative lengthening of telomeres ...................................................... 19 Telomerase and cancer ................................................................................ 19

Phosphatidylinosital-3-kinase signalling pathway ............................. 21

Class IA PI3K signalling pathway ................................................................ 21

Akt/PKB ............................................................................................................. 22

PTEN ................................................................................................................... 22

DJ-1 .................................................................................................................... 25 Function of DJ-1.............................................................................................. 25

DJ-1 and cancer .............................................................................................. 26

WT1 .................................................................................................................... 27

The WT1 gene, mRNA, and protein .......................................................... 27

WT1, the transcription factor ..................................................................... 28

WT1 targets ..................................................................................................... 28

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WT1 interacting proteins ............................................................................. 28

WT1 and cancer .............................................................................................. 28

cMyc .................................................................................................................. 30 The cMyc gene and protein ........................................................................ 30 The Myc-Max-Mad transcription network ............................................. 30

Regulation of cMyc by upstream signals ................................................ 31

Role of cMyc in cell growth and proliferation ....................................... 31

cMyc in apoptosis, telomerase activation and tumourigenesis ...... 31

Nijmegen breakage syndrome 1 (NBS1) ................................................ 33

NBS1 and double strand break repair ..................................................... 34

NBS1 and telomere stability ....................................................................... 34

AIMS OF THE THESIS..................................................................................... 36

MATERIALS AND METHODS....................................................................... 37

Materials ........................................................................................................... 37

RNA extraction and cDNA synthesis ......................................................... 37

Transient transfection .................................................................................. 38

siRNA transfection ............................................................................................... 38

Expression vectors ............................................................................................... 38

Real time PCR .................................................................................................. 38

Protein extraction .......................................................................................... 39

Western blot ................................................................................................... 39

Immunohistochemistry ................................................................................ 40

Immunofluorescence .................................................................................... 40

ChiP assay ......................................................................................................... 40

Telomerase activity ....................................................................................... 41

Genome-wide expression micro array .................................................... 41

DNA histogram analysis ............................................................................... 42

Cytotoxic assay ............................................................................................... 42

hTERT promoter activity assay ................................................................... 42

Statistical analysis .......................................................................................... 42

RESULTS AND DISCUSSION ........................................................................ 43

GENERAL CONCLUSIONS ............................................................................. 50

ACKNOWLEDGEMENTS ............................................................................... 53

REFERENCES .................................................................................................... 56

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ABSTRACT

Telomerase is a ribonucleoprotein complex that catalyses telomeric repeat addition at the ends of chromosomes. The catalytic subunit, hTERT, acts as a key determinant for telomerase activity control; the induction of hTERT expression is required for telomerase activity. hTERT participates in cellular immortalization and is elevated in certain malignant tissues. Several tumours exhibit telomerase activity, which contributes to the infinite proliferation capacity that promotes tumour progression.

Renal cell carcinoma (RCC) represents 2% of all adult malignancies and has a high mortality rate. The WHO classifies RCC into several sub-types based on cytogenetic aberrations and morphological features; the most prevalent sub-types are clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC). The aims of this thesis were to study the expression patterns of various signalling molecules, to elucidate the functional links among them, and to define the roles of these signalling molecules in the regulation of hTERT gene expression and telomerase activity in RCC. The first paper included in this thesis revealed mRNA overexpression of DJ-1 (a PTEN inhibitor), cMyc, and hTERT in clinical ccRCC samples compared to tumour-free kidney cortex tissues. Significant, positive correlations were detected for DJ-1, cMyc, and hTERT mRNA levels in ccRCC, but not in pRCC. In vitro knockdown of DJ-1 by siRNA in ccRCC cells induced downregulation of p-Akt, cMyc, hTERT, and telomerase activity. Forced overexpression of DJ-1 in an ovarian carcinoma cell line was followed by increased hTERT promoter activity, which appeared to be dependent on cMYC binding to the promoter. Collectively, the in vitro studies verified a functional link among DJ-1, cMyc, and hTERT as implied in the clinical ccRCC samples. The second paper included in this thesis demonstrated overexpression of NBS1 mRNA levels in ccRCC compared to the kidney cortex. NBS1 mRNA levels exhibited significant, positive correlations with DJ-1, cMyc, and S phase, but not with hTERT. In vitro experiments suggested that DJ-1 could regulate NBS1 gene expression. The role of the hTERT transcriptional repressor WT1 in RCC was evaluated in the third paper included in this thesis. ccRCC samples displayed low WT1 mRNA levels compared to kidney cortex samples. Interestingly, WT1 expression was negatively associated with hTERT and cMyc both of which were elevated in ccRCC. Forced overexpression of WT1 isoforms in a ccRCC cell line increased the expression of several negative transcriptional regulators of hTERT and diminished the expression of hTERT positive regulators. In consequence, hTERT mRNA levels and telomerase activity were reduced. Chromatin immunoprecipitation verified direct binding of WT1 to the cMyc, Smad3, and hTERT promoters. Taken together, these data suggested that in ccRCC, WT1 affects hTERT at the transcriptional level via a combined effect on both positive and negative regulators. In conclusion, DJ-1 can regulate hTERT and telomerase activity through the PI3K pathway encompassing PTEN, NBS1, p-Akt, and cMyc in ccRCC, but not in pRCC. WT1 negatively regulates hTERT and telomerase activity directly and indirectly through multiple pathways in ccRCC.

LIST OF ABBREVIATIONS

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LIST OF ABBREVIATIONS

aa amino acid ALT alternative lengthening of telomeres bHLH/LZ basic helix-loop-helix leucine zipper bP base pair ccRCC clear cell renal cell carcinoma chRCC chromophobe renal cell carcinoma DKC dyskeretosis congenita DNA deoxy ribo nucleic acid CpG cytosine precedes a guanine FCS fetal calf serum G1 gap1 hTERT human telomerase reverse transcriptase hTR human telomerase RNA HSP heat shock protein IL2 interleukin2 kbp kilo base pair kDa kilo Dalton KTS lysine, threoine, serine mRNA messenger RNA M1/M2 Mortality 1 /Mortality 2 mTOR mammalian Target of Rapamycin mTORC1 mammalian Target of Rapamycin Complex 2 NBS1 Nijmegen breakage syndrome 1 PCR polymerase chain reaction PDK1 phosphoionositide-dependent kinase-1 PI3K Phophatidylinositol-3-kinase PI Phophatidylinositol PIP2 phophatidylinositol diphosphatase PKB protein Kinase B POT1 Protection of telomeres1 PH pleckstrin homology PTEN Phosphatase TEnsin homolog on chromosome TEN pRCC papillary Renal cell carcinoma RCC renal cell carcinoma RAP1 Ras-proximate-1 RT-PCR real time polymerase chain reaction RTK receptor tyrosine kinase SV40 simian virus 40 siRNA small interference RNA ser serine S phase synthesis phase TPP1 Tripeptidyl-peptidase 1 TIN2 TRF1-interacting nuclear factor 2 TL telomere length T-loop telomere loop TNM tumour node metastasis TRF1 and 2 telomere repeat binding factor 1 and 2 thr threoine TRAP telomere repeat amplification protocol VEGF Vascular endothelial growth factor WT1 Wilm’s tumour

ORIGINAL ARTICLES

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ORIGINAL ARTICLES

This thesis is based on the following papers and manuscript referred in the text

by their Roman numerals.

Paper I

Sitaram RT, Cairney CJ, Grabowski P, Keith WN, Hallberg B, Ljungberg B, Roos G.

(2009) "The PTEN regulator DJ-1 is associated with hTERT expression in clear cell

renal cell carcinoma." Int J Cancer 125(4): 783-90.

Paper II

Degerman S, Sitaram RT, Ljungberg B and Roos G. (2010) “The NBS1 gene is

overexpressed and regulated by DJ-1 in clear cell renal cell carcinoma”

Manuscript

Paper III

Sitaram RT, Degerman S, Ljungberg B, Andersson E, Oji Y, Sugiyama H, Roos G, Li

A. (2010) "Wilms' tumour 1 can suppress hTERT gene expression and telomerase

activity in clear cell renal cell carcinoma via multiple pathways." Br J Cancer

103(8):1255-62.

INTRODUCTION

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INTRODUCTION

Renal cell carcinoma (RCC) includes diverse tumour types characterized by a

variety of genetic abnormalities. In general, RCC is asymptomatic at early stages;

most cases are diagnosed at an advanced stage and carry a poor prognosis. This

thesis focuses on the most common sub-type, clear cell RCC (ccRCC), and the

signalling pathways that impact telomerase activity, providing the tumour cells

with an “indefinite” lifespan. Elucidation of these pathways enables a better

understanding of the mechanisms leading to tumour development and

progression.

Renal cell carcinoma

Epidemiology, etiology, and risk factors

Globally, RCC represents 2% of all adult malignancies and has a high mortality

rate of 40%. RCC is the third most common urological malignancy, after prostate

cancer and carcinoma of the urinary bladder, and RCC is two times more

prevalent in men than in women [1, 2]. The incidence of RCC is higher in Europe

and North America than Asia and Africa; in the last three decades, the rate of

occurrence increasing 2% every year in Europe and the United States [3]. In

Sweden, around 1000 RCC cases are diagnosed every year [4], but the RCC

incidence has slightly been decreasing in Sweden and Denmark since 1980 [2].

The peak incidence of RCC occurs in the sixth and seventh decade of life [5, 6].

Epidemiological studies have identified several risk factors for RCC. Cigarette

smoking is not only implicated in the genesis of RCC, but also impacts the

prognosis. Overweight/obesity and a hyper-caloric diet are associated with risk

for RCC [7-9], and other probable risk factors include hypertension, occupational

hazards, hormonal factors, and reproductive factors [2].

INTRODUCTION

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Classification of RCC

According to Heidelberg classification, RCC encompasses different types of

tumour; more specific diagnoses are based on histopathological appearance,

specific genetic alterations, clinical behaviour, and therapeutic response [10].

The five most common RCC types are:

1. clear cell RCC (ccRCC)

2. papillary RCC (pRCC)

3. chromophobe RCC (chRCC)

4. Collecting duct RCC

5. Unclassified RCC

1. ccRCC

ccRCC is the most common sub-type of RCC, constituting 75-80% of RCC cases.

These tumour cells show abundant, clear cytoplasm (Figure 1A), and the tumour

exhibits a growth pattern that varies from solid, trabecular, and tubular or cystic

to rare focal areas of papillary growth. In addition to other genetic aberrations

(Table 1), the short arm of chromosome 3 is often deleted in ccRCC [10, 11].

Approximately 80% of ccRCC tumours carry a deletion of the tumour suppressor

gene vHL (von Hippel-Lindau), which plays a pivotal role in ccRCC pathogenesis

[12, 13]. ccRCC tumours are refractory to chemotherapy and have a poor

prognosis compared to other sub-types [14-16].

2. pRCC

The pRCC sub-type accounts for 5-10% of RCC cases; the tumour cells are small,

with scanty, moderate, or abundant cytoplasm (Figure 1B). The papillary growth

pattern predominates in pRCC, and its development is associated with multiple,

bilateral microscopic lesions. However, pRCC generally has a better prognosis

than ccRCC.

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3. chRCC

The chRCC sub-type accounts for 5% of RCC cases and includes a better

prognosis than ccRCC. The tumour cells contain pale or eosinophilic granular

cytoplasm, and electron microscopy reveals unevenly distributed vesicles in this

sub-type (Figure 1C).

4. Collecting duct carcinoma

Collecting duct carcinoma, also known as Bellini’s duct carcinoma, constitutes

only 1% of RCC diagnoses. This RCC sub-type mostly originates in the medulla

but may extend to the cortex. The morphology is heterogeneous, characterized

by uneven channels of atypical epithelium [10] and variable genetic

abnormalities [17].

5. Unclassified RCC

Unclassified tumours account for 3-5% of RCC cases, and do not qualify for a

place in any category. These tumours display variable morphologies and genetic

lesions, limiting the applicability of specific definitions [10].

Common genetic alterations in three main subtypes (ccRCC, pRCC and chRCC)

are summarized in Table 1 [10, 11].

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Table 1: Common genetic alterations in RCC

RCC Type Chromosomal aberrations

(chromosomes involved)

ccRCC Deletion (3p, 6q, 8p, 9p, 14q)

Duplication (5q22)

pRCC Trisomy (3q, 7, 8, 12, 16, 17, 20)

Deletion (Y)

chRCC Loss of heterozygosity

(1, 2, 6, 10, 13, 17)

Clinical features of RCC

Symptoms

At present, more than 50% of RCC patients are diagnosed from an incidental

radiological examination, since they exhibited no overt symptoms of RCC [5].

The most common symptoms - the classic triad hematuria, flank pain, and a

palpable flank mass suggest an advanced tumour stage. Other less-specific signs

are weight loss, fever, hypertension, hypercalcemia, night sweats, malaise, and

left side varicocele caused by obstruction of the testicular vein [18].

Tumour grade

The morphological grade of the tumour cell is linked to prognosis through the

tumour grading system [19], which is based on nuclear morphology and nuclear

size. An internationally approved grading system was presented by Fuhrman

(Table 2), which in turn was derived from the system documented by Skinner in

1971 [20, 21].

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Table 2: Fuhrman’s nuclear grading of RCC

Tumour stage

Tumour stage is the most important prognostic factor for predicting the survival

of RCC patients. The TNM staging system is the most common classification

system, and is applied to RCC (Table 3); T stands for primary tumour, N for

regional lymph node, and M for metastases status [22]. RCCs confined to the

kidney have a five-year survival of 91-100%, compared with 74-96%, 59-70%,

and 16-32% for TNM stages II, III, and IV, respectively [23].

Table 3: TNM staging of RCC (2002)

Stage TNM Definition

I T 1 N0 M0 7 cm tumour diameter, confined to kidney

II T 2 N0 M0 7 cm tumour diameter, confined to kidney

III

T3aN0 M0

T3b-cN0 M0

N1 M0

Extension to adrenal gland or perirenal fat, but not

beyond Gerota‘s fascia

Involvement of renal vein/vena cava

Involvement of single lymph node

IV T 4 N0-1 M1 Extension to other organs, beyond Gerota’s fascia

Distant metastases

Grade Nuclear size Appearance of nuclei

1 10 m Round, uniform, absent

2 15 m Irregular and small

3 20 m Irregular and prominent

4

20 m Multi-lobed, misshaped, prominent and

heavy chromatin clumps

INTRODUCTION

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Treatment

Surgical excision of a local tumour is the only curative treatment for RCC, and

generally includes open or laparoscopic radical nephrectomy or nephron-sparing

nephrectomy [24, 25]. Metastasectomy in RCC is recommended for solitary

metastases [26]. Ablative techniques such as radio frequency ablation [27] and

cryosurgical ablations [28] are used to treat small kidney tumours. For

metastatic RCC, cytokines such as the interferons and interleukin-2 have been

used as treatment, with a general response rate of approximately 20% of

metastatic RCCs, as well as significant side effects [1].

In recent years, several targeting agents have been developed such as Sorafenib,

Sunitinib being multikinase inhibitors, Bevacizumab a monoclonal antibody that

binds to VEGF-A targeting the VEGF pathway [29-32], and Temsirolimus and

Everolimus target the mTOR pathway [33]. These targeting agents can

significantly prolong the progression-free and overall survival rates for patients

with metastatic disease.

INTRODUCTION

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Telomere biology

The end replication problem

Muller coined the term “telomere” from the Greek words telos (end) and meros

(part) [34]. During the early 1960s, Hayflick and co-workers reported that

normal human diploid cells divided a limited number of times in vitro, i.e. the

“Hayflick limit”[35].

Olovnikov (1971) and Watson (1972) proposed the existence of an “end

replication problem” (Figure 2), because DNA polymerase is incapable of

replicating the extreme end of the linear chromosome [36, 37]. Olovnikov

proposed a theory called “Theory of Marginotomy”, in which the limited cellular

proliferation is due to progressive chromosomal shortening [36]; tumours were

suggested to possess an unknown mechanism to overcome this problem. In

vivo, attrition of telomere length was observed with age [38-40].

In 1979, Blackburn and Gall discovered the telomeric DNA repeat in the

protozoa Tetrahymena thermophilia [41]. Later, Blackburn and Greider

identified the ribonucleoprotein enzyme telomerase, which counteracts the end

replication problem by adding telomere repeats to the chromosome end [42,

43]. In 1989, Morin identified human telomerase in HeLa cells [44].

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Telomere structure and associated proteins

The telomere structure consists of G-rich telomere sequences and associated

proteins. In humans, the 5’-TTAGGG-3’ telomere strand forms a single-stranded

overhang 100-200 bp in length [45].

The telomeric DNA strand folds back to form a ring structure called the T-loop

(Telomere loop), which serves as a cap to protect the single-stranded overhang

from degradation [46, 47]. Telomeres can also adopt secondary DNA

conformations such as intermolecular G-quadruplexes, which can decrease

telomerase activity [48]. In addition to these DNA structures, several DNA

binding proteins are associated with the telomere. A collection of six proteins

(TRF1, TRF2, POT1, TIN2, TPP1, and RAP1), called the “Shelterins”, is essential

for telomere length regulation and telomere integrity [49, 50].

Initially, telomeres were considered to be transcriptionally silent, due to the

heterochromatic structures surrounding the chromosome end and the non-

coding telomere sequence. The genes located near the telomere and in sub-

telomeric regions are frequently silenced because of a “telomere position

effect” [51]. However, recent studies have revealed that telomere DNA

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transcribes to a non-coding RNA, TERRA (Telomere repeat containing RNA) that

seems to be involved in regulating telomere integrity [52].

The telomere hypothesis of aging and immortalization

The telomere length (TL) ranges from 4-14 kb in human somatic cells, and it

varies depending on a number of factors including heredity, cell type, age, and

epigenetic influences. The TL also varies among individual chromosomes [53-

55]. In vitro fibroblast cultures have illustrated the telomerase hypothesis of

cellular aging and immortalization (Figure 3), showing a close relationship

among telomere length, replicative capacity, and cellular senescence [53, 56,

57]. The average normal somatic cell, with no telomerase activity, loses

approximately 50bp of telomere sequence with each population doubling. After

a certain number of population doublings in culture, cells enter replicative

senescence at a critical telomere length called Hayflick limit or mortality stage 1

(M1-stage) [58]. At M1-stage, cells have a critical telomere length of

approximately 5-6 kb long [59], and show cell cycle arrest, low metabolic rates,

and exhibit -galactosidase expression together with morphological changes

[60]. The M1-stage can be bypassed by inactivating tumour suppressor genes

(p53 and, pRb) [61], leading to further cell divisions. As consequence, cells divide

further with progressive telomere loss. Finally, the cells reach Mortality stage-2

(M2-stage) or “Crisis’’, where a massive cell death occurs, and telomeres are

extremely short [62]. At this point, telomeres are no longer protecting the

chromosome ends, and signs of chromosomal instabilities such as anaphase

bridges and chromosome fusions are frequently observed [63, 64] (Figure 3).

Cells can occasionally overcome crisis and attain immortal status by counter-

balancing the unprotected telomeres with de novo formation of telomere tracts,

either by telomerase activation or by alternative lengthening of telomeres (ALT)

[65-67]. In cell types other than the fibroblasts used to elucidate the telomere

hypothesis, differences appear in the senescence stages and checkpoints,

indicating that the immortalization process is somewhat specific to cell type [68,

69].

INTRODUCTION

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The telomerase complex

Human telomerase has two main parts: human Telomerase RNA (hTR) and the

catalytic protein human Telomerase Reverse Transcriptase (hTERT). The

telomerase complex is also associated with and regulated by other proteins,

including dyskerin (DKC1), 14-3-3, and chaperones [70, 71]. hTR carries a

template complementary to the human telomere sequence, allowing it to

recognize and anneal to the telomeric DNA strand. hTERT then reverse

transcribes the complementary hTR templates and synthesizes telomeric

sequence [71-73].

Regulation of hTERT

During embryogenesis, telomerase is activated and expressed in most tissues,

but it is silenced in adult somatic cells [74]. Nevertheless, highly proliferative

tissues, such as stem cells, activated lymphocytes, and germ cells express

telomerase activity to stabilize telomeres. Telomerase-negative somatic cells

express hTR and DKC1, but not the catalytic subunit hTERT; immortal cells that

exhibit detectable telomerase activity express hTERT [73, 75-78]. Ectopic hTERT

INTRODUCTION

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expression restores telomerase activity in telomerase-negative cells [79],

demonstrating a positive association between hTERT and telomerase activity.

hTERT is therefore a key determinant of telomerase activity, and about 90% of

all tumours have detectable telomerase activity [80, 81].

The hTERT gene is localized on chromosome 5p15.33 and consists of 16 exons

and 15 introns [82]. The promoter region of hTERT is GC-rich, but lacks

traditional TATA and CAAT boxes. Several transcription factors have binding sites

on the hTERT promoter, enabling positive (cMyc, Hif1 , Sp1, estrogen, ETS1) or

negative (WT1, SMAD3, JunD, TGF , P53, Mad) regulation of hTERT [83-87] in a

cell type-specific manner. The hTERT promoter harbours a number of CpG sites,

indicating that methylation status also plays a key role in hTERT transcription

[85, 88]. Alternative splicing of hTERT mRNA contributes to the regulation of

telomerase activity in various cell types [89]. In RCC, full-length hTERT mRNA

expression is positively correlated with telomerase activity [90]. Finally, hTERT is

regulated at the post-transcriptional level by phosphorylation. Akt/PKB, in

association with the HSP90 chaperones, phosphorylates and activates hTERT,

resulting in telomerase activation [91-94].

Alternative lengthening of telomeres (ALT)

Some cells maintain their telomeres without telomerase [65, 95, 96]; these cells

have very long (>50 kb) and heterogeneous telomeres [97]. In these cells,

telomeres are maintained by ALT, a recombination-based event [98, 99]. Only a

small subset (10%) of tumours undergoes ALT, including mesenchymal and

neuroepithelial malignancies [97, 100, 101].

Telomerase and cancer

Transformed cells are immortalized during tumourigenesis, a necessary step for

unlimited replication. In the majority of cases, immortalization is achieved by

activation of telomerase and hTERT. hTERT activity is sufficient to immortalize

cells, but is not sufficient for transformation [40, 102]. Normal human epithelial

cells and fibroblasts are transformed by co-expression of hTERT, SV40, and H-ras

[103-106]. Telomerase activity is detected in 85-90% of malignancies, and a high

INTRODUCTION

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level of telomerase has been considered a negative prognostic marker in various

tumour types [107, 108]. In RCC, 80% of tumours show telomerase activity,

which is positively associated with hTERT mRNA levels [109].

Further, expression of full-length hTERT is linked to RCC tumour progression and

shows a positive association with cMyc [90]. Since hTERT seems to have a major

role in RCC progression, hTERT has potential as an RCC biomarker, and pathways

leading to hTERT activation are potential therapeutic targets.

INTRODUCTION

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Phosphatidylinositol-3-kinase (PI3K) signalling pathway

The phosphatidylinositol-3-kinase (PI3K) pathway is an essential signalling

pathway, facilitating basic cellular functions such as proliferation, growth,

migration, metabolism, and survival. Deregulation of this pathway can lead to

pathological conditions including cancer and metabolic disturbances [110-115].

The PI3Ks belong to a family of lipid kinases that catalyse the phosphorylation of

phosphatidylinositols (PIs) on the D3 position of the inositol ring. These enzymes

are divided into three classes based on substrate specificity and sequence

homology of the catalytic subunits [112].

PI3Ks operate near the inner surface of the cell membrane. The class I PI3Ks

phosphorylate the D3 site of PI(4)P and PI(4,5)P2 in vitro, but the preferred

substrate is PI(4,5)P2 (PIP2). In vivo, PI3K phosphorylation of PIP2 generates a

lipid second messenger, PI(3,4,5)P3 (PIP3), which activates downstream

components of the PI3K pathway [116]. The class I PI3Ks are divided into class IA

and class IB proteins, which are induced by tyrosine kinase receptors and G-

protein-coupled receptors, respectively [117]. Class II and class III PI3Ks are not

discussed in this thesis.

Class IA PI3K signalling pathway

The class IA PI3Ks are activated following the binding of growth factors to the

cell surface receptors, which have an internal receptor tyrosine kinase (RTK)

domain. These growth factors phosphorylate the tyrosine residues and change

the conformation of the RTK. The SH2 domain of the regulatory subunit (p85)

binds to the RTK, and allows the catalytic domain (p110) to interact with lipid

phosphotidine substrates [112]. Further, PI3K catalyses the phosphorylation of

PIP2 to PIP3 [110]. PIP3 possesses a specific PI interaction domain, PH

(Pleckstrin homology), which activates the PI3K pathway and recruits proteins

such as 3-PI-dependent kinase-1 (PDK-1) and Akt/PKB (protein kinase B) (Figure

4).

INTRODUCTION

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Akt/PKB

Akt/PKB belongs to the AGC kinase (AMP/GMP kinase and protein kinase C)

family and includes three highly conserved isoforms: Akt1 (PKBα), Akt2 (PKBβ),

and Akt3 (PKBδ). Akt protein consists of a PH domain, a kinase domain, and a

hydrophobic motif. The PH domain of PIP3 binds to the PH domain of Akt,

thereby inducing conformational changes in Akt. Further, PDK1 and mammalian

Target of Rapamycin Complex 2 (mTORC2) phosphorylate the thr308 and ser473

residues of Akt, respectively, and activate Akt [118, 119]. Following complete

activation, Akt has a wide range of targets that are involved in integral cell

functions.

In cell metabolism, Akt stimulates the glucose transporter (GLUT4) and then

translocates it to the cell membrane, which leads to an increase in glucose

uptake by muscles and adipose tissues [120]. Akt inhibits glycogen synthesis by

inactivating Glycogen Synthase Kinase (GSK) 3α and 3β [121]. Akt participates in

cell cycle progression from G1 to S phase by blocking the cell cycle inhibitors p27

and Rb2 [122]; Akt also indirectly promotes cell cycle progression by

stabilization of cyclin D1 via inhibition of GSK3β [123]. Akt positively regulates

cell survival by inactivating BAD (BCL2 antagonist of cell death) and enhances

mTORC1-mediated cell growth by phosphorylating and inhibiting the mTORC1

inhibitors TSC2 and PRAS40 [124-127]. Akt is involved in increasing telomerase

activity by forming an Akt-HSP90-hTERT complex in the nucleus that directly

phosphorylates and activates hTERT. The role of HSP90 in the complex is to

stabilize the Akt protein [91-94, 128, 129].

PTEN (Phosphatase TEnsin homolog on chromosome TEN)

PTEN, a lipid phosphatase, is a well-known tumour suppressor gene located on

chromosome 10q23.3 that is deleted in a variety of cancers [130, 131]. Under

normal physiological conditions, PTEN controls cell cycle progression, cell

survival, cell spreading and motility, endocytosis, release of angiogenic factors,

and regulation of translation [132]. The primary substrate of PTEN is PIP3, which

is a lipid second messenger of PI3K [133]. The growth factors activate PI3K that

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phosphorylates PIP2 to PIP3. PTEN reverses this process by dephosphorylating

PIP3 to PIP2, thereby negatively regulating the PI3K pathway [134-137].

Conversely, a loss-of-function mutation in PTEN leads to activation of the

PI3K/Akt pathway. Phosphorylation of PTEN stabilizes the protein, but reduces

its catalytic activity [138].

At the transcriptional level, the tumour suppressor p53 positively regulates

PTEN by binding to the PTEN promoter [139]. In addition, PTEN is regulated by

the Transforming Growth Factor β (TGF β) [140], RAS-MAPK, MEKK4, and JNK

pathways [141-144]. Recent studies have revealed that the proto-oncogene DJ-1

negatively regulates PTEN [145, 146]. At the post-translational level, protein-

protein interactions relocate PTEN to various cellular compartments, or cause

PTEN to become a target for degradation or stabilization [147, 148].Somatic

PTEN mutations occur in a large percentage of human cancers, with the highest

number found in endometrial cancer, brain, skin, and prostate cancer (reviewed

by[149]). In RCC, the PI3K/Akt pathway and its downstream targets, like mTOR

are active [150, 151]. PTEN activity is linked to aberrant PI3K/Akt activity;

although PTEN mutations are less frequently observed in RCC, PTEN protein

levels are often reduced and associated with RCC prognosis [152-155].

INTRODUCTION

24

INTRODUCTION

25

DJ-1

DJ-1/PARK7/RS is a proto-oncogene that is able to transform NIH3T3 cells

weakly on its own, moderately in association with cMyc, and strongly with H-ras

[156]. Recently, DJ-1 has been linked to the early onset of rare, autosomal

recessive Parkinson’s disease [157].

Human DJ-1 belongs to the DJ-1/ThiJ/Pfpl super family and encodes a 21-kDa

protein. The protein exists as a dimer [158] with a heat shock chaperone domain

[159, 160] and is expressed specifically in most tissues. DJ-1 is localized in the

cytosol; as the cell cycle progresses to S phase, the protein in part translocates

to the nucleus. The nuclear translocation might be due to other interacting

proteins [156].

Initially, the structure of DJ-1 was predicted from the structure of its bacterial

homologue, PH1704 protease [158]. The bacterial homologue PH1704 belongs

to ThiJ/Pfpl super family of proteins with proteolytic activity, suggesting that DJ-

1 acts as a protease [159, 161]. However, DJ-1 lacks protease activity because

the catalytic Cys-His-Glu triad in the PH1704 active site is missing [159, 161].

Under normal physiological conditions, DJ-1 exists as a dimer that forms a

hydrophobic patch at the dimer interface for interactions with other proteins, a

characteristic feature of chaperone proteins [159]. DJ-1 exhibits structural

similarity to Heat shock protein 31 (HSP31) [159]. A reducing environment

abrogates the chaperone activity of DJ-1 [162].

Function of DJ-1

CAP1/DJ-1 levels in rat epididymis are positively correlated with male fertility

[163], implicating DJ-1 in fertilization [164, 165]. Previously, DJ-1 was thought to

indirectly regulate androgen activity [166], but a recent study revealed that DJ-1

trancriptionally regulates androgen by binding to the androgen receptor [167].

Oxidative stress converts DJ-1 into a variant with a highly acidic isoelectric point

that quenches reactive oxygen species in cultured cells [168]. DJ-1 also indirectly

regulates oxidative stress by stabilizing NRF2, a master transcriptional regulator

of antioxidant enzymes such as Nqo1 and HO-1 [169]. Thus, DJ-1 prevents cell

INTRODUCTION

26

death from reactive oxygen species generated by oxidative stress, and may also

modulate apoptosis by regulating p53 [170-172].

DJ-1 and cancer

Various studies have provided evidence of an oncogenic function for DJ-1. For

example, DJ-1 is overexpressed in small lung cell carcinoma and ovarian

carcinoma, and elevated levels of DJ-1 autoantibodies were observed in breast

cancer patients [145, 173-175]. DJ-1 negatively regulates PTEN and activates the

PI3K/PKB pathway [145]. DJ-1 inactivates PTEN by proteolysis [145] or by direct

binding to PTEN [146]. DJ-1 also represses p53, thereby facilitating cell growth

[171, 172]. Finally, DJ-1 can activate cMyc via multiple pathways [176].

INTRODUCTION

27

WT1 (Wilms’ Tumour 1)

The WT1 gene was discovered as a tumour suppressor gene in a childhood

kidney neoplasm, later called Wilms’ tumour (WT) [177]. In addition to WT, a

heterozygous mutation of WT1 causes sporadic WAGR (Wilms’ tumour aniridia

genitor urinary abnormality and mental retardation), Denys-Drash syndrome,

(DDS) and Frasier syndrome [178]. During embryogenesis, WT1 is essential for

the development of kidneys, gonads, spleen, and mesothelial tissues; deletion of

WT1 results in an early embryonic death in transgenic mice [179]. The WT1-

deleted mice lack kidneys due to apoptosis of renal stem cells. It has been

shown that WT1 is involved in the development of the retina, the olfactory

system, and also in spermatopoiesis [180-182]. WT1 is also expressed in adult

tissues like including the urogenital system, the central nervous system, bone

marrow, and lymph nodes [183].

The WT1 gene, mRNA, and protein

WT1 has 10 exons, is located on chromosome 11p13, and encodes a 3 kb mRNA

that generates a 50 kDa protein [184]. The N-terminal domain of WT1 harbours

proline-glutamine rich sequences that are involved in protein/RNA interactions

and transcriptional regulation. The C-terminal domain contains four Cys2-His2

zinc fingers, which interact with RNA and proteins [184].

Two major alternative mRNA splicing events follow WT1 transcription. The first

splice occurs in exon 5 and encodes 17 amino acids. The second splice splits

exons 9 and 10, leading to the insertion of three amino acids (KTS). Alternative

splicing generates four protein isoforms, A (-/-), B (+/-), C (-/+), and D (+/+),

where ‘+’ represents the presence and ‘–’represents the absence of exon 5 and

the KTS inserts, respectively [185]. Formation of the four isoforms reflects the

complexity of WT1 function. The KTS- isoforms display better transcriptional

activity than the KTS+ isoforms [186-188] and are distributed throughout the

nucleus like other transcription factors, while the KTS+ isoforms are localized to

a small spot in the nucleus [189].

INTRODUCTION

28

WT1, the transcription factor

WT1 is a potent transcription factor with a wide variety of target genes [190].

WT1 can act as a transcriptional repressor or activator, a decision that is

influenced by the level of WT1 expression, the presence of isoforms (KTS+ or

KTS-), the position of the transcriptional start site, and cell type [187, 191-193].

WT1 targets

The WT1 promoter is auto-regulated, since high expression of WT1 levels

suppress its own promoter [194, 195]. WT1 transcriptional targets include

growth factors (e.g. TGF and CSF-1), growth factor receptors (e.g. insulin

receptor, IGF-IR, and EGFR), transcription factors (e.g. EGR, cMyc, Pax2, Dax-1,

and Sry) and other proteins (e.g. VEGF, ODG, MDR1, HSP70, P21, and Bcl2) [190,

196]. WT1 represses hTERT transcriptional activity in a kidney-derived cell line

[87].

WT1 interacting proteins

Several proteins have been identified as interacting partners to WT1. These

partners are also transcription factors or modulators of WT1 activity. Par4

(Prostate apoptosis response 4) interacts with the zinc finger motif of WT1, and

augmenting the transcriptional repressor function of WT1 [197]. Par4 also

interacts with the 17 aa isoform of WT1 and rescues ultraviolet radiation-

treated cells from apoptosis [197, 198]. CBP (CREB binding protein) interacts

with the zinc finger motif of WT1 and acts as a coactivator, enhancing its

transcriptional activity [199]. CBP also mediates the interaction between WT1

and p53, which contributes to transactivation and stability of the proteins [200].

WT1 also binds the p53 homologues p63 and p73 [201].

WT1 and cancer

WT1 plays a central role in carcinogenesis; however, WT1 seems to act

differently in different tumour types, indicating a form of cell-type specificity.

WT1 transcript levels were reduced in ccRCC samples compared to tumour-free

kidney cortex samples, indicating that WT1 acts as a tumour suppressor in adult

RCC [202]. Further, in one study only four out of twelve (33%) ccRCC samples

INTRODUCTION

29

showed WT1 protein expression [203]. High WT1 expression has been detected

in various tumours, including bone and tissue sarcoma, breast cancer, colorectal

cancer, and lung cancer [204-208]. A majority of acute leukaemia cases express

elevated levels of WT1 mRNA, and patients with low WT1 expression levels

experienced good prognoses (reviewed in [196]). In summary, depending on the

tumour type, WT1 can act either as a tumour suppressor or as an oncogene.

INTRODUCTION

30

cMyc

The Myc gene family consists of the proto-oncogenes cMyc, n-Myc, and l-Myc,

all of which are basic helix-loop-helix leucine zipper (bHLH/LZ) transcription

factors. cMyc is a cellular homologue of the viral oncogene v-Myc [209]. Under

normal physiological conditions, cMyc is ubiquitously expressed throughout

embryogenesis and in highly proliferative adult tissues. cMyc is also involved in

various integral cellular processes; deregulation of cMyc often leads to tumour

formation [210-213]. cMyc is differentially expressed in various human tumours,

including breast carcinoma, lung carcinoma, colon cancer, and RCC [214-218].

The cMyc gene and protein

cMyc is located on chromosome 8q24 and consists of three exons [219] that

encode two polypeptides of molecular weight 64 and 67 kDa [220]. The C-

terminal domain of cMyc harbours the bHLH/LZ motif that mediates protein–

protein interactions. This motif also binds DNA in a sequence-specific manner,

targeting the hexamer CACGTG, also known as the E-box [221]. The N-terminal

domain has highly conserved Myc Boxes (MBI - III) that transactivate cMyc

targets involved in cell proliferation, transformation, and apoptosis [222, 223].

The Myc-Max-Mad transcriptional network

cMyc forms a complex with other proteins and exerts cell type-specific

transcriptional activity via pol II. The key component of this network is Max (Myc

associated protein X), a protein that can dimerize with cMyc, Mad (Mxd), Mnt,

and Mga [212, 224]. All combinations of the complexes can bind promoter E-

boxes, but differential transcriptional effects depend on the cofactor of the Max

heterodimer. Under normal physiological conditions, Max and Mad have a slow

and constant turnover during the cell cycle, forming the Mad-Max (or Max-Mnt)

complex and repressing transcription [224-226]. Various mitogens upregulate

cMyc, which in turn replaces Mad in the Mad-Max heterodimer to form a cMyc-

Max complex [227]. This cMyc-Max activates transcription [228] (Figure 5). In

vivo, Mad-Max or Max-Mnt often predominate in resting or differentiating cells,

INTRODUCTION

31

whereas Myc-Max is predominant in proliferating cells [229].

Regulation of cMyc by upstream signals

cMyc has a complex promoter region that harbours binding sites for growth

factors, transcription factors, and other factors [230]. The Rac-dependent

pathway upregulates cMyc through Src kinase [231], while the TGF- receptor

can repress cMyc transcription via its direct mediator, SMAD3 [232].

The Wnt signalling pathway is implicated in cMyc upregulation [233]. In

addition, cMyc is upregulated by the PI3K pathway through NFkB signals [234].

Post-translational modifications such as phosphorylation can regulate stability of

the cMyc protein [235].

Role of cMyc in cell growth and proliferation

cMyc has a vital role in protein synthesis and growth in B-lymphocytes

[236].cMyc has a role in cell cycle progression from G1 to S phase through gene

activation and repression. Studies have shown that Myc-Max activates cyclin E/

CDK2 activity. Myc activates CDK4 [237] and cyclin D2, which in turn sequester

the CDK inhibitor p27 [227]. cMyc also contributes to cell cycle progression by

transcriptionally repressing the growth-arrest genes p15 [237] and p21 [238,

239]. Cells lacking both cMyc alleles prolonged their cell-doubling times, further

demonstrating cMyc participation in cell cycle progression [240].

cMyc in apoptosis, telomerase activation, and tumourigenesis

Several pathways have been suggested as the source of cMyc-induced

apoptosis. cMyc indirectly induces the expression of p19ARF

(an inhibitor of

MDM2 E3 ligase), which stabilizes p53 in mouse embryo fibroblasts [241]. cMyc

releases cytochrome c from mitochondria, an activity that is independent of

p19ARF

and p53 [242]. Furthermore, cMyc suppresses the pro-apoptotic proteins

Bcl2 and BCL-XL that contribute to the release of cytochrome c from

mitochondria [243].

cMyc recruits histone acetyl transferase, stimulates hTERT transcription [244],

and has also been implicated in inducing telomerase activity in mammary

epithelial cells and normal fibroblasts [245]. cMyc transcriptionally activates

INTRODUCTION

32

hTERT by binding to its promoter [246], while Myc-Max directly binds two E-

boxes on the proximal hTERT promoter [244, 247, 248]. Further, cMyc seems to

participate in stepwise upregulation of hTERT transcription in NBS T cell cultures

[83]. RCC shows a positive association between hTERT and cMyc [90].

INTRODUCTION

33

Nijmegen breakage syndrome 1 (NBS1)

The Nijmegen breakage syndrome 1 (NBS1) protein participates in double-

strand break recognition and repair, signal transduction in the cell cycle

checkpoint system, and telomere maintenance [249]. NBS1 is located on

chromosome 8q21 and contains 16 exons that encode a 754 aa protein (Nibrin

or NBS1 or p95) with a molecular weight of ~95 kDa [250-252]. NBS1 displays

weak homology to the Saccharomyces cerevisiae repair protein Xrs2. Human

NBS1 is one of the main components of the MRN complex, which also includes

MRE11 and RAD50 [253].

Homozygous or heterozygous biallelic mutations in NBS1 cause Nijmegen

breakage syndrome (NBS), a rare, autosomal recessive genetic disorder that

occurs mainly among Eastern Europeans [254]. NBS patients exhibit low birth

weights and length, and reduced head circumference [255], but the most

prominent feature is microcephaly [256, 257]. In mice, a null mutation of NBS1

causes poor development of embryonic and extra-embryonic tissues, leading to

early embryo death. In Nbs1 -/- mice, embryonic fibroblasts show a proliferation

defect [258-261].

The majority of NBS patients are homozygous for a 5 bp deletion (657del5), a

frame shift that results in a truncated protein (p26) [252]. In addition to the N-

terminal truncated (p26) protein, a N-terminal lacking protein fragment (p70)

can be produced. p70 is capable of interacting with MRE11 and retains some of

the vital functions of the full-length protein [262]. p26 is abundantly expressed

in NBS patients and in individuals heterozygous for the 657del5 mutation. p70

has been found in lymphoid cells but not in fibroblasts [252, 262, 263].

The full-length NBS1 protein (p95) has three functional regions: N-terminal,

central, and C-terminal. The N-terminal region consists of two functional

domains, a forkhead-associated (FHA) domain and a breast cancer carboxy

terminal (BRACT) domain [250, 251, 264]. Mutation of these domains causes

impairment in MRN foci formation and hypersensitivity to ionization radiation

INTRODUCTION

34

[265, 266]. The central region of the NBS1 protein harbours two

phosphorylation sites, ser278 and ser343, which are phosphorylated by the

Ataxia Thelangiectasia mutated (ATM) protein and control the S phase

checkpoint response to ionizing radiation [267]. The C-terminal region has two

binding domains for Mre11 and ATM; deletion of this domain leads to defective

MRN complex formation, increased radiation sensitivity, and altered cell cycle

checkpoint control [262, 268].

NBS1 and double-strand break repair

Although NBS1’s role in double-strand break repair has not been clearly

elucidated, NBS1 is known to serve as a molecular chaperone by binding H2AX

through the N-terminal FHA/BRACT domains. RAD50 and MRE11 translocate to

the nucleus and form MRN complexes proximal to a double-strand break [264,

269, 270]. The MRN complex then activates downstream targets that regulate

DNA repair and cell cycle checkpoints, such as ATM, p53, structure maintenance

protein (SMC1), and BRCA1 [249, 257, 271-273].

NBS1 and telomere stability

In mammalian cells, TRF2, Mre11, and RAD50 stabilize the T-loop throughout

the cell cycle [274, 275]. Indirect immunofluorescence demonstrated interphase

telomere occupancy by Mre11 and RAD50, but NBS1 was only associated with

TRF2 and telomeres in S phase, implicating NBS1 in telomere replication [274].

Indeed, primary fibroblasts from NBS patients showed an increased rate of

telomere shortening in vitro compared with cells from unaffected individuals.

Introducing either NBS1 or hTERT alone did not restore telomere length, but co-

expression of hTERT and NBS1 markedly increased telomere length [276].

Stabilized telomere length occurs in immortalized T cells derived from NBS

patients [263], most likely due to the presence of NBS1 (p70), a variant protein

that contains the Mre11 binding domain, which seems to function in telomere

elongation [263].

In telomerase-negative cancer cells, telomere length is maintained by ALT

(described above). NBS1 seems to participate in the ALT pathway (reviewed in

INTRODUCTION

35

[249]). NBS1 was first identified as a putative tumour suppressor gene due to

the high incidence of malignancies in NBS patients with defective NBS1 protein

and the observation of low NBS1 expression in breast carcinomas [277]. During

mouse development, NBS1 expression was elevated in highly proliferative cells

[278], and increased NBS1 expression was observed in head and neck squamous

cell carcinoma [279], uveal melanoma [280], and micro-satellite stable

colorectal cancer [281]. Overexpression of NBS1 contributes to transformation

and tumourigenesis through the PI3K pathway [282, 283] and to cell

proliferation through the Ras/Raf/MEK/ERK cascade [258]. Taken together,

these lines of evidence suggest an oncogenic role for NBS1 in various cancers.

AIMS OF THE THESIS

36

AIMS OF THE THESIS

The main goal of this thesis was to investigate the signalling pathways that

directly or indirectly are involved in the regulation of hTERT and telomerase

activity in RCC.

Specific Aims

Paper I

To investigate the functional role of DJ-1 in RCC, with a special focus on hTERT

expression and telomerase activity.

Paper II

To evaluate NBS1 expression, and its association with DJ-1, cMyc, cell

proliferation, and hTERT in ccRCC.

Paper III

To characterize WT1 as a transcriptional regulator of hTERT in ccRCC.

MATERIALS AND METHODS

37


Materials

The tumour materials used in this thesis were collected between 1985 to 2003

under a protocol approved by the Human Ethics Committee of the medical

faculty, Umeå University. The samples were obtained from the tumour bearing

kidney, after permission from the patient with signed consent. All specimens

were reviewed and confirmed by a pathologist. Tumour stage was classified

according to the TNM classification system 2002 [22] and tumour grading was

performed according to Skinner et al [21]. The follow-up medical records of the

patients were retrospectively updated by the urologist and used for survival

analysis. For paper I 176 RCC samples (153 ccRCC and, 23 pRCC) and 49 normal

kidney cortex samples, for paper II 213 RCC (175 ccRCC, 27 pRCC and 11 chRCC)

and 52 normal kidney cortex samples were used. In paper III 73 ccRCC samples

and 26 normal kidney cortex samples were analyzed. One part of the sample

was used for RNA expression analysis and one part for immunoblotting. For

immunohistochemistry formalin fixed and paraffin embedded tissues were used

in study I and II (DJ-1 and NBS1).

The cell lines A498, TK-10, 786-0 (ccRCC cell lines), T47D1 (breast cancer cell

line) and FADU, derived from a hypopharyngeal squamous cell carcinoma, were

cultured in DMEM 1X (with 10% FCS) (GIBCO, Stockholm, Sweden). The ovarian

cancer cell line A2780 was cultured in RPMI 1640 (with 10% FCS) (GIBCO,

Paisley, UK). All cell lines were kept in 5% CO2 at 37° C except for FADU which

was kept at 10% CO2

RNA extraction and cDNA synthesis (Paper I, II and III)

Total RNA was extracted from both clinical and in vitro study samples by the

TRIzol method (Invitrogen, Stockholm, Sweden) according to the manufacturer’s

protocol. Concentration of total RNA from the clinical sample was measured by

spectrophotometry at 260nm, and the RNA quality verified by agarose

electrophoresis. Total RNA from in vitro experiments was measured by


38

Nanodrop spectrophotometer (Nanodrop, Thermo Scientific, Wilmington, DE,

USA).

cDNA was synthesized by reverse transcription of total RNA using random

hexamers (Applied Biosystem, Sundbyberg, Sweden) Superscript reverse

transcriptase kit (Invitrogen) according to the manufacturer’s instructions.

Transient transfection

siRNA transfection (Paper I and II)

In order to knock down DJ-1, pooled siGENOME SMART pool siRNA

(100nM/well, with 105cells/well) (Dharmacon, Chicago, IL, USA) was transfected

using Lipofectamine 2000 (Invitrogen).

NBS1siRNA (mRNA target 5’-AAUGAUCAGUCGAUCAGCCGA-3’, 200pmol/well,

with 105cells/well) was transfected into cells using Dharmafect 1 (Dharmacon).

As control, non-specific control siRNA (Dharmacon) was used with

Lipofectamine 2000 (Invitrogen). Cells were collected for further analysis after

24,48,72 and 92h respectively.

Expression vectors (Paper I, II and III)

Expression vectors DJ-1/Park7 (Origene Technologies, Rockville, MD, USA, 800

ng/ well), NBS1 (Origene Technologies, 1000ng/ well), WT1 A, and WT1D

(obtained from professor Oji, Osaka University, 1000ng/well) and pcDNA 3.1(+)

(Invitrogen, 1000 ng/ well) empty vector were transfected into cells

(105cells/well) by FUGENE 6 (Roche Diagnostics Corp. Indiana polis, IN, USA).

Real time PCR (Paper I, II and III)

DJ-1, cMyc, hTERT and PBGD mRNA levels were quantified using Light cycler

FastStart DNA master SYBR green I Kit (Roche Diagnostics) in a Light cycle

instrument (Roche Diagnostics, GmbH, Mannheim, Germany). Primer sequences

and detailed PCR protocols are described in papers I and II.

WT1 and -actin were quantified using Taqman technology, and run on the ABI

prism 7000 Sequence Detection System. PCR protocol, primer and probe

sequences are described in paper III. SMAD3, ETS1 and AP2α were analyzed by


39

TaqMan assay according to manufacturer's protocol and run on the ABI prism

7000 Sequence Detection System.

Quantitative real-time PCR method (Tel-PCR) [284] was used to measure

telomere length. Detailed protocol and information on primers used described

in Paper I. The Tel-PCR provides a relative telomere length value (T/S).

Protein extraction (Paper I, II and III)

Fresh frozen clinical samples were homogenized and sonicated twice for 15

seconds each in Tris lysis buffer, containing Nonidet 40 (0.5%), Sodium

deoxycholate (0.5%), SDS (0.1%), Tris-HCl (50nM, pH 7.5), NaCl (150nM), EDTA

(1mM, pH8.0), Sodium fluoride (1mM), Leupeptin (5mg/ml), Aprotinin (5mg/ml)

and phenyl methyl sulphonyl fluoride (1mM). Samples were centrifuged, and the

supernatant collected and stored at -80° C.

For in vitro experiments, proteins were extracted using CHAPS lysis buffer (3-[3-

cholamidopropyl) dimethyl- ammonio]-1propane sulphate. Collected samples

were incubated with CHAPS lysis buffer for 30 minutes on ice and then

centrifuged at 14,000 rpm for 30 minutes at 4° C, where after the supernatant

was collected. The protein content in both clinical and in vitro experiment

samples was determined using the bicinchoinic acid (BCA) protein assay (Pierce,

Rockford, IL, USA) according to the manufacturers’ protocol.

Western blot (Paper I, II and III)

Proteins (10 -20 g/ l) were separated by SDS-PAGE (10%), and transferred to

membrane (Hybond-ECL Amersham Biosciences, Buckinghamshire, UK) by semi-

dry transfer method. Membranes were blocked with 5% milk in TBS with 0.1 %

Tween for 60 minutes at room temperature, and then probed with primary

antibody overnight at 4° C. The antibodies were detected by horseradish

peroxidase conjugated anti-rabbit or anti-mouse antibodies, and visualized by

chemi-luminescence detection system (ECL-advances, Amersham Biosciences).

Primary antibodies used are described in the respective papers.


40

Immunohistochemistry (Paper I and II)

Immunohistochemistry was performed to detect the DJ-1 and NBS1 proteins.

Formalin fixed and paraffin embedded ccRCC and pRCC samples were stained in

a semiautomatic staining machine (Vantana ES,Ventana Tuscon, AZ, USA) using

iVIEW dab detection kit (Vantana) with or without pre-treatment with

endogenous biotin blocking kit (Ventana). For DJ-1 a primary monoclonal

antibody (Zymed labpratories, San Francisco, CA, USA) was used in 1:400

dilution, and for detection of NBS1, a monoclonal antibody against NBS1 (ab398,

Abcam, Cambridge, UK) was used in 1:250 dilution.

Immunofluorescence (Paper III)

Fresh cultured cells were fixed on slides with formaldehyde (3%) and sucrose

(2%) in PBSA, and permeabilized by glycine (0.1%) treatment, and blocked with

normal goat serum (2%) and TritonX-100 (0.2%) in PBSA. Cells were thereafter

incubated with WT1 antibody (1:100, Dako) overnight, washed with Triton-X

(0.2%), and probed with secondary antibody Alexa Flour 488 rabbit anti-mouse

IgG (H+L) (Molecular Probe Inc., Eugene OR, USA; 1:500). DAPI staining was used

for nuclear visualization.

ChIP assay (Paper III)

Chromatin immunoprecipitation (ChIP) was performed using a Chromatin

Immunoprecipitation Kit (Upstate Millipore, Billerica, MA, USA) according to the

manufacturer´s manual. Antibodies used were anti-WT1 antibody (C-19, Santa

Cruz Biotechnology Inc, Santa Cruz, CA, USA) or normal rabbit IgG (Cell Signalling

technology Inc, Danvers, MA, USA). The PCR protocol and primer sequences for

the hTERT, SMAD3 and cMyc promoters are described in paper III. PCR products

were run on 1% agarose gel, ethidium bromide stained DNA and observed in an

Ultraviolet Trans-illuminator (Specroline, Westbury, NY, USA).


41

Telomerase activity

Paper I

In this study, 500 ng of CHAPS extract proteins were used to measure the

relative telomerase activity by TRapeze XL telomerase detection kit (Chemicon

International, Temecula, CA) according to the manufacturer’s guidelines.

Phosphor imager and Image-Quant software (Molecular Dynamics, Sunnyvale,

CA, USA) were used to quantify band intensities. The relative telomerase activity

was expressed as a total product generated (TPG).

Paper III

250ng of CHAPS extract was used to measure telomerase activity using

quantitative telomerase detection kit (QTD kit, Allied biotech Inc, Ijamsville, CA,

USA). We followed the procedure according to manufacturers' protocol, using

700-sequence system (Applied Biosciences) to perform qRT-PCR. The standard

curve was generated by TSR control provided by the kit, and telomerase activity

was evaluated using 700 SDS system software (Applied BioSystem).

Genome-wide expression micro array (Paper III)

Total RNA was used to generate cRNA as described in the Illumina Total Prep

RNA amplification kit (Ambian Inc., Austin TX, USA). Biotinylated cRNA was

hybridized to the human HT12 Illumina Bead chip gene expression array as

mentioned in the manufacturer’s protocol (Illumina, San Diego, CA, USA). The

arrays were scanned by an Illumina Bead array Reader. Data was normalized and

analyzed using Bead studio 3.2 software. By Metacore software (GeneGo Inc, St

Joseph, MI, USA) cell signalling pathways and networks were evaluated. The

quantile algorithm normalized the samples. Differentially expressed genes were

identified by fold change and with the Illumina custom differential expression

algorithm (described in the Illumina gene Expression module user guide) to

identify ≥2 fold changes and statistically (p<0.01) differentially expressed genes.

Genes with signals below background levels were omitted.


42

DNA histogram analysis (Paper I and II)

DNA staining with propidium iodide was performed as described [285]. The

samples were analysed in a FACScan flow cytometer (Becton Dickinson Immuno-

cytometry Systems, San Jose, CA) and DNA index (DI) was calculated. A tumor

was denominated diploid (DI = 1) when only one peak was detected and

aneuploid when two (or more) separate peaks were found. The percentage of

cells in various cell cycle phases was calculated by the Cellfit software using the

RFIT evaluation model (Becton Dickinson).

Cytotoxic assay (Paper I)

Cytotoxic effects produced by transfection experiments were determined using

MTT based TOX1 In Vitro Toxicology Assay Kit (Sigma-Aldrich, St. Louis, MO). The

protocol followed the manufacturer’s manual and percent viable cells was

calculated.

hTERT promoter activity assay (Paper I)

Wild type hTERT (pGL3 hTERT 24/9, 250ng) or E-box mutant hTERT (pGL3 hTERT

3c, 250ng) reporter vectors were co-transfected with DJ-1 plasmid (Origene

Technologies, 40ng and 80ng) using Superfect transfection reagent (Quigen,

Crawley, UK) in a 96 well multiplex format and performed in quadruplicate. Cells

were analyzed after 48 for luciferase expression using Luciferase assay system

(Promega Southampton, UK). Experiments were repeated three times.

Statistical analysis (Paper I, II and III)

SPSS software (Version 14 for paper I, PASW 18 for paper II and Version 15 for

paper III) was used to perform statistical analysis. Mann-Witney U test was used

to calculate differences in the expression of two independent variables.

Spearman correlation test (statistical significance p<0.05) was used to test the

correlation between two variables. Survival times were illustrated by Kaplan-

Meier curves and survival times were compared using log-rank test.

RESULTS AND DISCUSSION

43


Paper I

The PTEN regulator DJ-1 is associated with hTERT expression in clear cell renal cell carcinoma

The main objective of this study was to investigate the expression of DJ-1 and its

potential effects on the PI3K/PKB pathway and hTERT expression in RCC. We

found that DJ-1 mRNA and protein levels were higher in ccRCC than in the

normal kidney cortex, suggesting that DJ-1 is overexpressed in ccRCC. These

results are in line with previous reports of DJ-1 upregulation in other cancers

[173-175, 286]. We also observed over-expression of cMyc and hTERT mRNA

levels in ccRCC samples. These findings were also in agreement with previous

reports, showing upregulation of cMyc [90] and hTERT [90, 109, 287] in ccRCC.

We detected significant, positive correlation between the DJ-1, cMyc, and hTERT

mRNA levels in ccRCC, indicating functional links between these factors. To

elucidate possible mechanisms behind these associations, we used the ccRCC

cell line A498 for in vitro experiments. Transfecting A498 cells with siRNA

directed against DJ-1 resulted in reduction of DJ-1 expression and suppression of

p-PTEN, p-Akt, p-GSK-3 , and cMyc protein levels. This siRNA treatment also

reduced cMyc and hTERT mRNA levels and telomerase activity. Thus, reducing

DJ-1 expression resulted in inhibition of the PI3K/Akt (PKB) pathway, triggering

activation of GSK-3 , which previously has been shown to induce cMyc

destabilization via phosphorylation of thr58 [235, 288]. Knockdown of DJ-1 thus

repressed the cMyc level in ccRCC cells through down regulation of PI3K/Akt

(PKB)/pGSK-3 . We also demonstrated that forced overexpression of DJ-1

increased hTERT promoter activity in an ovarian cancer cell line. This promoter

assay revealed that the E-boxes in the hTERT promoter were required for DJ-1-

induced hTERT activation, indicating that cMyc was required for this

downstream effect of DJ-1 on hTERT. A previous study had also documented

that cMyc can regulate hTERT transcription by binding to the E-boxes of the


44

hTERT promoter [289]. Hence, our combined results substantiated that DJ-1 via

the PI3K/Akt (PKB) pathway can exert downstream effects on hTERT and

telomerase activity via cMyc.

Previous studies have shown that p-Akt phosphorylates and thereby activates

the hTERT protein, leading to increased telomerase activity [93, 128]. In our

study, since DJ-1 activated Akt (PKB), it is also possible that the enhanced

telomerase activity partly was due to post-transcriptional modification of hTERT

by phosphorylation.

In ccRCC, the expressions of DJ-1, cMyc, and hTERT mRNA were linked to neither

tumour stage nor survival. Excluding DJ-1, these findings are in line with

previous studies on RCC in which cMyc demonstrated no independent

prognostic value [290] and the hTERT mRNA level was not associated with stage

or presence of metastasis [90]. Another study showed that telomerase activity

was not correlated with stage or grade in RCC [291].

In pRCC samples, the DJ-1 expression level did not differ from normal kidney

cortex, suggesting that DJ-1 is not functionally active in pRCC. cMyc and hTERT

levels were elevated in pRCC compared to the kidney cortex samples, but there

was no significant correlation between them. Hence, the activity of a DJ-1-

PI3K/Akt (PKB)-cMyc pathway could not be documented in pRCC, a finding that

is not surprising given that ccRCC and pRCC are characterized by different

genetic aberrations [292-295]. In conclusion, this study suggests that DJ-1

regulates hTERT through a putative PI3K-cMyc pathway in ccRCC, but not in

pRCC.


45

Paper II

The NBS1 gene is overexpressed and regulated by DJ-1 in clear cell renal cell carcinoma

NBS1 participates in various cellular functions, such as DNA damage response,

DNA replication, telomere maintenance, and cell cycle progression. Recently,

NBS1 was shown to contribute to transformation via the PI3K pathway. The

main objective of this study was to elucidate the role of NBS1 in intracellular

signalling in RCC with regard to the PI3K pathway and thereby also with regard

to telomerase maintenance, since hTERT is a downstream target of Akt/PKB.

In the present study, compared to normal kidney cortex samples, NBS1 mRNA

levels were increased in pRCC samples, while both mRNA and protein levels

were increased in ccRCC samples. These observations were in line with reports

of NBS1 overexpression in other cancers, such as non-small cell lung carcinoma

[283], microsatellite-stable colorectal cancer [281], uveal melanoma [280], and

head and neck squamous cell carcinoma [279].

The PI3K pathway and its downstream targets are activated in a variety of

human malignancies [296-299], including ccRCC [154, 300, 301]. A recent study

demonstrated that NBS1 regulates the PI3K pathway by direct interaction with

the p110 subunit of PI3K [282]. We previously showed that DJ-1, a negative

regulator of PTEN, is upregulated and potentially activates the PI3K pathway in

ccRCC [301]. In the present study, NBS1 displayed a positive association with DJ-

1. Since both DJ-1 and NBS1 are involved in regulation of the PI3K pathway, the

data suggested a possible functional link between them. To verify this, we used

the ccRCC cells A498 and FaDu cells for in vitro experiments. In these cells,

neither suppression nor overexpression of NBS1 changed the DJ-1 expression

level; since NBS1 had no effect on DJ-1, it was possible that NBS1 was regulated

by DJ-1. To investigate the effect of DJ-1 on NBS1, we repressed DJ-1 by siRNA in

A498 cells, which resulted in downregulation of NBS1. In addition,

overexpression of DJ-1 in 786-0 cells led to upregulation of NBS1. Collectively,


46

our in vitro experiments suggested that NBS1 operates downstream of DJ-1, but

the details of the interaction between DJ-1 and NBS1 remain unclear.

We detected a positive association between NBS1 and cMyc. Previously, cMyc

was reported to transcriptionally regulate NBS1 [302], but our in vitro data

revealed that NBS1 acts upstream of cMyc in ccRCC. This finding implies that the

interaction between NBS1 and cMyc may be cell type-specific. Additional

experiments are needed to confirm the interaction between NBS1 and cMyc in

ccRCC.

We also showed that NBS1 activated Akt/PKB in the FaDu cell line, but not

convincingly in the A498 cell line. Recent studies have shown that NBS1 can

directly interact with PI3K and activate Akt/PKB [282, 283], suggesting that NBS1

may function differently in ccRCC than in other cell types, regarding its effect on

PI3K and Akt/PKB activation.

NBS1 mRNA levels were also positively correlated with S phase. This result is in

accordance with a previous study showed that NBS1 is required for IGF-1-

induced cellular proliferation through the Ras/Raf/MEK/ERK cascade [258]. In

contrast, we observed no association between S phase and cMyc, a surprising

result since cMyc is a proliferation-associated oncogene and is supposed to play

an essential role in the proliferation of ccRCC cells. The reason for this

contradictory observation is unknown, but in ccRCC NBS1 may be involved in

undefined pathways that affect tumour cell proliferation.

hTERT is one of the downstream targets of PI3K, leading to activation of

telomerase via hTERT phosphorylation by Akt/PKB [93, 128, 303]. Our previous

study showed that DJ-1 regulates hTERT in a cMyc-dependent manner in ccRCC

[301]. In the present study, we detected no significant correlation between

NBS1 and hTERT mRNA levels in ccRCC, thereby indicating that NBS1 is not

involved in activation of hTERT in ccRCC.

In pRCC, we detected no correlations between NBS1 and other parameters, such

as DJ-1, cMyc, and S phase. These results may point to a non-functional DJ-1-

NBS1-cMyc pathway in pRCC, partly explained by the fact that ccRCC and pRCC


47

are different from each other [10, 11, 293, 295]. The pRCC tumour collection

studied was also relatively small compared to the ccRCC collection, which may

obscure potential connections.

In conclusion, the present study demonstrates that NBS1 is upregulated in

ccRCC and pRCC compared to tumour-free kidney cortex. Further, NBS1

expression correlated with proliferation, cMyc expression, and DJ-1 expression

in ccRCC, and experimental data suggesting that DJ-1 acts upstream of NBS1 and

cMyc.


48

Paper III

Wilms' tumour 1 can suppress hTERT gene expression and telomerase activity in clear cell renal cell carcinoma via multiple pathways

The purpose of this study was to investigate the functional aspects of WT1

regarding hTERT regulation and telomerase activity in ccRCC.

We demonstrated that WT1 mRNA and protein levels were lower in ccRCC

samples compared to tumour-free kidney cortex. Few previous studies have

explored WT1 expression in ccRCC. Campbell et al. demonstrated aberrant WT1

expression in four out of five ccRCC samples [304], an observation in contrast to

our findings. Recent study showed, low WT1 transcription levels in ccRCC

compared to in normal kidney [203], and another study reported that only 33%

(4 out of 12 samples) of ccRCC samples were positive for the WT1 protein [202].

These latter reports are in concordance with our findings and suggest that WT1

can act as a tumour suppressor gene in ccRCC.

Several positive and negative regulators of hTERT transcription have been

identified; among these, WT1 has been implicated as a cell type-specific

transcriptional repressor of hTERT through its biding sites on the hTERT

promoter [87]. We observed a significant negative link between WT1 and hTERT

mRNA levels in ccRCC, suggesting that WT1 may negatively regulate hTERT. To

demonstrate this potential regulatory ability, we overexpressed WT1 in a kidney

cancer cell line using expression vectors for WT1A and WT1D; WT1 subsequently

repressed hTERT mRNA levels and telomerase activity. Immunofluorescence of

transfection experiments demonstrated nuclear localization of the WT1 protein.

Previously, the cytoplasmic sequestration of WT1 led to upregulation of hTERT

and telomerase activity in IL-2-induced HTLV1 T cells, indicating that nuclear

localization of WT1 may be a prerequisite for repression of hTERT and

telomerase activity [305]. In addition, chromatin immunoprecipitation verified

direct binding of WT1 to the hTERT promoter, an observation in agreement with

a previous report [87].


49

cMyc is a well-known transcriptional activator of hTERT [289]. Like other

investigators, we detected overexpression of cMyc in ccRCC, as well as its

positive association with hTERT levels [90, 301]. On the other hand, WT1 can

have both stimulatory [306] and repressive effects on cMyc transcription [307].

In the present study, WT1 displayed a negative correlation with cMyc expression

in ccRCC. Transfection experiments and chromatin immunoprecipitations

confirmed that WT1 downregulated cMyc mRNA and protein levels by directly

binding the cMyc promoter. Collectively, these results indicate that WT1 can

transcriptionally regulate cMyc and hTERT in parallel.

Gene expression array analysis revealed that forced expression of WT1 induced

expression changes in various known and novel targets of WT1 that included

hTERT transcriptional regulators not previously identified as WT1 targets. GM-

CSF2 regulates hTERT transcription both positively and negatively, in

combination with other factors [308]; we also observed upregulation of CSF2 by

WT1, implicating it as a negative regulator of hTERT transcription in ccRCC.

Similarly, ETS1 has been associated with both positive and negative hTERT

regulation [309], and since ETS1 was upregulated by WT1 overexpression, it

seems to function as an hTERT repressor in ccRCC. Furthermore, inhibitors of

hTERT transcription such as IRF1 [310], JUN (AP1), and SMAD3 [311] were

upregulated by WT1, whereas NFX1, an activator of hTERT, was repressed [312].

Differentially expressed genes such as SMAD3, ETS1, and AP-2 were confirmed

by real-time PCR. In conclusion, WT1 plays a tumour suppressive role in ccRCC,

and seems to regulate hTERT gene expression through multiple pathways.

GENERAL CONCLUSIONS

50

GENERAL CONCLUSIONS

Regulation of hTERT is a complex process involving a large number of signalling

molecules and transcription factors. The PI3K pathway and its downstream

targets, including hTERT, are activated in RCC. In ccRCC, we showed that the

proto-oncogene DJ-1 regulates transcription of hTERT via the PI3K-PKB-cMyc

pathway by inhibiting PTEN. In addition, DJ-1 may also regulate hTERT protein

activity through phosphorylation by pAkt. The existence of a DJ-1-PI3K-Akt/PKB-

cMyc-hTERT pathway in clinical ccRCC samples was verified by in vitro

experiments using cell lines.

NBS1 is upregulated and involved in the tumourigenesis of e.g. head and neck

cancer by direct interaction with the PI3K pathway. Our study also detected

upregulation of NBS1 in ccRCC, and demonstrated positive links with DJ-1

expression, cMyc expression, and tumour cell proliferation. Experimental data

indicated that DJ-1 acts upstream of NBS1 and cMyc, and NBS1 acts upstream of

cMyc; the mechanisms underlying these interactions require elucidation in

additional experiments. NBS1 expression was not correlated with hTERT

expression, implying that NBS1 does not participate in hTERT regulation in

ccRCC. We present a tentative network diagram based on these data collected

from ccRCC samples in Figure 6.

GENERAL CONCLUSIONS

51

Our study suggested that WT1 functions as a tumour suppressor in ccRCC, and

we further showed that WT1 can transcriptionally repress hTERT and cMyc, and

transcriptionally activate SMAD3 in ccRCC. We also demonstrated that WT1

induces other target genes involved in hTERT regulation. Hence, we conclude

that WT1 not only regulates hTERT directly, but also through additional

pathways (Figure 7).

In conclusion, this thesis presents novel data regarding regulation of the hTERT

in ccRCC, which also further increases its complexity.

GENERAL CONCLUSIONS

52

ACKNOWLEDGEMENTS

53

ACKNOWLEDGEMENTS

I owe my deepest gratitude to the people who supported and contributed throughout my thesis. It is my pleasure to thank them without whom this thesis would not have been possible.

Göran Roos, my supervisor, whose encouragement, guidance and support from the initial to the final stage motivated me to complete this thesis. Thanks for giving me the opportunity to be the part of your research project and it gives me the privilege to say that you are the ocean of knowledge, I am proud to be your student. Besides, you are the person who I admire and look up to whether on the personal front or at work. You gave me a feel at home working atmosphere, which made me accomplish my goal without any obstacles. Well, few words are not enough to express my gratitude towards you. You have been and always will be my inspiration!

Börje Ljungberg, co-supervisor, I am grateful for your advice, constant support and help whenever I needed despite your busy schedule; it is really great to have you as my co-supervisor.

Karin Nylander, co-supervisor, thanks for all the help giving me the valuable feedbacks. I am thankful to your lab members for helping me borrow antibodies and other stuffs. I am honored to have you as a chairperson for my thesis defence.

Aihong Li, it was very informative and pleasant working with you, I learnt a lot from you. Thank you very much for everything.

Nicol Keith, thanks for all the nice discussions, valuable suggestions and help. Cairney thank you for your input. Professor Y.Oji, Professor H. Sugiyama, I thank you for the collaboration.

Bengt Hallberg, thanks for involving me in your journal club and all the help. Ylva Hedberg, thanks for all your help during the beginning stage of my PhD. Terry, Åsa, Karin B, thanks for keeping everything in order and making it much smooth much easier for me. Ulla-britt, you taught me most of the techniques when I joined the lab, undoubtedly you are an excellent teacher. Bodil, ever charming, thanks with the cell culture work, I enjoyed working with you, and it is nice to have you around in the lab. I would also like to thank my former lab members Agneta, Eva, Teresa, Lars, Simon, Gautam, and Josephine.

My present lab members: Sofie, thanks for the collaboration and all the help you did during my thesis work. Kattis, thank you very much for teaching me real time PCR and all the help, best of luck with your future. Pia, you gave me an enormous support in the lab, thank you for sharing your knowledge in the lab meetings. Emma, thanks for helping me with MS Word and also your input in my publication. Magnus and Ullrika, it was nice to share the office room with you

ACKNOWLEDGEMENTS

54

guys, good luck to both of you. Pawel thanks for your contribution to the first paper. Former lab member Inger, I wish you all the best. Linda, the new member of the lab, thank you very much for reading my thesis and giving me the valuable feedback. Gudrun, Solvig, Britt-Inger and Kerstin, from Urology, you all were very much helpful to me, thank you. Thomas, Hani, Jowel, Johan, Vincy, Ana, Sofia, Emma, Ana Birve, Ritu, Nina ,Per, Linda, Xaolion,Karin and other members of the corridor, thank you all for making a lively atmosphere. Elisabeth Sauer-Eriksson, thanks a lot for the help and support when I needed.

I would like to pay my deep respect to my teachers Dr.Srinivas Gowda, Dr. Mukund Gajendragad and Dr. Ramachandra for giving me the constant guidance and helping me to choose the right path in career.

Zaki, without you my stay in Umeå would not have been the same. We got along very well from the day one we met; the time we spent together is always cherishable. Fun days, travels, IKSU, partying and the discussions we had on almost all the topics under the Sun. The list goes on…I gained a lot from you. My best wishes to you and Freschta. Chandru, no matter what, you are there for me in all aspect of my life. Late Mohan, you might not be there with us, I miss you. Raama, I had your great companionship during our unforgettable college days. MTB thanks for your help during the tough time, I always remember that.

Bala & Uma, thanks for being around and sharing with us the feeling of nativity in all terms. Good luck to both of you. Kiran & Sireesha, you have always been very warm to us and have always given us the opportunity to share your joy and very nice time in gatherings. Deepa & Giri, thanks for all the help especially during our wonderful trip to Austria, which was incredible. Satish & Namith thanks for helping us during the Swiss trip. Mari & Devi, Pradeep & Padmini, Deepak & Rathi, Srinivas Organti, Kalyan, Vijay, Nagaraju Gorre, Ramesh Tati, Hanumakumar, I wish you all the greatest future and happiness.

Anubhav alias Thaakur, first of all I have to thank you for having enormous patience for helping me with thesis corrections. You sat with me for long hours for the corrections keeping your own work aside. Without a word you were there for me whenever I needed your help. I have known you for long time now. I will always cherish the time when we had all Hansi mazaak, never ending discussions, long walks, IKSU and FOOD, thanks for everything. Manah, I am overwhelmed by the fact of you being a sincere devotee of Lord Krishna and thanks for giving me an opportunity to take part in Parama Karuna Ashram gatherings, which made me longing less for my home country. Erik Ramos, it is really kind of you to help me with MS Word.

Here comes the part where I must mention the names of those who made my days in Umeå enthusiastic, relaxing, fun filled and energetic. NL members, I can not thank you enough. Munender alias Munnabhai an ever happy go lucky guy and a wonderful person, thanks man for designing my thesis cover page, a great piece of work and I enjoyed watching movies with you, which was

ACKNOWLEDGEMENTS

55

unforgettable. I wish you good luck with your thesis. Sharvani, a principled lady, thanks for always being so hospitable, kind, helpful and supportive in all aspects. Dinesh, enemies also fails to hate you, applauds to your optimism, and for facing new challenges that you choose to do in life no matter what. Gaurav, ever since we met you have been my friend you are one of them whom I can ever depend on. I miss your company. Renu & Pari thanks for the good company. Sanju & Roxy, happy couple, I wish you both good luck with your future and it was nice having you guys around in parties and trips. Krishna a person who is like a midnight sun (no offence to Dinesh he.. he..) is very disciplined, well organized, I still remember your spontaneity from doing things like ´now or never´ to bringing fruits right away from Maxi during our ongoing party itself. I wish you all, the very best of luck from the bottom of my heart. I hope we all will meet up once again in life with extended families.

I am immensely grateful to my father, the late Sri. Sitaram and mother Smt. Sharada Sitaram without whom I could have never achieved the same. I consider my life to be complete if I can ever make you proud of me. My sister Uma, brother-in-law Narendra and nephew Achintya, thanks for being supportive and understanding me throughout. I am sincerely grateful to my in-laws Sri. Srinivasaiah.T.R and Smt. Manjula, Vinay and Madhura for providing me all the support and encouragement during my PhD. At this point I would also like to remember my relatives Laxminarayan, LaxmiDevi, Suresh, Meenakshi, Shubha, Harsha, Suma, Hemant, Sachchu, Byju, Vikas, Varsha and kids Skanda, Maanya and little Smera.

Finally, my beloved wife Vidya, well honestly I do not have enough words to thank you, you are the one of the best thing happened in my life. I do not think I would finish this work without your support and love. I love you for ever.

Thank you all

Ravi

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56

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