Graduate Department of Molecular and Medical Genetics,University of Toronto

IDENTIFICATION OF 6p22 ONCOGENES

IN RETINOBLASTOMA

By Marija Orlic-Milacic

September 2007

ABSTRACT

The study of retinoblastoma, the malignant tumor of the retina, has set the fundamentals

of cancer genetics and the genetics of familial cancer syndromes through discovery of the first

tumor suppressor gene RB1. Retinoblastoma is an excellent disease model of the multistep

nature of cancer. The initiation events, leading to the loss of function of both alleles of the RB1

gene, hence named mutation 1 and 2 (M1 and M2) are well characterized, as well as the

number of recurrent chromosomal aberrations that are positively selected for during tumor

progression. Recurrent regions of chromosomal gain and loss are hypothesized to carry

oncogenes and tumor suppressor genes, respectively, which are targeted by mutational events

M3-Mn.

One of the most frequently gained chromosmal regions is the short arm of chromosome

6, 6p, with the minimal region of gain mapping to the chromosomal band 6p22. In this thesis,

through comparative expression analysis of retinoblastoma and healthy retina, the number of

candidate 6p22 oncogenes is narrowed to two genes, DEK and E2F3. The functional analysis

of the oncogenic potential of DEK and E2F3 in retinoblastoma cell lines through RNA

interference, shows that both genes display oncogenic properties, since the knockdown of any

of the two adversely affects the growth of retinoblastoma when 6p22 genomic gain is present.

In addition, it is shown that, besides increase in the genomic copy number of 6p22, some

retinoblastoma cell lines exhibit translocations between chromosmal arms 6p and 6q, with a

recurrent translocation breakpoint at 6p22, in the vicinity of DEK and E2F3 loci, emphasizing

the involvement of chromosmal band 6p22 in the etiology of retinoblastoma. Based on the

work presented, DEK and E2F3 are the promising new targets for treatment and prevention of

retinoblastoma.

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TABLE OF CONTENTS

CHAPTER I: Introduction

1. INTRODUCTION AND OBJECTIVES 4

2. RETINOBLASTOMA GENETICS 6

2.1. The RB1 mutation and Knudson’s hypothesis 8

2.2. Oncogenes and tumor suppressor genes 9

2.3. Multistep model of retinoblastoma development: M1-Mn events

3. RECURRENT CHROMOSOMAL ABERRATIONS IN

RETINOBLASTOMA

3.1. Chromosomal regions of gain and loss identified by cytogenetic analysis

and comparative genomic hybridization

3.2. Isochromosome 6p

4. MINIMAL REGION OF GAIN AT 6P22

4.1. Mapping of the minimal region of 6p gain to chromosomal band 6p22 by

several CGH studies

4.2. Application of QM-PCR to narrow 6p22 region of gain to 0.6 Mb

5. CANDIDATE 6P22 ONCOGENES

5.1. DEK and other genes within 0.6 Mb minimal region of gain

5.1.1. DEK

5.1.1.1. DEK in transcriptional regulation

5.1.1.2. DEK in chromatin architecture maintenance

3

5.1.1.3. DEK and mRNA splicing

5.1.1.4. DEK and cancer

5.1.1.5. DEK and autoimmune diseases

5.1.2. AOF1

5.1.3. TPMT

5.1.4. NHLRC1

5.1.5. KIF13A

5.1.6. NUP153

5.2. E2F3 and other genes at 6p22.

5.2.1. E2F3

5.2.1.1. E2F3 in cell cycle control

5.2.1.2. E2F3 in transcriptional regulation

5.2.1.3. E2F3 and cancer

5.2.2. MBOAT1

5.2.3. ID4

5.2.4. IBRDC2

CHAPTER II: A detailed expression analysis of genes mapping to

the 6p22 minimal region of gain

1. ABSTRACT

2. INTRODUCTION

3. MATERIALS AND METHODS

3.1. Clinical samples

4

3.2. Cell lines

3.3. Mouse tumors

3.4. Antibodies

3.5. RNA extraction and reverse transcription

3.6. Real-time RT-PCR

3.7. Protein extracts

3.8. Immunoblotting

3.9. Immnufluorescent staining

4. RESULTS

4.1. mRNA expression of genes within the 6p22 minimal region of gain in

retinoblastoma

4.2. Correlation of genomic gain and overexpression

4.3. mRNA expression of Dek, Kif13a, Nup153 and E2f3 in mouse

retinoblastoma

4.4. Developmental regulation of Dek, Kif13a, Nup153 and E2f3 expression

4.5. Protein expression of candidate genes withing the 6p22 region of gain

4.6. Immunofluorescent staining for DEK and E2F3

5. DISCUSSION

6. ACKNOWLEDGEMENTS

5

CHAPTER III: Oncogenic potential of DEK and E2F3 in

retinoblastoma

1. ABSTRACT

2. INTRODUCTION

3. MATERIALS AND METHODS

3.1. Cell lines

3.2. Viral constructs

3.3. Antibodies

3.4. Infection and selection procedures

3.5. Test for the presence of replication competent lentivirus

3.6. Growth rates

3.7. RNA extraction and reverse transcription

3.8. Protein isolation

3.9. Real-time RT-PCR

3.10. Immunoblotting

3.11. Immunofluorescent staining and quantification of positive cells

3.12. BrdU incorporation

4. RESULTS

4.1. Screening Sigma Mission shRNA Library vectors for efficiency in DEK

and E2F3 knockdown in HeLa cells

4.2. DEK and E2F3 knockdown in retinoblastoma cell lines

6

4.3. Growth rates, proliferation index and caspase 3-dependent apoptosis of

retinoblastoma cell lines infected with anti-DEK shRNA-producing

lentiviral vectors

4.4. Growth rates, proliferation index and caspase 3-dependent apoptosis of

retinoblastoma cell lines infected with anti-E2F3 shRNA-producing

lentiviral vectors

5. DISCUSSION

CHAPTER IV: Novel 6p rearrangements a recurrent translocation

breakpoints in retinoblastoma identified by SKY and mBand

analyses

1. ABSTRACT

2. INTRODUCTION

3. MATERIALS AND METHODS

3.1. Cell lines and metaphase preparation

3.2. FISH

3.3. SKY

3.4. mBand analysis

4. RESULTS

4.1. Chromosome 6 rearrangements

4.1.1. Retinoblastoma cell line RB1021

7

4.1.2. Retinoblastoma cell line RB247c

4.1.3. Retinoblastoma cell line RB383

4.1.4. Retinoblastoma cell line Y79

4.1.5. Overall pattern of chromosome 6 rearrangements

4.2. Spectral karyotype analysis

5. DISCUSSION

6. ACKNOWLEDGEMENTS

CHAPTER V: DiscussionChapter Outline1. DEK AND E2F3 AS TARGETS OF 6P CHROMOSOMAL GAIN IN

RETINOBLASTOMA

2. EVIDENCE THAT DEK AND E2F3 EXPRESSION IN

RETINOBLASTOMA IS NOT THE CONSEQUENCE OF THE LOSS OF

FUNCTION OF RB1

3. DECREASE OF DEK OR E2F3 LEVEL NEGATIVELY AFFECTS THE

GROWTH OF RETINOBLASTOMA WHEN 6P GENOMIC GAIN IS

PRESENT

4. RECURRENT TRANSLOCATION BREAKPOINT AT 6P IN

RETINOBLASTOMA AS A MECHANISM OF ONCOGENE

ACTIVATION

5. FUTURE DIRECTIONS

5.1. Function of DEK and E2F3 in retinoblastoma

8

5.2. Identification of 6p translocation breakpoint

5.3. Mutational analysis of DEK and E2F3 in retinoblastoma

APPENDICES

REFERENCES

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CHAPTER I: Introduction

1. INTRODUCTION AND OBJECTIVES

The study of retinoblastoma, a rare childhood malignancy, established the fundamental

principles of genetic predisposition to cancer and cancer development, and led to the discovery

of the first tumor suppressor gene RB1. Retinoblastoma is initiated by the loss of function of

both alleles of the RB1 gene, in accordance with the Knudson’s two hit hypothesis. Mutations

targeting the RB1 gene are named mutational event 1 (M1) and mutational event 2 (M2). M1

and M2 are rate-limiting events in retinoblastoma formation and may be sufficient for the

development of a benign retinal tumor, retinoma. However, retinoblastoma tumors contain

additional recurrent genetic alterations, named mutational events M3-Mn, that target other

genes involved in disease development. Regions of recurrent chromosomal gain and loss in

retinoblastoma indicate locations of a fraction of potential oncogenes and tumor suppressor

genes, respectively, targeted by M3-Mn events. Identification of these genes is necessary for

elucidation of the molecular genetics of retinoblastoma and for invention of novel therapeutic

and prevention approaches.

Short arm of chromosome 6, 6p, is gained in approximately 50% of retinoblastoma

tumors, and is the second most frequently gained genomic region. The gain usually occurs

through the formation of an isochromosome i(6p). It is hypothesized that 6p harbors an

oncogene(s) involved in retinoblastoma development.

This thesis addresses the following points:

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1. Identification of the oncogene targeted by 6p22 gain through detailed comparative

expression analysis of genes in the minimal region of gain in retinoblastoma and healthy retina,

described in Chapter II.

2. Examination of the oncogenic potential of two 6p22 candidate genes, DEK and E2F3 in

retinoblastoma, described in Chapter III.

3. Investigation of chromosome 6 rearrangements besides copy number increase in

retinoblastoma by spectral karyotyping (SKY), mBand analysis for chromosome 6, and

fluorescence in situ hybridization (FISH) for DEK and E2F3, described in Chapter IV.

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2. RETINOBLASTOMA GENETICS

2.1. The RB1 mutation and Knudson’s hypothesis

Retinoblastoma belongs to familial cancer syndromes. 35-45% of retinoblastoma cases are

hereditary, and the predisposition to the disease is transmitted as a dominant Mendelian trait.

Over 60% of patients with the hereditary form of the disease develop bilateral multifocal

tumors, with an average number of three tumors per individual [1], a smaller percentage of

patients develop unilateral tumors (25-40%), and a very small percentage of disease gene

carriers remain unaffected (1-10%). Patients with a nonhereditary form of the disease always

develop unilateral monofocal tumors, and these patients are, on average, older at the time of

disease presentation than the patients with the genetic predisposition to retinoblastoma [1]. By

studying the statistical distribution of the number of tumors per patient with respect to the

existence of genetic predisposition, and by taking into account the probability of occurrence of

genetic mutations, Knudson showed that two mutational events were necessary for

retinoblastoma formation. This theory became famous as the ‘two-hit hypothesis’. In hereditary

cases, the first mutation occurs in the germline cell, and the second mutation occurs at the

somatic level. In nonhereditary cases, both mutations are somatic [1].

In several independent studies conducted in late 1970s, it was found that a small percentage

of patients with hereditary retinoblastoma had deletions within the long arm of chromosome 13

in their white blood cells, with the chromosomal band 13q14 consistently missing [2-8]. The

deletion of 13q14 was subsequently reported in hereditary tumors in which the deletion was

not detectable in white blood cells [9] and nonhereditary tumors [10, 11].

By linkage analysis, it was found that the retinoblastoma susceptibility gene, named RB1,

co-segregated with the gene encoding esterase D (ESD) [12, 13]. The linkage to ESD and study

12

of restriction fragment length polymorphisms (RFLPs) at 13q14 led to the elucidation of the

cellular recessive nature of the RB1 gene [14-16].

The RB1 gene was cloned by hybridization of genomic probes from 13q14 region to RNA

isolated from normal retina and retinoblastoma, which identified a ~4.7 kb transcript that was

absent in a fraction of retinoblastoma tumors. The cDNA probe was subsequently used to clone

the gene itself [17-19]. The identity of the isolated gene was subsequently confirmed by

identification of point mutations in RB1 cDNA in both retinoblastoma [20] and other cancers

[21], and functional studies confirmed the tumor suppressor role of the RB1 gene [22].

RB1 codes for a 110 kDa protein, pRB , which was found to be one of the key regulators of

the cell cycle progression, reviewed by Weinberg in 1995. In G0 and early G1 cells, pRB is

present in a hypophosphorylated form. The hypophosphorylated pRB binds the cell cycle

promoting E2F transcription factors – E2F1, E2F2 and E2F3, and represses transcription of

their target genes. In late G1, at the restriction point when the cell decides to commit to

division, pRB becomes hyperphosphorylated predominantly by cyclin D-dependent kinases,

CDK4 and CDK6. The phosphorylation of pRB releases the transcriptional block, and E2F1,

E2F2 and E2F3 are able to induce the transcription of genes needed for subsequent stages of

the cell cycle [23].

Besides controlling the major G1 checkpoint of the metazoan cell cycle, pRB is also

involved in cellular differentiation through promotion of cell-cycle exit and expression of

tissue-specific genes, and it is also implicated in the regulation of cellular senescence, reviewed

by Liu in 2004 [24].

2.2. Oncogenes and tumor suppressor genes

13

RB1 gene was the first identified member of a class of genes named tumor suppressors.

Tumor suppressor genes function as negative regulators of neoplastic disease, and the loss of

their function contributes to tumor development. When exogenously expressed in cancer cells,

tumor suppressor genes can cause the loss of tumorigenic potential [25].

The second tumor suppressor identified, and the most famous besides RB1 is p53. Germline

mutations in p53 are the underlying cause of the Li-Fraumeni familial cancer syndrome. The

main function of p53 is to act as a sensor of DNA damage and invoke a protective response

either by induction of growth arrest or apoptosis. p53 has therefore been named “the guardian

of the genome” and is the most frequently mutated gene in human neoplasms [26].

Contrary to tumor suppressor genes, oncogenes function in a dominant manner, and

positively regulate neoplastic growth. Introduction of oncogenes into nontransformed cells, in

the presence of two copies of normal alleles, can result in the acquisition of the transformed

phenotype, which may manifest itself through the loss of density-dependent growth, loss of

contact inhibition, reduced growth factor dependence, anchorage independent growth,

immortality, and/or abnormal differentiation [25].

Oncogenes were first identified in the early 1970s as part of the genome of acutely

transforming retroviruses [27]. It was subsequently discovered that retroviral oncogenes were

derived from normal cellular genes, named proto-oncogenes [28, 29].

Oncogenes may become activated through mutations that increase their expression level,

such as increase in the gene copy number or translocation of the coding sequence of the proto-

oncogene to a promoter of another gene. MYC was the first oncogene found to be amplified in

human neoplasms [30], and amplifications of many other oncogenes, such as MYB [31], KRAS

[32], and MDM2 [33] have since been documented. In Burkitt’s lymphoma, MYC is

translocated to promoters of immunoglobulin genes, resulting in its aberrant expression [34].

14

Alternatively, oncogenes may become activated through mutations that result in increased

activity of their protein product, or the acquisition of novel functions that are the consequence

of translocations that result in the formation of fusion proteins. For example, point mutations in

the HRAS proto-oncogene may affect the GTP-ase function of its protein product, resulting in a

constitutively active protein [35]. BCR/ABL fusion protein is the result of a translocation

between chromosomes 9 and 22, which leads to the formation of the Philadelphia chromosome

in chronic myelogenous leukemia. The BCR sequence present in the fusion protein serves as a

direct activator of the tyrosine kinase activity of ABL, thereby enabling the fulfillment of its

oncogenic role [36].

2.3. Multistep model of retinoblastoma development: M1-Mn events

NO: 1. Armitage P., Doll R. The age distribution of cancer and a multi-stage theory of carcinogenesis. Br J Cancer. 1954 Mar;8(1):1-12.2. Armitage P., Doll R. The age distribution of cancer and a multi-stage theory of carcinogenesis. Int J Epidemiol. 2004 Aug 19.

One of the first authors to propose the multistep nature of cancer development was

Ashley, who calculated that the common cancers were produced by 3-7 mutations, depending

on a specific cancer [37]. The multistep model of cancer development was supported by in

vitro studies which showed that introduction of one activated oncogene was insufficient to

induce transformation of primary rat embryo fibroblasts [38]. In vivo studies showed that

introduction of an activated oncogene through construction of transgenic mice led to cancer

formation only after a substantial latent period, and that only few clonal tumors were formed,

supporting the hypothesis that mutations in additional genes were necessary for tumorigenesis

[39, 40].

Colorectal cancer is an excellent model of multistep nature of tumor formation in

humans. Colorectal cancers evolve from small benign colon polyps (adenomas) over a period

15

of several years, and most tumors were shown to contain mutations in several oncogenes and

tumor suppressor genes [41].

By karyotype analysis of retinoblastoma tumors, it was shown that most tumors

contained recurrent chromosomal aberrations besides aberrations involving chromosomal band

13q14 [42, 43]. In addition, a fraction of retinoblastoma tumors was shown to contain MYCN

oncogene amplification [43, 44]. It was therefore proposed that the mutational events that

target RB1 gene (M1 and M2) were the initiating steps in retinoblastoma formation, but that

mutations in additional genes (M3-Mn) were necessary for tumor formation [45]. This is

supported by the recent finding of Dimaras et al. that a benign retinal tumor, retinoma, results

from inactivating mutations in both alleles of the RB1 gene (Figure 1.1), indicating that M1 and

M2 are not sufficient for malignant tumor development, but, in accordance with the Knudson’s

hypothesis, are rate-limiting events in retinoblastoma pathogenesis (Dimaras et al. manuscript

in preparation).

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RECURRENT CHROMOSOMAL ABERRATIONS IN

RETINOBLASTOMA

3.1. Chromosomal regions of gain and loss identified by cytogenetic

analysis and comparative genomic hybridization

The first karyotype studies of human retinoblastoma tumors were conducted in the early

1980s and consistently reported gain of chromosomal arm 6p through the formation of an

isochromosome i(6p), genomic gain of chromosomal arm 1q, and occasionally reported

deletions within chromosomal arm 13q [11, 42, 43, 46]. Frequent loss of chromosome 16 in

retinoblastoma was first reported in the study of 76 retinoblastoma tumors conducted by

Pogosianz and Kuznetsova [47].

Comparative genomic hybridization (CGH) was used to study genomic copy number

changes in retinoblastoma tumors in more detail. This technique requires less material than the

standard cytogenetic analysis, does not require live cells, and enables identification of smaller

regions of gain and loss compared to classic chromosomal banding techniques [48]. In five

independent CGH studies of retinoblastoma tumors, the most frequent genomic gains were

reported at chromosomal arms 1q, 6p and 2p, while the most frequent region of genomic loss

was reported at the chromosomal arm 16q [49-53].

In the study by Bowles et al. it was proposed that gains of 1q and 6p tend to occur

together, while gain of 2p showed negative correlation with the loss of 16q [54].

KIF14, a mitotic kinesin gene, was proposed as the candidate 1q oncogene in

retinoblastoma [55]. MYCN is the candidate 2p oncogene in retinoblastoma [43, 44], and high

level MYCN amplification is detected in 5-10% of retinoblastoma tumors [43, 56]. CDH11, a

17

member of the cadherin gene family was proposed as the candidate 16q tumor suppressor gene

in retinoblastoma [57].

3.2. Isochromosome 6p

The gain of the short arm of chromosome 6 represents one of the two most frequent

recurrent genomic gains in retinoblastoma, besides 1q gain. In the study by Cano et al.

genomic gain of 6p was associated with a more malignant phenotype of retinoblastoma,

expressed through the undifferentiated histology and invasion of the optic nerve [58]. Increase

in the copy number of 6p is usually achieved through the formation of an isochromosome

i(6p), which consists of two 6p chromosomal arms joined by a centromere, first reported by

Kuznetsova et al. [42][59]. The i(6p) is considered to be one of retinoblastoma hallmarks, since

it is present in the majority of tumors, and is rarely reported in other malignancies, such as

acute lymphoblastic leukemia [60] and meningeal melanoma [61].

Several mechanisms of isochromosome formation in cancer cells were proposed, and the

most widely accepted theory is the one of transverse instead of longitudinal centromere

division [62, 63]. Retinoblastoma tumors with 6p gain usually contain two normal

chromosomes 6 and one isochromosome, making them tetrasomic for the 6p region. Squire et

al. therefore proposed that mitotic nondisjunction must precede isochromosome formation

[59]. Horsthemke et al. performed a detailed analysis of the mechanism of isochromosome 6p

formation by determining the dosage of polymorphic alleles on chromosome 6 in

retinoblastoma tumors [64]. They found that the dosage of polymorphic alleles on

chromosomal arm 6p in tumors with 6p gain was 3:1, while the dosage of polymorphic alleles

on 6q arm in the same tumors was 1:1. re. In the second model, mitotic nondisjunction results

in the trisomy of chromosome 6, which is followed by intrachromosomal chromatid exchange

18

[64]. Both proposed mechanisms would result in the formation of two daughter cells, one with

isochromosome 6p and the other with isochromosome 6q, and the cell with i(6p) would

undergo positive selection [64].

Some retinoblastoma tumors are trisomic for 6p region [59], while some contain gains of

smaller portions of 6p [50-53], indicating, as proposed by Squire et al. [59] that gain of 6p and

not isochromosome formation per se is the critical event in retinoblastoma development.

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4. MINIMAL REGION OF GAIN AT 6p22

4.1. Mapping of the minimal region of 6p gain to chromosomal band 6p22

by multiple CGH studies

In order to identify the target of 6p gain in retinoblastoma, it was necessary to narrow down

the region of interest to focus on a limited number of candidate oncogenes. A small fraction of

retinoblastoma tumors with 6p gain do not gain the entire 6p arm. Since CGH has higher

resolution than the classical cytogenetic banding techniques, it was possible to delineate these

smaller regions of gain at 6p. In the study by Herzog et al. the minimal region of gain can be

mapped to 6p22-p25, based on 2/11 tumors with 6p gain [50]. In the study by Chen et al. the

region of gain was narrowed to chromosomal band 6p22, based on 5/23 tumors with 6p gain

[51]. In the study by Lillington et al. the region of gain was narrowed to 6p22-p23, based on

5/27 tumors with 6p gain [52]. In the study by van der Wal et al. the region of gain was

narrowed to 6p21.3-p22, based on 1/6 tumors with 6p gain [53]. If these CGH studies are

combined, it is obvious that the overlapping region for the four informative studies is the

chromosomal band 6p22.

4.2. Application of quantitative multiplex PCR to narrow 6p22 region of

gain to 0.6 Mb

Chromosomal band 6p22 spans ~15 Mb and contains over two hundred genes. To facilitate

the identification of the putative 6p22 oncogene, Chen et al. opted for the approach of reducing

the number of candidate genes by defining a smaller and hence more manageable minimal

region of gain (MRG) within 6p22 chromosomal band. The technique of the quantitative

multiplex PCR (QM-PCR) was applied, in which the copy numbers of UniSTS markers across

20

6p22 were determined in seventy retinoblastoma tumors. The UniSTS marker at 10q21 was

used as an endogenous control, since genomic copy number changes in this region have not

been reported in retinoblastoma [65]. It was proposed that the most frequently gained UniSTS

markers (authors used the copy number of 3 as the cutoff value for genomic gain) would define

a novel minimal region of gain. At the time, the number of available UniSTS markers was

limited, since the sequencing of the human genome was still not completed. In the first round

of QM-PCR, the authors used five UniSTS markers and identified the initial 5 Mb MRG

between UniSTS markers SHGC-130608 and WI-22629 (overlapping with the E2F3 locus).

The hotspot marker of gain was identified as SHGC-103950. In the second round of QM-PCR,

the authors determined the copy numbers of the hotspot UniSTS marker and additional four

UniSTS markers within 5 Mb MRG, two centromeric and two telomeric to SHGC-103950.

This resulted in defining an MRG that spanned only 0.6 Mb, between UniSTS markers WI-

19208 (overlapping with the NUP153 locus) and X64229 (overlapping with the DEK locus).

The hotspot marker of gain remained the same, and the authors subsequently cloned a novel

kinesin gene KIF13A as the gene that overlapped with SHGC-103950 [65].

Grasemann et al. used QM-PCR analysis of 76 retinoblastoma tumors to determine

genomic copy numbers of 13 UniSTS markers across 6p22, one UniSTS marker at 6p23 and

one at 6p21. Two UniSTS markers, one at 6q15 and one at 10q22 were used as endogenous

controls, since retinoblastoma tumors are usually disomic for 6q chromosomal arm and

changes in copy number of 10q have not been reported. Based on QM-PCR results, tumors

were divided into three groups: tumors with no 6p gain, tumors with gain of entire 6p,

consistent with the formation of i(6p), and tumors with partial gain of 6p. Most tumors with

partial 6p gain showed normal copy numbers for 6p21 and 6p23, confirming the location of the

MRG at 6p22. The most frequently gained UniSTS markers in tumors with partial 6p gain

were the ones that overlapped with DEK and E2F3 loci, with the hotspot markers of gain

21

overlapping with the DEK locus. The MRG was therefore identified as the 2.5 Mb region

between DEK and E2F3, and did not include KIF13A locus [66].

In the conclusion, DEK locus was the only overlap between minimal regions of 6p22 gain

in retinoblastoma identified in two independent QM-PCR studies (Figure 1.2) [65, 66].

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5. CANDIDATE 6p22 ONCOGENES

5.1. DEK and other genes within 0.6 Mb minimal region of gain

0.6 Mb minimal region of gain identified by the QM-PCR in the study performed by Chen

et al. (Figure 1.2) includes six genes. Going from the centromere, these genes are DEK

(overlapping with the UniSTS marker X64229, the hotspot marker of gain in the study

performed by Grasemann et al.), AOF1, TPMT, NHLRC1, KIF13A (the gene overlapping with

the hotspot marker of gain SHGC-103950 in the study by Chen et al.), and NUP153

(overlapping with the UniSTS marker WI-19208).

5.1.1. DEK

DEK codes for an abundant nuclear phosphoprotein implicated in cancer and in

autoimmune diseases, with suggested roles in transcriptional regulation, chromatin architecture

and mRNA splicing [67].

DEK protein contains two DNA binding regions: the SAF (scaffold attachment factor) box,

between amino acids 149 and 187, and another DNA binding region at the C-terminus,

between amino acids 270 and 350, which is also involved in DEK-DEK protein interactions

and therefore named multimerization domain (Figure 1.3) [68]. The NMR structure of the C-

terminal domain of DEK from amino acids 309 to 375 shows structural homology to the

E2F/DP family of transcription factors, since it has the topology of the winged helix motif

present in E2F and DP proteins, which is responsible for binding to the major groove of

dsDNA [69].

The multimerization domain contains multiple phosphorylation sites, and phosphorylation

of this region affects DNA-binding properties of DEK, as well as protein-protein interactions

23

in vitro [70]. Protein kinase CK2 phosphorylates the C-terminal DNA-binding region of DEK

in vitro and in vivo. The phosphorylation fluctuates during the cell cycle, reaching a moderate

peak in G1 [70]. In addition to phosphorylation, DEK also undergoes acetylation in vivo on

lysine residues within the first 70 N-terminal amino acids. Acetylation decreases the affinity of

DEK for DNA elements within the promoter and stimulates accumulation of DEK in

interchromatin granule clusters, which are known to contain RNA-processing factors [71]. This

suggests that post-translational modifications of DEK may explain the diverse roles that DEK

is implicated to play in the nucleus.

DEK promoter contains binding sites for transcription factors NF-Y and YY1, which were

confirmed by electromobility shift assays and directional mutagenesis [72]. Both NF-Y and

YY1 are associated with the stimulation of cellular proliferation [73-75] and cancer [76-78],

and YY1 transcriptional activity is suppressed by interaction with pRB [79]. Wise-Draper et al.

provided evidence that DEK expression was regulated by the pocket family of proteins [80], as

previously suggested [72]. DEK was recently reported as one of the genes that are part of the

signature of E2F3 overexpression in mouse embryonic fibroblasts [81], and binding of E2Fs to

DEK promoter in vivo and E2F-stimulated DEK expression was demonstrated [82].

5.1.1.1. DEK in transcriptional regulation

The first indication that DEK could be involved in transcriptional control was the discovery

that DEK binds to pets site of the HIV-2 enhancer which plays a crucial role in the response of

the HIV-2 enhancer to T-cell stimulation [83] and that DEK binding was regulated through a

signalling cascade involving protein kinase C and phosphoprotein phosphatase-2A [84].

DEK was also shown to interact with AP-2 and act as a transcriptional co-activator of

AP-2 downstream promoters [85]. AP-2 is a transcription factor that modulates the function

of MYC and is known to interact with pRB [86, 87] and YY1 [88]. DEK was also reported to

act as a transcriptional co-repressor in repressing NF-B activity [89].

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5.1.1.2. DEK in chromatin architecture maintenance

The role of DEK in chromatin architecture maintenance was first implicated by several

studies that used SV40 minichromosomes as a model system [90]. DEK was shown to induce

changes in chromatin topology through reducing the number of negative supercoils in a process

that was not DNA sequence specific and was not associated with histone displacement, but

required interaction of DEK with histone H2A/H2B dimers [91]. DEK preferentially binds to

supercoiled and four-way junction DNA in vitro, and it can bind protein-free DNA [92]. DEK

was shown to induce positive supercoils into topologically fixed circular DNA substrates and it

facilitates intermolecular ligation of linear DNA molecules by DNA ligase in vitro [93].

The majority of DEK protein in the nucleus was reported to be associated with chromatin

through oligonucleosomes, with DEK being present on both transcriptionally active and

inactive chromatin regions [94]. However, levels of DEK were shown to be 2- to 3-fold higher

in promoter-proximal sites of expressed genes. Induction of CD21 expression in B lymphocyte

cell line led to DEK accumulation on the CD21 promoter, suggesting that DEK accumulation

was functionally linked to gene expression [95]. DEK exhibits an inhibitory effect on p300-

and PCAF-mediated histone acetyltransferase activity and transcription, resulting in histone H3

and H4 hypoacetylation [96].

5.1.1.3. DEK and mRNA splicing

DEK was shown to associate with the SRm160 splicing coactivator in vitro, and it was also

detected in vivo in splicing factor-containing nuclear speckles upon the concentration of

SRm160 in these structures [97]. Immunodepletion of DEK did not affect the level of splicing

of pre-mRNAs, suggesting that DEK may be providing a function that is not directly related to

splicing activity [97]. This is contradicted by the finding that DEK enforces 3’ splice site

discrimination, and that DEK and its phosphorylation are required for intron removal, but not

for splicing complex assembly [98].

25

DEK interacts with splicing complexes through SR proteins, and remains bound to exon-

product RNA after splicing, suggesting that it is involved in the control of postsplicing steps of

gene expression [97]. This is supported by the finding that DEK is the part of the protein

complex deposited on mRNA 20-24 nt upstream of exon-exon junctions by the splicosome

[99]. The exon-exon junction complex is involved in mRNA export to the cytoplasm and in the

regulation of nonsense-mediated mRNA decay [100].

5.1.1.4. DEK and cancer

DEK was identified as the gene translocated to the nucleoporin CAN (NUP214) locus on

chromosome 9 in ~1% of acute myeloid leukemia (AML) patients [101], and this cytogenetic

rearrangement was found to be associated with an unfavourable prognosis [102]. The

translocation breakpoints always occur in the single intron of DEK gene, between codons 349

and 350, and a single intron of CAN gene, between codons 812 and 813 [101, 103], resulting in

the transcription of a chimeric 5.5 kb mRNA, containing the 5’ coding region of DEK and 3’

coding region of CAN, in which the ORFs of DEK and CAN are merged without disruption of

their original reading frames [101]. The resultant DEK-CAN fusion protein has a predicted

molecular mass of 165 kDa, and localizes to the nucleus [101].

DEK is overexpressed in AML in the absence of DEK-CAN translocation [104, 105], and

becomes down-regulated when AML cells are treated with a differentiation inducer [106].

DEK is overexpressed in the cell line derived from the aggressive phase of the T cell large

granular lymphocyte leukemia compared to the cell line derived from the chronic phase of

leukemia in the same patient [107]. DEK mRNA is expressed at a higher level in immature

than mature hematopoietic cells, with expression level correlating with cellular proliferation

[108]. DEK is also expressed in response to erythropoietin stimulation of peripheral blood

mononuclear cells [109].

26

DEK was reported to be overexpressed in hepatocellular carcinoma (HCC), with DEK

levels increasing with the histological grade [110, 111]. DEK expression was shown to be S-

phase dependent in HCC cell lines and that DEK was down-regulated upon differentiation

induction [110]. However, in HeLa cells the amount and localization of DEK do not change

during the cell cycle [94].

Overexpression of DEK was also reported in glioma [112], melanoma [82, 113], colon

cancer [82], larynx cancer [82], prostate cancer [114] and bladder cancer [82, 115]. In bladder

cancer, DEK was overexpressed in invasive compared to the early stage tumors [115], and

DEK locus was found to overlap with the hotspot marker of 6p22 genomic gain in bladder

cancer identified by QM-PCR [116]. In neuroblastoma, DEK was identified as the main tumor

antigen, which is capable of eliciting T cell response [117].

Grasemann et al. identified DEK as one of the two candidate 6p22 oncogenes in

retinoblastoma based on genomic copy number analysis of 6p22 loci by QM-PCR and

microarray RNA expression analysis [66]. In Chapter II of this thesis a similar finding is

presented, based on quantitative real-time RT-PCR analysis and protein expression analysis of

DEK in retinoblastoma and healthy retina [118]. In the Appendix 1 of this thesis it is shown

that DEK is overexpressed in retinoblastoma compared to the adjacent benign retinoma tumor

in 50% of examined cases (Dimaras et al. manuscript in preparation).

DEK is upregulated in mouse mammary epithelial cells transformed by the antisense-

mediated knockdown of the candidate tumor suppressor gene p33ING1, and downregulated in a

time-dependent manner upon p33ING1 overexpression from a recombinant adenoviral vector

[119].

DEK was proposed to act as the senescence inhibitor in cervical cancer cells infected with

the oncogenic HPV viruses [94, 120]. Wise-Draper et al. provided the first in vitro evidence of

the oncogenic activity of DEK. DEK overexpression in HeLa cells from a recombinant

27

adenovirus vector results in the increased number of colonies formed in the soft agar [80], and

DEK overexpression in primary human keratinocytes significantly prolonged their life span

[80]. DEK was also reported to protect both HPV positive cancer cells and primary human

cells from apoptotic death through inhibition of the p53 pathway [121], and DEK was

implicated in the p53-induced cell cycle arrest [122].

5.1.1.5. DEK and autoimmune diseases

DEK is an autoantigen in several autoimmune disorders: juvenile rheumatoid arthritis -

JRA [123-125], scleroderma [124], systemic lupus erythematosus [126-129], sarcoidosis [126,

128], adult rheumatoid arthritis [128], systemic sclerosis [128], and polymyositis [128]. DEK

binds to the conserved Y-box regulatory sequences in class II MHC gene promoters in a gene-

and allele-specific manner, suggesting that DEK could be involved in the pathogenesis of JRA

and other autoimmune disease by differential regulation of class II MHC expression [130]. The

role of DEK in inflammation is supported by the recent finding that DEK is actively secreted

by macrophages and found in the synovial fluid of patients with JRA, where it acts as an

extracellular chemoattractant [96].

5.1.2. AOF1

AOF1 gene codes for the amine oxidase (flavin containing) domain 1. Amine oxidases are

involved in the metabolism of monoamines, diamines and polyamines which are produced

endogenously, or absorbed through diet or as xenobiotic substances [131].

5.1.3. TPMT

TPMT gene codes for the thiopurine-S-methyltransferase, a cytosolic enzyme that catalyzes

methylation of thiopurine drugs which are used in the therapy of cancer and autoimmune

28

diseases. TPMT has been widely studies in pharmacogenetics, since the polymorphisms in this

gene determine the ability of patients to tolerate thiopurine drugs [132].

5.1.4. NHLRC1

NHLRC1 codes for a putative E3 ubiquitin ligase with a ring finger domain and six NHL

motifs, and is mutated in lafora progressive myoclonus epilepsy [133].

5.1.5. KIF13A

KIF13A codes for a kinesin gene involved in intracellular trafficking from the Golgi

complex to plasma membrane [134]. Chen et al. cloned human KIF13A as the gene that

overlapped with the hotspot marker of 6p22 genomic gain in retnoblastoma identified by QM-

PCR [65]. Although KIF13A is overexpressed in some retinoblastoma tumors, in Chapter II of

this thesis it is demonstrated that not all tumors with 6p gain overexpress KIF13A, excluding it

as the 6p22 oncogene candidate [118].

5.1.6. NUP153

NUP153 codes for the nucleoporin 153, a part of the nuclear pore complex. NUP153 is

involved in nuclear export and import and in nuclear membrane remodeling during mitosis

[135].

5.2. E2F3 and other genes at 6p22

The minimal region of gain identified in the QM-PCR study of 76 retinoblastoma tumors

by Grasemann et al. (Figure 1.2) includes, going from the centromere, E2F3 (overlapping with

29

the second most frequently gained UniSTS marker G67592), MBOAT1, ID4, IBRDC2, DEK

(overlapping with the hotspot marker of gain X64229).

5.2.1. E2F3

E2F3 gene belongs to the E2F family of transcription factors, which act as downstream

effectors of the pocket protein family and play a major role in cell cycle control. Similarly to

E2F1 and E2F2, E2F3 codes for a potent transcriptional activator that interacts with pRB, acts

as a promoter of cell cycle progression, and its expression is cell cycle regulated. The

oncogenic role of E2F3 in human malignancies has emerged in recent years [136]. E2f3 has

recently been implicated in the regulation of neuronal migration [137].

E2F3 gene was cloned in 1993 from a human cDNA library, by low-stringency

hybridization with the E2F1 cDNA probe [138]. It was demonstrated that E2F3 binds E2F

consensus sites in a manner similar to E2F1, and that it interacts with pRB in vivo [138].

E2F3 activity is negatively regulated by pRB and Rb1-deficient neuronal precursor cells

exhibit significant enhancement of E2f1 and E2f3 activity throughout differentiation[139].

E2f3 locus in mouse was found to code for two different transcripts, E2f3a and E2f3b.

E2f3a corresponds to the classical E2f3 – its expression is cell cycle regulated and it plays a

cell cycle promoting role. On the contrary, E2f3b is expressed in quiescent cells and is thought

to be involved in Rb1-dependent transcriptional repression of genes needed for exit from G0

[140]. E2f3a and E2f3b are transcribed from two different promoters, with the promoter of

E2f3b mapping to the first intron of E2f3a. Therefore, the first exon in E2f3a is different from

the first exon in E2f3b transcript, resulting in the absence of the N-terminal region and Cyclin

A binding site from E2f3b protein [140]. He et al. showed, however, that E2f3b was a

physiological target of Cyclin A [141]. By analysis of promoters used for transcription of

E2f3a and E2f3b, it was found that E2f3a promoter contained E2f-binding sites that were

30

shown to be involved in transcriptional repression during G0, most likely through the

formation of E2f-pRB complexes. E2f3a promoter also contains E-elements, which are known

Myc-binding sites, and Myc was shown to positively regulate E2f3a transcription. E2f3b

promoter contains Sp1 and Ets binding sites, and lacks E2f and Myc-binding sites, which is

constistent with the constant presence of E2f3b transcript in both quiescent and proliferating

cells (Figure 1.4) [142].

E2f3-/- mice, created by Humbert et al., are born at 1/3 of the expected frequency, implying

important role of E2f3 in normal embryonic development. Embryonic fibroblasts derived from

E2f3-/- mice show impairment in mitogen-induced transcriptional activation of numerous E2f-

responsive genes, including B-Myb, Cyclin A, Cdc2, Cdc6 and Dhfr [143]. Apart from

significant growth retardation, surviving E2f3-/- mice have no obvious histopathological defects,

but their life span is significantly reduced in comparison to wild type littermates, due to a

congestive heart failure. The phenotype of E2f3+/- mice is in between phenotypes of E2f3+/+ and

E2f3-/- mice, suggesting dose-dependency of E2f3 loss [144].

5.2.1.1. E2F3 in cell cycle control

Expression of either E2F1, E2F2 or E2F3 alone is sufficient to induce S-phase entry in

quiescent rat fibroblasts, independently of DP1 expression [145]. In primary human T

lymphocytes E2F3 protein is absent in arrested cells and the expression becomes detectable

only at G1/S transition [146]. Likewise, in mouse fibroblasts E2f3 protein is present at a very

low level or absent in G0 and G1 cells, and the levels increase during G1/S transition, with

E2f3 protein produced in S-phase cells being 40-fold more stable compared to E2F3 protein

isolated from asynchronously growing cells [147]. In myeloid cells, however, overexpression

of E2F3 alone is not sufficient for S-phase induction, but also requires the presence of IL-3

[148], suggesting that the cell cycle stimulating role of E2F3 may be tissue specific.

31

Leone et al. showed that the increase in transcription of E2F1, E2F2 and E2F3 in late G1 is

limited to the cells that are transitioning from the quiescent state into actively proliferating

state. Once the cells are actively proliferating, transcription levels of E2F1, E2F2, and E2F3 do

not change at different stages of the cell cycle, suggesting that the transcription of E2F1, E2F2

and E2F3 is growth regulated, but not cell cycle regulated [149]. However, protein levels of

both E2F1 and E2F3 fluctuate during the cell cycle in actively proliferating cells, and E2F3,

but not E2F1, DNA-binding activity in actively proliferating cells is cell cycle regulated,

suggesting the involvement of posttranscriptional regulation [149], possibly through ubiquitin-

dependent protein degradation [150, 151]. CyclinA/CDK2-dependent phosphorylation is the

proposed mechanism of regulation of DNA binding activity of E2F3 [149]. Both E2F1 and

E2F3 share the CyclinA/CDK2-binding motif, and it was demonstrated that binding of

CyclinA/CDK2 to the N-terminus of E2F1 leads to phosphorylation of the E2F1-associated

DP1 protein, resulting in the inactivation of E2F1 DNA-binding activity [152-155]. Decline in

E2F3 DNA-binding activity during the S-phase coincides with accumulation of Cyclin A-

dependent kinase activity, while the reappearance of the E2F3 DNA-binding activity in the

next G1 coincides with the decline in Cyclin A-dependent kinase activity as cells go through

G2/M [149]. Consistent with the cell cycle dependent regulation of E2F3 but not E2F1 DNA-

binding activity, it was shown that E2F3 but not E2F1 is necessary for the S-phase induction in

actively proliferating rat fibroblasts [149].

Through experiments conducted on E2f1-/-, E2f2-/- or E2f3-/- primary mouse embryo

fibroblasts it was found that E2f2 and E2f3 were required for Myc-induced S-phase entry [156].

Triple knockout of E2f1, E2f2 and E2f3 genes results in elevated levels of p21Cip1 protein,

leading to decrease in cyclin-dependent kinase activity and, consequently, decrease in pRB

phosphorylation, which completely abolishes the ability of mouse embryonic fibroblasts to

enter S phase, progress through mitosis and proliferate [157].

32

E2f3 is proposed as the link between DNA and centrosome duplication cycles, since

inactivation of E2f3 in mouse embryo fibroblasts results in the disruption of centrosome

duplication cycle, leading to centrosome amplification, mitotic spindle defects and aneuploidy

[158].

5.2.1.2. E2F3 in transcriptional regulation

E2F3 belongs to a helix-loop-helix family of transcription factors. It binds to DNA as a

heterodimer, in association with DP proteins, and its DNA-binding activity is restricted to

G1/S transition and S-phase of the cell cycle [147].

Cell cycle promoting E2Fs (E2F1, E2F2 and E2F3) stimulate transcription of a number of

genes needed for DNA replication, such as DNA polymerase (POLA2), PCNA and

ribonucleotide reductase (RRM1), and their role resembles the role of SWI4/6/MBF

transcription factors in budding yeast and the role of Cdc10 in fission yeast. E2F3 is involved

in the regulation of MYB expression [159], and was implicated as the main activator of CDK2

transcription [160].

E2F3 and E2F2 were shown to activate CDC6 transcription at G1/S transition through

interaction with YY1 or RYBP transcription factors [161]. E2Fs, including E2F3, interact with

SP1 transcription factor both in vitro and in vivo, and this interaction may be needed to

transmit an activation signal from SP1 to the basic transcription machinery [162]. By yeast

two-hybrid screen and subsequent immunoprecipitation studies, it was found that E-box

binding factor TFE3 specifically interacts with E2F3, through marked box domain of E2F3,

leading to the synergistic activation of transcription of the p68 subunit of the DNA polymerase

[163]. TFE3 was also found to be involved in the regulation of cyclin E expression in an

E2F3-dependent manner [164].

33

E2F1, E2F2 and E2F3 were shown to bind to MYCN promoter in neuroblastoma cell lines.

Inhibition of E2F activity through overexpression of p16INK4a tumor suppressor led to decreased

MYCN expression in neuroblastoma cell lines with MYCN amplification [165].

Along with E2f1 and E2f2, E2f3 positively regulates the expression of the fibroblast

growth factor receptor-2 (Fgfr2) in mouse fibroblast NIH 3T3 cells [166].

In mouse embryonic fibroblasts, E2f1, E2f2 and E2f3 positively regulate the expression of

survivin, a candidate oncogene implicated in tumor progression and resistance to therapy in

various cancer types [167].

E2f3b was proposed as the repressor of p19Arf tumor suppressor gene transcription in

normal cells. In cells with abnormal E2f3 levels, p19Arf expression is increased, either due to

loss of transcriptional repression by E2f3b, when E2f3 levels are low, or induction of p19Arf

transcription by E2f3a, thereby controlling for abnormal cellular proliferation [168].

E2F3 positively regulates that transcription of a polycystronic cluster of mi-R-17-92

miRNAs which is oncogenic in the mouse model of Burkitt’s lymphoma [169, 170]. miR-20

miRNA from this cluster was found to regulate translation of E2F1 and E2F3 by binding to 3’

UTRs of their mRNAs. E2F3 is negatively regulated by miR-34a miRNA, which has a

proposed tumor suppressor role in neuroblastoma [171].

5.2.1.3. E2F3 and cancer

The first indication of the oncogenic potential of E2F3 came from the study in which E2f3

was overexpressed from a retroviral vector in mouse fibroblast cell line NIH 3T3. E2f3, as well

as E2f1 and E2f2 tested positive in the anchorage independent growth assay, by enabling

growth of stably infected NIH 3T3 cells in soft agar. In addition, these three E2f factors also

tested positive in the saturation-density growth assay, since the NIH 3T3 cells that

overexpressed any of the three E2fs grew to much higher saturation density than the control

sample [172].

34

Overexpression of either E2F1, or E2F2 or E2F3 in rat fibroblasts results in the

overcoming of the G1 cell cycle arrest. induced by p16INK4A tumor suppressor gene [145],

relieves the mitogen requirement for entry into S-phase of the cell cycle [173], and leads to the

significant shortening of the G0 and G1 phase [173].

In E2f3 -/- mouse fibroblasts, E2f3 is not required for immortalization, but is rate-limiting

for proliferation of the resulting tumor cell lines [143].

Knocking out E2f3 in Rb1-/- background inhibits aberrant proliferation in lens and central

nervous system of mouse embryos, and reduces aberrant proliferation in the peripheral nervous

system by 65% [174, 175].

p19ARF, a mouse homologue of human tumor suppressor gene p14INK4a, which suppresses

growth of cells lacking p53 [176, 177], was found to complex with E2f1, E2f2 and E2f3 and

reduce their levels, through a mechanism involving protein degradation, suggesting that this is

the mechanism of p19ARF tumor suppressor function when p53 is absent [178].

In Rb1+/- mice, knockout of E2f3 significantly prolongs the life span by suppressing the

development of pituitary tumors that are the most frequent cause of premature death. However,

Rb+/-; E2f3-/- mice show an increased incidence of medullary thyroid carcinomas [179].

Overexpression of E2f3 from an inducible promoter in Rb1+/+ mice leads to hyperproliferation

of melanotrophs in the pituitary gland, but is not sufficient for pituitary tumor initiation, since

the hyperproliferating cells enter an irreversible senescence-like state [180].

E2f3 inactivation in Rb-/- chimeric mice completely suppresses the pulmonary

neuroendocrine hyperplasia which is thought to represent the preneoplastic lesion of the small-

cell lung carcinoma [181]. E2f1, E2f2 and E2f3 are required for oncogene-mediated

transformation of mouse embryonic fibroblasts, and E2f1-3 mediated negative regulation of the

p53-p21Cip1 tumor suppressor axis is critical for cell cycle progression and cellular proliferation

[182].

35

E2f3a was found to be weakly oncogenic on its own when overexpressed from a keratin-5

promoter in transgenic mice, leading to an increased number of epidermal malignancies [183].

E2F3 overexpression in human neoplasms was first reported in Wilms’ tumor [184], and

was also reported in ovarian cancer [185] and lung cancer [186].

By array CGH, E2F3 was identified as the part of the 6p22 amplicon in bladder cancer

[187], with the portion of tumors that overexpress E2F3 increasing with the histopathological

grade [188, 189], E2F3 overexpression being strongly associated with the expression of the

proliferation marker Ki67 [189]. Knockdown of E2F3 by siRNA in bladder cancer cell line

containing the 6p22 amplicon reduced the level of BrdU incorporation and the rate of cellular

proliferation [190, 191].

E2F3 is overexpressed and is an independent prognostic indicator in prostate cancer [192].

Overexpression of E2F3a boosts BrdU incorporation in prostate cancer cell lines with the

functional loss of RB1, but has not effect on BrdU incorporation in prostate cancer cell lines

with intact RB1 [191].

E2F3 was found to map to the minimal region of 6p22 gain in retinoblastoma identified by

Grasemann et al., and was found to be overexpressed in retinoblastoma at the mRNA level

[66]. In Chapter II of this thesis, E2F3 overexpression in retinoblastoma at both mRNA and

protein level is shown [118]. And in chapter III…much more data…

Check out Wenzel P. L., Wu L., de Bruin A., Chong J. L., Chen W. Y., Dureska G., Sites E., Pan T., Sharma A., Huang K., Ridgway R., Mosaliganti K., Sharp R., Machiraju R., Saltz J., Yamamoto H., Cross J. C., Robinson M. L., Leone G. Rb is critical in a mammalian tissue stem cell population. Genes & development. 2007 Jan 1;21(1):85-97. Highly relevant

5.2.2. MBOAT1

36

MBOAT1 is a newly discovered gene that codes for the protein involved in transport of

organic compounds across plasma membrane [193].

5.2.3. ID4

ID4 is the member of the ID family of proteins which function as negative regulators of

basic helix-loop-helix transcription factors, and are implicated in cellular proliferation,

differentiation and cancer, with different roles being ascribed to different ID factors [194]. ID4

is a putative tumor suppressor in colorectal cancer [195], leukemia [196], lymphoma [197] and

breast cancer [198, 199]. 2006).

5.2.4. IBRDC2

IBRDC2 was recently identified as the gene that codes for a novel p53-regulated protein,

p53RFP, which acts as a switch between p53-dependent growth arrest and apoptosis, and is an

apoptosis stimulator [200].

37

CHAPTER II:

Expression analysis of 6p22 genomic gain in retinoblastoma

This majority of this chapter was published as follows:

Orlic, M., Spencer, C.E., Wang, L., and Gallie, B.L. Expression analysis of 6p22 genomic

gain in retinoblastoma. (2006). Genes Chromosomes Cancer. 45(1):72-82

Section 4.5 is in preparation by Pajovic et al., while section 4.8 in preparation by Dimaras et al.

38

1. ABSTRACT

To identify gene(s) targeted by 6p22 genomic gain, present in more than 50%

retinoblastoma tumors, we used real-time RT-PCR to quantify the expression of seven genes in

normal human retina and retinoblastoma. Six genes are located in the quantitative multiplex

PCR-defined 0.6 Mb minimal region of gain at 6p22 (DEK, AOF1, TPMT, NHLRC1, KIF13A,

and NUP153), and E2F3 is 2 Mb away from the minimal region of gain on 6p22. E2F3, DEK,

KIF13A, and NUP153 were most frequently overexpressed in retinoblastoma with 6p genomic

gain, compared with the normal adult human retina. E2F3 and DEK mRNA levels were

increased in all human tumors showing 6p22 gain, as well as in mouse retinoblastoma induced

by SV40 large T antigen expression in developing retina, compared with the normal controls

(adult human retina and 7-day-old mouse retina, respectively). Only DEK showed statistically

significant correlation of expression and genomic copy number (P = 0.019). E2F3 and DEK,

but not NUP153, showed developmental regulation. E2F3 and DEK mRNA overexpression

was always associated with protein overexpression, determined by immunoblotting or

immunofluorescent staining of primary tumors, relative to the adjacent normal retina. E2F3

was strongly expressed in actively proliferating cells, while DEK was overexpressed in all

tumor cells. Taking into account the proliferation-promoting role of E2F3, implication of E2F3

in bladder and prostate cancer, and the translocation and overexpression of DEK in leukemia,

we conclude that either DEK or E2F3 (or both) are targeted by the 6p22 gain in retinoblastoma.

39

2. INTRODUCTION

Retinoblastoma, the cancer of developing retina, is initiated by two mutational events,

M1 and M2, which result in the loss of function of both alleles of the RB1 tumor suppressor

gene. Additional mutational events (M3-Mn) in other tumor suppressor genes or oncogenes

may be important for the development of malignancy and disease progression [45]. One of the

two most frequent genomic gains, discovered in the early 1980s by karyotypic studies [43], is

the gain of the short arm of chromosome 6 (6p), usually in the form of isochromosome 6p [59].

In CGH studies [49-52, 201], 6p gain was present in 50-70% retinoblastoma tumors, with the

minimal region of gain (MRG) mapping to chromosome band 6p22 [51].

Using quantitative multiplex PCR (QM-PCR), we further narrowed the MRG on 6p22 to

a 0.6 Mb region spanning the UniSTS markers X64229 and WI-19208 [65]. A novel kinesin

family member KIF13A (named RBKIN) was cloned as the gene that overlapped with the

hotspot marker of gain, SHGC-103950, which was gained in >60% tumors. Initial studies of

KIF13A showed high mRNA levels in retinoblastoma and decreased growth rates of two

retinoblastoma cell lines upon KIF13A antisense treatment [65]. However, quantitative

expression analysis of the whole MRG was needed to determine whether KIF13A was the only

gene affected by the genomic gain.

We hypothesized that the gene(s) targeted by the 6p22 genomic gain to contribute to

cancer progression would be overexpressed at the mRNA and protein levels in human

retinoblastoma tumors with documented 6p22 gain. Developmentally regulated expression and

overexpression in mouse retinoblastoma tumors would further support a role of the gene in

retinal cancer.

We now show that KIF13A is not the most likely gene at 6p22 to be involved in

progression to retinoblastoma after loss of both RB1 alleles. The DEK oncogene and E2F3,

40

which is 2 Mb away, are the most highly overexpressed 6p22 genes in human retinoblastoma

tumors. Similar high expression of Dek and E2f3 is observed in the murine retinoblastoma that

arises in transgenic mice (TAg-RB mice) that express the SV40 large T antigen transgene from

an unidentified integration into chromosome 4 [202]. Expression of both of these genes is

developmentally regulated in the mouse retina, with higher levels in the immature retina.

Therefore, we identify E2F3 and DEK as the most likely 6p22 oncogenic candidates.

41

3. MATERIALS AND METHODS

3.1. Clinical samples

The use of retinoblastoma tumors for research was approved by parental consent and

the Research Ethics Board of the Wellesley Hospital, the Hospital for Sick Children, the

University Health Network (UHN) and the University of Toronto. Tumor specimens were

obtained from patients treated by enucleation of the affected eye. Research studies were

conducted after completion of all clinical tests. Anonymous use of normal human retinas,

isolated from eyes obtained from the Eye Bank of Canada, was approved by the Research

Ethics Board of the UHN.

3.2. Cell lines

Four retinoblastoma cell lines (Y79, WERI, RB247, and RB1021) were grown for

DNA, RNA, and protein harvesting at 37°C, in 5% CO2. The media used was Iscove's

medium, with 15% fetal clone serum, 10 g/ml insulin, 55 M -mercaptoethanol, and 100

U/ml penicillin-streptomycin.

3.3. Mouse tumors

Tumors were obtained from transgenic mice (TAg-RB mice) that were originally

developed as a pituitary adenoma model, expressing SV40 large T antigen from the LHB

promoter. One line of mice in which the SV40 large T antigen is expressed in an inner nuclear

layer retinal cell from a transgene integration in an unidentified locus on chromosome 4

develops aggressive, multifocal retinal tumors that histologically and immunologically

42

resemble human retinoblastoma [202]. All animals were studied using protocols approved by

the Animal Care Committee of the Ontario Cancer Institute.

3.4. Antibodies

Antibodies used for immunoblotting of E2F3 (specific for E2F3a), KIF13A, and

NUP153 were purchased from Santa Cruz Biotechnology, Santa Cruz, CA (sc-879, sc-16787,

and sc-20590). Antibodies used for immunofluorescent staining of E2F3 and immunoblotting

and immunofluorescent staining of DEK were purchased from Upstate, Charlottesville, VA

(05-551) and BD Biosciences, Franklin Lakes, NJ (610948). Antibodies used for

immunofluorescent staining of PCNA and TUBB immunoblotting were purchased from Sigma

Aldrich, St. Louis, MO (P 8825 and T 4026). Anti-DEK rabbit serum used for

immunofluorescent staining of Dek in mouse retina and retinoblastoma was developed at the

University of Konstanz, Germany and kindly provided by Dr. Ferdinand Kappes. Anti-TAg

antibody used for immunofluorescent staining of large T antigen of the SV40 was purchased

from Santa Cruz Biotechnology, Santa Cruz, CA, USA (…..). Rabbit anti-goat IgG AP-

conjugated antibody was purchased from Sigma-Aldrich (A-4062), goat anti-rabbit IgG AP-

conjugated antibody was purchased from Bio-Rad Laboratories, Hercules, CA (170-6518), and

goat anti-mouse IgG AP- and HRP-conjugated antibodies were purchased from Santa Cruz

Biotechnology (sc-2008 and sc-2005). Horse biotinylated anti-mouse IgG antibody and anti-

rabbit IgG antibody, used for immunofluorescent staining, were purchased from Vector

Laboratories, Burlingame, CA (BA-2000 and ___, respectively). NBT/BCIP Stock Solution

(Roche Applied Science, Mannheim, Germany; 1681451) was used for detection of AP-

conjugated secondary antibodies. HRP-conjugated secondary antibodies were detected by

43

Western LightningTM Chemiluminescence Reagent Plus (Perkin-Elmer Life Sciences,

Wellesley, MA; NEL104).

3.5. RNA extraction and reverse transcription

Total RNA was prepared by Trizol extraction except for samples RB462 and RB858,

for which it was prepared by the guanidium isothiocyanate-phenol chloroform method. Single-

stranded cDNA was produced by reverse transcription of the total RNA with SuperScript II

reverse transcriptase (Invitrogen, Carlsbad, CA) and random hexamer primers.

3.6. Real-time RT-PCR

Real-time RT-PCR analyses of E2F3, DEK, AOF1, TPMT, NHLRC1, KIF13A, and

NUP153 were performed using TaqMan® chemistry. TaqMan Gene Expression Assays for

human and mouse E2F3 (Hs01076037_m1, Mm01138828_m1, specific for E2F3a transcript),

DEK (Hs00180127_m1, Mm00662582_m1), KIF13A (Hs00223154_m1, Mm00660179_m1),

and NUP153 (Hs00185037_m1, Mm00723665_m1), human AOF1 (Hs00400708_m1), human

TPMT (Hs00795478_s1), human NHLRC1 (Hs01112790_s1), human HPRT

(Hs99999909_m1), and mouse Tbp (Mm00446973_m1) were purchased from Applied

Biosystems, Foster City, CA. After validation of equivalent amplification efficiency for human

E2F3, DEK, AOF1, TPMT, NHLRC1, KIF13A, and NUP153 relative to HPRT endogenous

control and mouse E2f3, Dek, Kif13a, and Nup153 relative to Tbp endogenous control through

amplification of dilution series, real-time RT-PCR was performed in 12.5 l reaction volumes

in 384-well clear plates on an ABI Prism SDS 7900HT, in triplicate. Reaction mixture

contained 1× TaqMan primer-probe mix, 1× TaqMan Universal PCR Master Mix including

AmpErase UNG (Applied Biosystems), and 10 ng of first-strand RT product. UNG was

44

activated by a 2-min incubation at 50°C, followed by a 10-min incubation at 95°C to activate

the polymerase, and 40 cycles of 15 sec at 95°C and 1 min at 60°C. Ct relative expression

values, normalized to HPRT and Tbp for human and mouse samples, respectively, were

calculated using the SDS 2.1 software and calibrated to the mean Ct value of all normal adult

human retinas for human retinoblastoma, normal mouse retina at P7 for mouse retinoblastoma,

and whole eye for mouse developmental expression. Microsoft Excel was used to plot the data.

3.7. Protein extracts

Whole cell protein extracts of normal human retinas, retinoblastoma tumors, and cell

lines were prepared by repeated freezing and thawing of mechanically dissociated tissue or

harvested cells in 1× Lysis Buffer (20 mM HEPES, pH 7.2, 100 mM KCl, 1 mM MgCl2, 0.1

mM EDTA, 7% glycerol, 100 M sodium orthovanadate, 0.2 mM PMSF, and 1 g/ml of each

leupeptin and aprotinin).

3.8. Immunoblotting

Immunoblotting for DEK, KIF13A, and NUP153 was performed by PAGE of 60 g of the

whole cell protein extract, followed by transfer of resolved proteins to a PVDF membrane. For

E2F3, 20 g of the whole cell protein extract was loaded. TUBB was used as the loading

control. DEK, E2F3, and TUBB primary antibodies were used at 1:200 dilution in 1%

BSA/TBST. KIF13A and NUP153 primary antibodies were used at 1:100 dilution in 5%

Blotto/TBST. Incubation with primary antibodies was carried out for 1 hr at room temperature.

Anti-mouse IgG secondary antibodies were used at 1:5,000 dilution in 1% BSA/TBST. Anti-

goat IgG secondary antibody was used at 1:20,000 dilution in 1% BSA/TBST. Incubation with

secondary antibodies was carried out for 30 min at room temperature.

45

3.9. Immunofluorescent staining

Enucleated eyes were fixed in 4% formaldehyde, paraffin embedded, and sectioned at 5

m. Prior to the immunofluorescent staining, paraffin removal from microscope slides and

tissue hydration was performed by xylene (two times of 10 min each) and ethanol washes (two

times of 5 min each in 100% ethanol, once for 2 min in 95%, 70%, and 50% ethanol) and 5

min incubation in 1× TBS. For the heat-mediated antigen retrieval, slides were put in a PBS

citrate solution, and heated in a microwave pressure cooker for 16 min at the power level 10

and 5 min at the power level 7. Blocking was carried out for 30 min at room temperature, in a

humidified chamber, using a 10% DAKO Protein Block (X0909) solution in 1% BSA/TBST.

Primary antibodies and biotinylated secondary antibodies were used at 1:200 dilution in 1%

BSA/TBST, with 10% Antibody Diluent (DAKOCytomation, Glostrup, Denmark; S3022), and

the incubation was performed for 1 hr at room temperature. Streptavidin-Alexa488 was used as

the detection reagent for E2F3 and DEK staining. Streptavidin-Alexa594 was used as the

detection reagent for PCNA staining. DAPI was used to visualize nuclei of retinal cells. Slides

were mounted using the DAKOCytomation Fluorescent Mounting Medium (S3023).

46

4. RESULTS

4.1. mRNA expression of genes within the 6p22 minimal region of gain in

retinoblastoma

To determine the genes overexpressed in human retinoblastoma with genomic gain of

6p22, we studied the mRNA expression of 6 genes (DEK, AOF1, TPMT, NHLRC1, KIF13A,

and NUP153) in the MRG and E2F3, which is 2 Mb away from the MRG (Figure 2.1) in

normal adult human retina and retinoblastoma, using real-time RT-PCR. E2F3 was included in

the analysis because it is a 6p22 gene whose role in the RB1 pathway of cell-cycle control is

well established [140, 142, 203], it lies within the initial 5-Mb MRG determined by the QM-

PCR, and recent reports implicate it as an oncogene in bladder and prostate cancer [188, 189,

192].

Overexpression was scored when a gene was expressed in retinoblastoma more than

three SDs above the mean of normal adult retina (Table 2.1, Figure 2.2). Overexpression was

detected for E2F3 in 13/13, for DEK in 11/13, and for NUP153 in 9/13 retinoblastoma tumors

and cell lines. The increase in expression ranged from 4- to 33-fold for NUP153, 4- to 35-fold

for DEK, and 4- to 360-fold for E2F3. NHLRC1 was overexpressed in 7/13, AOF1 and KIF13A

were overexpressed in 6/13, and TPMT was overexpressed in 3/13 retinoblastoma tumors and

cell lines.

4.2. Correlation of genomic gain and overexpression

Statistical analysis was performed to determine the correlation between the genomic

copy number of the hotspot marker, SHGC-103950, and the relative level of gene expression

determined by real-time RT-PCR in human retinoblastoma. Of the seven examined genes, only

47

DEK showed statistically significant correlation (P = 0.019) between the genomic gain and the

transcript level increase (Table 2.2).

Only E2F3, DEK, and NUP153 were overexpressed, relative to the normal adult human

retina, in all tumors (5/5) with documented 6p22 genomic gain (Table2.1, Figure 2.2). E2F3

was also overexpressed in all (4/4) tumors and cell lines with normal genomic copy numbers of

the hotspot marker, SHGC-103950. DEK was overexpressed in 2/4 tumors with normal

genomic copy number of SHGC-103950. TPMT was overexpressed in only 1/5, and AOF1 and

NHLRC1 were overexpressed in only 2/5 tumors and cell lines with SHGC-103950 genomic

gain. KIF13A expression was increased in 3/5 tumors with documented 6p22 genomic gain.

However, in the cell line RB1021, which has five copies of the SHGC-103950 marker, there

was only a threefold relative increase in the amount of KIF13A transcript (Table 2.1). In

summary, only three genes, E2F3, DEK, and NUP153, were overexpressed at the mRNA level

in all retinoblastoma tumors with genomic gain of 6p22.

4.3. mRNA expression of Dek, Kif13a, Nup153 and E2f3 in mouse

retinoblastoma

The structure of the 6p22 MRG is conserved in the mouse genome, occupying 0.4 Mb

on mouse chromosome 13. E2f3 maps to the same chromosome, but is 17 Mb away from the

region homologous to the human 6p22 MRG. We examined the expression of three MRG

genes (Dek, Nup153, and Kif13a) and E2f3 in retinoblastoma tumors obtained from TAg-RB

transgenic mice. RNA isolated from the retina at the postnatal day 7 (P7) was used as the

control, since large T antigen starts to express at P8. We found that expression of Dek was

increased in 3/3 TAg-RB tumors (four- to fivefold range), relative to normal P7 mouse retina.

E2f3 levels were approximately twofold higher in all examined tumors than in the normal P7

48

mouse retina (Figure 2.3). Nup153 showed no difference in expression levels between normal

P7 retina and retinoblastoma, while Kif13a levels were fivefold lower in tumors, compared to

the P7 control (Figure 2.3). These results are consistent with only Dek and E2f3 being

oncogene candidates in mouse retinoblastoma.

4.4. Developmental regulation of Dek, Kif13a, Nup153 and E2f3 expression

Since oncogenes are often developmentally regulated [25], we applied real-time RT-

PCR to examine mRNA expression of Dek, Kif13a, Nup153, and E2f3 in the mouse retina

isolated from 15- and 18-day-old embryos (E15 and E18), at the day of birth, and second and

seventh postnatal day (P0, P2, and P7), and from adult mouse (AMR).

E2f3, Dek, and Kif13a demonstrate expression patterns consistent with developmental

regulation. Nup153 mRNA levels do not vary significantly during mouse retinal development

(Figure 2.4).

4.5. Immunofluorescent staining for Dek in mouse retinoblastoma

Expression of Dek in the cell of origin of mouse retinoblastoma and in mouse

retinoblastoma tumors was examined by double immunofluorescent staining for Dek and large

T antigen of the SV40 virus (TAg). At P9, one day after the expression of TAg is first detected

by immunofluorescent staining, Dek expression is also detectable in the TAg-expressing cells,

at the level similar to the level observed in adjacent TAg-negative cells of the inner nuclear cell

layer. This indicates that the cell of origin of mouse retinoblastoma expresses Dek (Figure 2.5).

At four weeks of age, TAg positive retinal cells form small tumors in the inner nuclear cell

layer of the retina. Double immunofluorescent staining for Dek and TAg in these tumors shows

49

that a portion of tumors overexpress Dek at the protein level, compared with the adjacent

unaffected retina (Figure 2.6).

4.6. Protein expression of candidate genes within the 6p22 region of gain

To determine the genes overexpressed at the protein level, we examined DEK, KIF13A,

NUP153, and E2F3 expression in human retina and retinoblastoma by immunoblotting. E2F3

was overexpressed in 10/10 retinoblastoma tumors and cell lines, relative to the normal adult

human retina. A moderate increase in KIF13A protein level was detected in 4/10

retinoblastoma tumors and cell lines, compared with the healthy retina control (Figure 2.7).

Elevated DEK protein was evident in 2/9 retinoblastoma tumors and cell lines, while NUP153

was overexpressed in 1/9 retinoblastoma tumors, relative to the normal adult human retina

(Figure 2.7). Tumor RB2175 overexpressed mRNA of all examined genes, but showed

increased protein expression only for E2F3 (Figure 2.7, Table 2.3). We conclude that, on the

basis of immunoblotting results, only E2F3 is overexpressed at the protein level in all tumors

with genomic gain of 6p22.

4.7. Immunofluorescent staining for DEK and E2F3 in retinoblastoma

We compared the spatial expression of DEK and E2F3, the two most promising 6p22

oncogene candidates, in normal human retina and retinoblastoma by immunofluorescent

staining of five eyes enucleated to treat advanced retinoblastoma. In the normal retina, DEK is

expressed in all three retinal cell layers: ganglion cell layer (GCL), inner nuclear cell layer

(INL), and the outer nuclear cell layer (ONL), with the highest expression in photoreceptor

cells (Figure 2.8A). A weak signal for E2F3 was detectable in all three retinal cell layers of the

normal retina (Figure 2.8B). Retinoblastoma stained more strongly for both DEK and E2F3

50

than the adjacent normal retina (Figure 2.8 and 2.8 D). More tumor cells were DEK positive

than E2F3 positive. We compared the proliferative state of tumor cells with E2F3 expression

by double staining for E2F3 and the proliferation marker, PCNA. DAPI was used to visualize

nuclei of all cells present in the section. Since high E2F3 expression coincided with PCNA

expression in tumor cells, we concluded that E2F3 expression is limited to actively

proliferating cells, while DEK is highly expressed in all tumor cells regardless of proliferative

status (Figure 2.8D). E2F3 staining of tumor cells was limited to the nucleus, suggesting that

E2F3 is in its functionally active state in these cells.

Immunofluorescent staining of the enucleated eye from which the unilateral tumor

RB2175 was isolated showed high expression of DEK protein in retinoblastoma cells relative

to the adjacent normal retina (Figure 2.9). This result is consistent with the mRNA expression

(Figure 2.2) and inconsistent with the immunoblot where the signal for DEK in this tumor was

less intense than the signal obtained from the normal adult human retina (Figure 2.7).

4.8. Immunofluorescent staining for DEK and E2F3 in retinoma

In approximately 15% of eyes enucleated to treat retinoblastoma, an underlying benign

tumor retinoma, which precedes retinoblastoma, is detected (Dimaras et al., manuscript in

preparation). Retinoma harbors the same M1 and M2 mutations in the RB1 gene as the adjacent

retinoblastoma. Compared to retinoblastoma cells, retinoma cells are more regularly shaped

and less densely packed (Figure 2.10.A), and they do not express the proliferation marker Ki67

(Dimaras et al., manuscript in preparation). The expression of DEK and E2F3 was compared

between retinoblastoma and the adjacent retinoma in four enucleated eyes, to examine if the

increase in DEK and E2F3 levels is the consequence of the loss of function of RB1.

51

While DEK is expressed in retinoma cells, its levels are significantly higher in

retinoblastoma cells in 2/4 enucleated eyes (Figure 2.10.B). E2F3, however, is not expressed in

retinoma but shows high expression in retinoblastoma in all four samples examined (Figure

2.10.C).

These results indicate that increased levels of DEK and E2F3 are not the consequence of

M1 and M2 events, but are directly or indirectly caused by M3-Mn events.

52

5. DISCUSSION

To identify the putative oncogene(s) targeted by 6p22 genomic gain in more than 50% of

retinoblastoma tumors, we have examined the expression of six genes mapping to the 0.6 Mb

6p22 MRG (DEK, AOF1, TPMT, NHLRC1, KIF13A, and NUP153), and E2F3 (Figure 2.1) in

normal retina and retinoblastoma of human and mouse origin.

Using real-time RT-PCR, it was found that only three of the seven examined genes,

DEK, NUP153, and E2F3, are overexpressed in all retinoblastoma tumors and cell lines with

documented genomic gain of 6p22 (Figure 2.2, Table 2.1). KIF13A, initially identified as the

6p22 oncogene candidate because of its overlap with the hotspot marker of gain, SHGC-

103950 [65], does not show increase in mRNA and protein levels in all tumors and cell lines

with 6p22 gain (Tables 2.1 and 2.3). Therefore, we exclude KIF13A as an oncogene candidate

in retinoblastoma. mRNA levels of three other MRG genes, AOF1, TPMT, and NHLRC1,

similar to KIF13A, are not elevated in tumors with genomic gain of 6p22, which eliminates

them from being potential oncogenes in retinoblastoma.

E2F3 and DEK homologues are subject to developmental regulation of expression in the

mouse retina, with high levels in the immature tissue, a feature shared by the majority of proto-

oncogenes [25]. Nup153 expression is not developmentally regulated, whereas maximal Kif13a

mRNA level is in adult retina, inconsistent with a proto-oncogene associated with proliferation

or differentiation of developing retinal cells.

The potentially important role of E2F3 and DEK in retinoblastoma is further supported

by real-time RT-PCR results on mouse retinal tumors. In TAg-RB mice, inactivation of RB

family proteins through interaction with the SV40 large T-antigen mimics the initiation steps in

human retinoblastoma (loss of both RB1 alleles). TAg-RB mice develop a discrete number of

retinal tumors, with a delay between the start of the large T antigen expression and tumor

53

formation, suggesting that additional independent mutational events are required for full

malignant transformation. We have shown that murine TAg-RB retinoblastoma show a

decrease in expression of candidate tumor suppressor genes CDH11 [57] and NGFR (p75NTR)

[204], similar to human retinoblastoma. We examined the expression of three genes from the

6p22 MRG, which is conserved on the mouse chromosome 13 (Dek, Kif13a, and Nup153), and

the expression of E2f3 (also on mouse chromosome 13). Only E2f3 and Dek were

overexpressed in murine retinoblastoma, with Dek showing higher increase in expression than

E2f3.

E2F3 protein overexpression was detected in all retinoblastoma tumors and cell lines

examined by immunoblotting (Figure 2.5B), and high expression of E2F3 and DEK proteins in

retinoblastoma was shown by immunofluorescent staining of five paraffin-embedded tumor

samples. Immunofluorescent staining for DEK in retinoblastoma was in accordance with the

real-time RT-PCR. However, for one tumor (RB2175), there was a discrepancy between data

obtained by immunoblotting and immunofluorescent staining. RB2175 is a unilateral

retinoblastoma initiated by a W195X point mutation in the RB1 gene, which showed high

mRNA levels of E2F3, DEK, KIF13A, and NUP153, consistent with genomic gain of the 6p22

hotspot marker, SHGC-103950. Only E2F3 protein was overexpressed compared to normal

adult human retina on immunoblot. KIF13A and NUP153 proteins were expressed at the same

level as in retinoblastoma as in normal adult human retina, while the level of DEK protein on

immunoblot was lower (Figure 2.5). However, immunofluorescent staining of sections of the

eye from which RB2175 was obtained showed high DEK expression in the tumor (Figure 2.7).

It is possible that DEK protein in this tumor is not detected with equal efficiency when it is in a

denatured state (immunoblot) as when it is not completely denatured (immunofluorescent

staining).

E2F3 is a cell-cycle promoting gene [140, 142], which has been recently implicated as

54

the target of 6p22 genomic gain in bladder cancer [188, 189], and whose overexpression was

shown to be a valuable prognostic indicator in prostate cancer [192]. Although E2F3

expression is regulated by the E2F-pRB complex [142], increase and variability in expression

seen in retinoblastoma tumors cannot be attributed solely to the loss of RB1. E2F1 and E2F2,

which are similarly repressed by the E2F-pRB complex, did not show increased expression in

retinoblastoma, and the cervical carcinoma cell line C33A, which lacks functional pRB, does

not overexpress E2F3 (data not shown).

DEK codes for a nuclear protein [94] sharing structural similarity with the E2F family

[69]. DEK is involved in chromatin remodeling [91-93] and possibly transcriptional regulation

[85] and mRNA splicing [97]. It is translocated to the NUP214 (CAN), forming a DEK-CAN

fusion protein, in acute myeloid leukemia [101]. DEK mRNA overexpression has also been

reported in a number of malignancies, including leukemia [104], melanoma [113],

hepatocellular carcinoma [110], and malignant brain tumors [112], and its genomic locus was

found to overlap with the hotspot marker of 6p22 gain in bladder cancer [116]. However, the

mechanism of DEK action in human cancers has not yet been elucidated.

A recent paper from Wu et al., reported overexpression of ID4 gene, which maps

between E2F3 and DEK, in bladder cancer [205]. However, we did not detect ID4

overexpression in retinoblastoma by immunoblotting (data not shown).

Of the seven examined genes, only DEK shows statistically significant correlation

between the increase in 6p22 genomic copy number and the relative increase in the mRNA

level (Table 2.2). E2F3 mRNA levels in a number of retinoblastoma cell lines and primary

tumors with and without the genomic 6p22 gain are within the 15-fold increase range reported

for dividing cells [140]. E2F3 is the only gene that shows the complete agreement between

increase in protein detected by immunoblot and increase in mRNA transcript level.

The discrepancy in DEK protein assessed by immunoblotting and immunofluorescent

55

staining in retinoblastoma RB2175 is not resolved1. Primary

retinoblastoma tumors highly expressed DEK in all tumor cells, while high E2F3 expression is

limited to actively proliferating cells, identified by PCNA staining, consistent with the cell-

cycle regulation of E2F3 expression [142]. We therefore conclude that both E2F3 and DEK are

promising targets of the 6p22 genomic gain in retinoblastoma. Further functional and

mutational analyses will show which of these two genes has oncogenic properties in human

retinal tumors, or whether they act synergistically.

Since 6p gain is one of the early mutational events in retinoblastoma development [58],

targeting the putative 6p22 oncogene in RB1 germline mutation carriers could be valuable in

preventing tumor formation, or reducing the number of tumors. Gain of 6p22 may have

prognostic significance [58]. Therefore, the 6p22 oncogene could also be a novel therapeutic

target.

1 The discrepancy between DEK protein levels assessed by immunoblotting and immunofluorescent

staining was the result of a technical problem. As suggested by Dr. Ferdinand Kappes, through

personal communication, the NaCl level in the protein extract buffer we used was not high enough to

separate DEK from genomic DNA during protein extraction. By making high salt protein extracts of

human retinoblastoma cell lines and normal retina, DEK overexpression was confirmed by

immunoblotting (Figure 2.11).

56

6. ACKNOWLEDGEMENTS

We thank Ella Bowles and Tim Corson for providing us with data on 6p22 genomic

copy numbers. We thank Jean McKay from Retinoblastoma Solutions for helping us to obtain

the paraffin block of RB2175, and we especially thank Dr. Valerie White and Dr. Katherine

Paton from the Vancouver General Hospital for providing us with the sections of the RB2175

paraffin block. We thank Dr. Joan O'Brien's laboratory in San Francisco for providing us with

TAg-RB mice. We are grateful to Dr. Sanja Pajovic for intellectual input and technical

assistance and Dr. Vivette Brown for technical advice.

57

CHAPTER III:

Oncogenic Potential of DEK and E2F3 in Retinoblastoma

This chapter will be submitted to Oncogene as follows:

Orlic, M., Khodadoust M., Kappes F., Markovitz D. and Gallie, B.L. Oncogenic potential

of DEK and E2F3 in retinoblastoma. (2007). Oncogene.

58

1. ABSTRACT

To test the oncogenic potential of DEK and E2F3, two candidate 6p22 oncogenes in

retinoblastoma, we have knocked down expression of either DEK or E2F3 in two

retinoblastoma cell lines, Y79 and RB247c, using recombinant lentiviral vectors. Y79 does has

two copies of 6p, two copies of DEK and E2F3 genes, does not overexpress DEK, but

overexpresses E2F3. RB247c has four copies of 6p, along with four copies of DEK and E2F3

loci, and it overexpresses both genes. Our hypothesis was that the decrease in the amount of

6p22 oncogene product in retinoblastoma cell line with 6p22 genomic gain would adversely

affect its growth rate. The results of our study show that knocking down either DEK or E2F3

does not significantly affect the growth of Y79, but affects the growth of RB247c, when

compared with the control viral vector that produces no shRNA. E2F3 knockdown results in

the slower growth of RB247c, while DEK knockdown results in massive cell death and

negative growth, with expression of proliferation/apoptosis markers supporting different

mechanism of action. We conclude that both 6p22 candidate genes exhibit oncogenic

properties in retinoblastoma cell line with 6p genomic gain, with DEK having a more profound

effect.

59

2. INTRODUCTION

Gain of chromosomal arm 6p is a frequent genetic aberration in retinoblastoma, present

in approximately 50% of tumors. By comparative genomic hybridization [50-53] and

subsequent quantitative multiplex PCR [65, 66], the minimal region of gain at 6p in

retinoblastoma was narrowed down to chromosomal band 6p22, and by expression studies two

genes, DEK and E2F3, were identified as candidate oncogenes [66, 118]. DEK and E2F3 are

frequently gained together, and are both overexpressed at the mRNA and protein level in

retinoblastoma tumors with 6p gain [66, 118]. DEK shows a statistically significant correlation

between increase in mRNA level and 6p22 genomic copy number [118]. E2F3 is

overexpressed in all examined retinoblastoma tumors, but shows a higher level of expression

when 6p22 genomic gain is present [66, 118].

Both DEK and E2F3 behave as oncogenes in in vitro assays. Infection of HeLa cells

with adenovirus overexpressing DEK results in the increased number of colonies formed in soft

agar [80]. Effect of E2F3 overexpression from a retroviral vector in NIH 3T3 cells is the same

[172]. E2F3 is also weakly oncogenic on its own in vivo, when overexpressed from a keratin 5

promoter in transgenic mouse model [183]. DEK was identified as the gene translocated to the

nucleoporin CAN in acute myeloid leukemia, resulting in the formation of a fusion DEK-CAN

protein in 1% of AML patients [101]. DEK is also overexpressed in AML patients without

DEK-CAN fusion [104, 105], and in a number of other malignancies [82, 110-115]. E2F3 was

found to be amplified and overexpressed in bladder cancer [188, 189], and its expression level

is an independent prognostic indicator in prostate cancer [192]. Oncogenic potential of E2F3

was proven in bladder and prostate cancer cell lines. Knockdown of E2F3 results in the

decreased growth rate of bladder cancer cell lines when 6p22 amplicon is present [190, 191],

60

and overexpression of E2F3 stimulates the proliferation of RB1-/- prostate cancer cell lines

[191].

Two recent studies bring up a possible connection between DEK and E2F3, suggesting

that E2F3 is one of the regulators of DEK expression [81, 82].

In this study, we have used lentiviral vectors of the third generation to knock down

DEK or E2F3 expression in retinoblastoma cell lines. Two retinoblastoma cell lines were

chosen for the study: Y79 and RB247c. Y79 does not posses the 6p gain, and has two copies of

DEK and E2F3 loci by SKY, locus specific FISH (Paderova et al. manuscript under review)

and gene specific QM-PCR [54]. DEK mRNA level is slightly increased in Y79, but the

increase is not statistically significant [118]. E2F3, however, is significantly overexpressed

[118]. RB247c has four copies of 6p, and four copies of DEK and E2F3 loci, by SKY, locus

specific FISH (Paderova et al. manuscript under review) and gene specific QM-PCR [54]. Both

DEK and E2F3 are overexpressed in this cell line [118].

We show that knocking down either DEK or E2F3 does not significantly affect the

growth rate of retinoblastoma cell line Y79, when compared with the control vector, but

impairs the growth of RB247c. While E2F3 knockdown results in a slower growth of RB247c,

DEK knockdown results in a massive cell death and negative growth rate. We conclude that

both DEK and E2F3 show oncogenic properties in retinoblastoma, with DEK having a more

profound effect.

61

3. MATERIALS AND METHODS

3.1. Cell lines

Retinoblastoma cell lines, Y79 and RB247c were grown in Iscove’s modified

Dulbecco’s medium, supplemented with 15% Fetal Clone Serum III, 10 g/ml insulin, 55 M

beta-mercaptoethanol, and 100 U/ml penicillin/streptomycin. HEK 293 FT, human embryonic

kidney cell line used for lentivirus production, was grown in the retinoblastoma cell line

medium if used for infection of retinoblastoma cell lines. HeLa cells and HEK 293 FT cells

used for infection of HeLa cells were grown in DMEM H21, supplemented with 10% Fetal

Bovine Serum and 100 U/ml penicillin/streptomycin. All cells were kept at 37 C, in 5% CO2.

3.2. Viral constructs

Third generation lentiviral constructs were used to knock down expression of genes of

interest, or as control vectors. Three packaging vectors were pRSV rev, pMDLg/pRRE and

pHCMV-G (Figure 3.1.A), providing rev protein, viral proteins gag and pol, and envelope

protein G of the vesicular stomatitis virus (VSV-G), respectively. The plasmid construct

KH1_GFP was used for producion of control lentivirus LV-EMPTY, which expressed

enhanced green fluorescent protein (EGFP) as the selection marker and produces no shRNA

(Figure 3.1.B). Anti-DEK shRNA lentiviruses LV-DEK1450 (targeting DEK mRNA at

nucleotide position 1450) and LV-DEK1775 (targeting DEK mRNA at nucleotide position

1775) used in Y79 cell line were produced from plasmid constructs KH1_GFP_DEK1450 and

KH1_GFP_DEK1775, respectively (Figure 3.1.B). The efficiency of LV-DEK1450 and LV-

DEK1175 in DEK knockdown was demonstrated in HeLa cells and will be published

elsewhere. Five plasmids that express viral genome coding for anti-E2F3 shRNA and

62

puromycin resistance gene were purchased from Sigma Mission shRNA library:

PLKO.1_E2F3_753, PLKO.1_E2F3_1002, PLKO.1_E2F3_1051, PLKO.1_E2F3_1214 and

PLKO.1_E2F3_2879 (targeting E2F3 mRNA at nucleotide position 753, 1002, 1051, 1214 and

2879, respectively). Another five plasmids purchased from Sigma Mission shRNA library code

for anti-DEK shRNA and puromycin resistance gene: PLKO.1_DEK_523, PLKO.1_DEK_832,

PLKO.1_DEK_1003, PLKO.1_DEK_1165, and PLKO.1_DEK_2065 (targeting DEK mRNA

at nucleotide position 523, 832, 1003, 1165 and 2065, respectively). Control vector used with

Sigma Mission shRNA library constructs was PLKO.1_PURO, coding for puromycin

resistance gene and no shRNA.

3.3. Antibodies

Antibodies used for immunoblotting of E2F3 and TUBB were purchased from Upstate,

Charlottesville, VA, USA (05-551) and Sigma Aldrich, St. Luis, MO, USA (T 4026),

respectively. Rabbit anti-DEK serum used for immunoblotting was developed in ___ lab. Anti-

BrdU antibody was purchased from Pharmingen __. Goat anti-mouse IgG HRP-conjugated

antibody and goat anti-rabbit IgG AP-conjugated antibody were purchased from Bio-Rad

Laboratories, Hercules, CA, USA (__ and 170-6518, respectively). Antibodies used for

immunofluorescent staining of Ki67, BrdU, Cyclin B1 and activated Caspase 3 were purchased

from _____. Horse biotinylated anti-mouse IgG antibody and horse biotinylated anti-rabbit IgG

antibody, used for immunofluorescent staining were purchased from Vector Laboratories,

Burlingame, CA, USA (BA-2000 and __, respectively). NBT and BCIP, used for detection of

AP-conjugated secondary antibodies, were purchased from ___. HRP-conjugated secondary

antibodies were detected by Western LightningTM Chemiluminescence Reagent Plus (Perkin-

Elmer Life Sciences, Wellesley, MA, USA, NEL104).

63

3.4. Infection and selection procedures

Five-day infection protocol was used for lentivirus production and infection of target

cells. On the first day, 293 FT cells were seeded in 25 cm2 filter cap tissue culture flasks, so

that they were 50-70% confluent at the time of transfection. On the second day, 293 FT cells

were transfected with 2 g of each of the packaging plasmids, and 2 g of one of the viral

RNA-producing plasmids. Transfections were performed using Lipofectamine 2000 reagent

(Invitrogen). On the third day, medium on transfected 293 FT cells was replaced with fresh

medium, to remove transfection complexes. On the fourth day, virus-containing supernatant

was harvested from 293 FT cells, filtered through 0.45 m filter, and used to infect target cells

from which their regular growth medium was removed. Fresh medium was added to virus-

producing 293 FT cells, and the infection was repeated on the fifth day, to increase the

infection efficiency. On the eighth day, the virus-containing medium was removed from target

cells and replaced with the fresh growth medium if the selection marker was EGFP, or with the

fresh medium containing 2 g/ml puromycin, if puromycin resistance was the selection

marker. Cells stably infected with EGFP-expressing lentiviral vectors were sorted on flow

cytometer ___ in Princess Margaret Hospital, and were 90-95% EGFP-positive after sorting

(data not shown). Puromycin resistant cells were selected in accordance with the

manufacturer’s instructions for one week prior to start of further experiments, as determined

from the puromycin selection curve (data not shown), and were grown in the puromycin-

containing growth medium throughout the experiments.

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3.5. Test for the presence of replication competent lentivirus

Although described viruses are replication deficient by definition, all stably infected cells

were tested for the presence of replication competent lentivirus (RCL test), as requested by the

Biosafety Committee of the University Health Network. 30% of medium harvested from stably

infected cells was used to infect naïve HeLa cells plated on 6-well plates (one well per original

infection). After infection, cells were grown for two passages. Medium harvested from infected

HeLa cells after the second passage was used to infect naïve HeLa cells. Cells were grown for

two passages after second infection and examined for the presence of either GFP positive

clones, or for the presence of puromycin-resistant colonies. No RCL activity was detected (data

not shown).

3.6. Growth rates

Retinoblastoma cell lines were grown for eight (RB247c) or ten days (Y79), an

empirically determined time frame of exponential growth for individual cell lines (data not

shown), on 96 well plates. 8000 cells were plated per well, in duplicate for each time point.

Cells were stained with trypan blue and live cells were counted in quadruplicates for each well

using the hemocytometer. Growth rate for each treatment was determined from the slope of the

logarithmic growth curve. Microsoft Excel was used to plot the data, fit the growth curve and

determine the curve slope and goodness of the fit.

3.7. RNA extraction and reverse transcription

Total RNA from stably infected cells selected by puromycin resistance or EGFP

expression was isolated using the Trizol procedure. Single-stranded cDNA was produced by

reverse transcription of 1 g of the total RNA with SuperScript II reverse transcriptase

65

(Invitrogen, Carlsbad, CA, USA) and random hexamer primers (Invitrogen, Carlsbad, CA,

USA).

3.8. Protein isolation

Whole cell protein extracts were made by cell lyses through repeated freeze and thaw

cycles in extraction buffer containing 20 mM HEPES pH 7.2, 0.5 M NaCl, 1 mM MgCl2, 0.1

mM EDTA, 7% glycerol and Roche protease inhibitor coctail.

3.9. Real Time RT-PCR

Real time RT-PCR was performed using the TaqMan chemistry. TaqMan gene

expression assays for human DEK (Hs00180127_m1), E2F3 (Hs01076037_m1) and HPRT

(Hs99999909_m1) were purchased from Applied Biosystems, Foster City, CA, USA, and the

experiment was performed as previously described [118]

3.10. Immunoblotting

Immunoblotting was performed by PAGE of 20 g of the whole cell protein extract

on10% precast polyacrylamide gels from ___, followed by transfer of resolved proteins to a

PVDF membrane. DEK, E2F3 and TUBB primary antibodies were used at 1:500 dilution in

1% BSA/TBST. Incubation with primary antibodies was carried out for 1 hr at room

temperature. Anti-mouse IgG HRP-conjugated and anti-rabbit IgG AP-conjugated secondary

antibodies were used at 1:10000 dilution in TBST. Incubation with secondary antibodies was

carried out for 30 min at room temperature.

66

3.11. Immunofluorescent staining and quantification of positive cells

Tissue culture grown retinoblastoma cell lines were centrifuged to remove the growth

medium, resuspended in warm 1% agarose/PBS and after agarose solidified, fixed with 4%

PFA, paraffin embedded and sectioned at 5 m. Paraffin removal, heat-mediated antigen

retrieval, and blocking procedures were carried out as previously described [118]. Primary

antibodies against Ki67, Cyclin B1 and activated Caspase 3, and biotinylated secondary

antibodies were used at 1:200 dilution in 1% BSA/TBST with 10% Antibody Diluent

(DAKOCytomation, Glostrup, Denmark; S3022). Incubation with primary antibodies was

carried out overnight at 4C, and the incubation with secondary antibodies was carried out for 1

hr at room temperature. Streptavidin-Alexa594 was used as the detection reagent. DAPI was

used to visualize nuclei of retinoblastoma cells. Slides were mounted using the

DAKOCytomation Fluorescent Mounting Medium (S3023). Pictures of stained cells were

taken at 200-fold magnification, in 5-10 randomly selected visual fields, containing at least 100

cells total. Percentage of cells expressing a marker of interest was determined as a tota number

of marker-positive DAPI stained cells over the total number of DAPI positive cells. Therefore,

only cells with relatively intact nuclei were counted.

3.12. BrdU incorporation

Stably infected cells were pulsed with BrdU to determine the proliferation index. BrdU

was added to the growth medium at the final concentration of ___ ug/ml. Cells were incubated

in the BrdU containing medium for 2 h. After incubation, the cell suspension was put on poly-

D-lysine coated microscope slides, fixed with cold 70% ethanol, DNA denatured by treatment

with 2.5 M HCl stained for BrdU in accordance with the anti-BrdU antibody manufacturer’s

instructions. BrdU positive cells were quantified as described above.

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4. RESULTS

4.1. Screening Sigma Mission shRNA Library vectors for efficiency in

DEK and E2F3 knockdown in HeLa cells

HeLa cells stably infected with anti-DEK or anti-E2F3 shRNA expressing lentiviruses

from Sigma Mission shRNA Library were screened for DEK and E2F3 expression,

respectively, using the real-time RT-PCR. The constructs most effective in knocking down

DEK were shown to be PLKO.1_PURO_DEK832 and PLKO.1_PURO_DEK1003, causing

80% reduction in DEK mRNA, and PLKO.1_PURO_DEK1165, causing >90% reduction in

DEK mRNA expression. compared with the control vector LV-EMPTY (Figure 3.2.a). The

constructs most effective in knocking down E2F3 mRNA were shown to be

PLKO.1_PURO_E2F3_1214 and PLKO.1_PURO_E2F3_2879, resulting in 80% and 70%

reduction in E2F3 mRNA expression, respectively, compared with the control vector LV-

EMPTY (Fugure 3.2.b). Constructs PLKO.1_PURO_DEK832 and PLKO.1_PURO_DEK1165

were used to knock down DEK in RB247c and Y79, while the constructs

PLKO.1_PURO_E2F3_1214 and PLKO.1_PURO_E2F3_2879 were used to knock down

E2F3.

4.2. DEK and E2F3 knockdown in retinoblastoma cell lines

Real-time RT-PCR analysis of retinoblastoma cell lines stably infected with anti-DEK

shRNA expressing lentiviruses reduced DEK expression by 70-80% in Y79 by lentivirus LV-

DEK1450, and 40-50% bu LV-DEK1175 compared to Y79 infected with the control vector

LV_EMPTY (Figure 3.3.a). Knockdown at the protein level was confirmed by western blot

(Figure 3.3.b).

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RB247c cell line did not survive sorting by flow cytometry. Therefore, viral constructs

with puromycin resistance selection marker were used to knock down DEK in this cell line.

Lentiviruses PLKO.1_PURO_DEK832 and PLKO.1_PURO_DEK1165 knock down DEK

70% and 90%, respectively, by real-time RT-PCR in RB247c, compared with RB247c infected

with the empty vector PLKO.1_PURO (Figure 3.3 e). The same viruses result in 50% and 90%

DEK knockdown, respectively, in Y79, by real time RT-PCR, compared with Y79 infected

with the control PLKO.1_PURO vector. (Figure 3.3 c). DEK knockdown in RB247c and Y79

was confirmed by western blot (Figures 3.3.f and 3.3.d).

Infection with PLKO.1_PURO_E2F3_1214 or PLKO.1_PURO_E2F3_2879 led to

~90% reduction in E2F3 mRNA level in both Y79 and RB247c (Figures 3.3.g and 3.3.i), by

real-time RT-PCR. The reduction of E2F3 protein product level in Y79 and RB247c was

confirmed by immunoblotting (Figures 3.3.h and 3.3.j). By immunoblotting, it seems that

PLKO.1_PURO_E2F3_1214 is more efficient in E2F3 knockdown, and this finding is

supported by the greater biological effect of PLKO.1_PURO_E2F3_1214 infection, described

below. The shRNA produced by PLKO.1_PURO_E2F3_2879 maps to the 3’ UTR of E2F3

mRNA, and this could potentially explain the observed difference.

Since it was recently suggested that E2F3 positively regulates DEK expression [81, 82],

we examined expression of DEK upon E2F3 knockdown with the vector

PLKO.1_PURO_E2F3_1214. No change in DEK mRNA or protein level was detected in either

Y79 or RB247c (Figure 3.4).

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4.3. Growth rates, proliferation index and caspase 3-dependent apoptosis

of retinoblastoma cell lines infected with anti-DEK shRNA-producing

lentiviral vectors

Knocking down DEK in Y79 by LV-DEK1450 and LV-DEK1775 led to no significant

decrease in the growth rate, compared with Y79 infected with the control virus LV_EMPTY

(Figure 3.5.a). This was confirmed by knocking down DEK by PLKO.1_PURO_DEK832 and

PLKO.1_PURO_DEK1165, in comparison with the control virus PLKO.1_PURO (Figure

3.5.b), and further supported by the lack of change in the proliferation index measured by

either Ki67 staining or BrdU incorporation (Figures 3.5.c and 3.5.d). No significant change in

the number of CYCB1 positive cells or activated Caspase 3 positive cells was evident (Figures

3.5.e and 3.5.f). In RB247c, however, DEK knockdown resulted in decrease in viable cell

numbers over time and, hence, a negative growth rate (Figure 3.5.g), supported by a decrease

in proliferation index determined Ki67 staining or BrdU incorporation (Figures 3.5.h and

3.5.i). No change in CYCB1 expression was observed (Figure 3.5.j). Expression of activated

Caspase 3 was elevated in a dose-dependant manner, with the more efficient vector

PLKO.1_PURO_DEK1165 resulting in higher percentage of activated Caspase 3 positive cells

compared to the less efficient PLKO.1_PURO_DEK832 (Figure 3.5.k).

4.4. Growth rates, proliferation index and caspase 3-dependent apoptosis

of retinoblastoma cell lines infected with anti-E2F3 shRNA-producing

lentiviral vectors

Knocking down E2F3 in retinoblastoma cell line Y79 did not significantly affect the

growth rate or proliferation index, when compared to the control vector PLKO.1_PURO

(Figures 3.6.a, 3.6.b and 3.6.c). There was no significant change in the number of CYCB1

70

positive cells or activated Caspase 3 positive cells. (Figures 3.6.d and 3.6.e). In RB247c,

knocking down E2F3 with a less potent construct PLKO.1_PURO_E2F3_2879 did not cause a

decrease in the growth rate, but the construct, PLKO.1_PURO_E2F3_1214 led to a

significantly slower growth (Figure 3.6.f). No change in the proliferation index was observed

after treatment with either PLKO.1_PURO_E2F3_1214 or PLKO.1_PURO_E2F3_2879

(Figures 3.6.g and 3.6.h), but the number of CYCB1 positive cells was dramatically increased

after treatment of RB247c (Figure 3.6.i) and increased expression of activated Caspase 3 was

evident (Figure 3.6.j).

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5. DISCUSSION

Gain of the short arm of chromosome 6 is present in >50% retinoblastoma tumors, by

karyotype studies [11, 42, 46, 59], comparative genomic hybridization [49-53, 201] and

quantitative multiplex PCR [65, 66]. The minimal region of gain maps to chromosomal band

6p22 [51], and two 6p22 genes, DEK and E2F3 were identified as candidate oncogenes in

retinoblastoma in two independent studies [66, 118]. E2F3 is overexpressed in all

retinoblastoma tumors examined, but its levels are higher in tumors with 6p gain [66, 118].

DEK expression level correlates with the genomic copy number of 6p [118].

Both DEK and E2F3 have been proposed as oncogenes in other cancer types [101, 188,

189], and they both behave as oncogenes in functional studies [80, 172, 190, 191]. Therefore, it

was necessary to functionally examine the putative oncogenic role of these two genes in

retinoblastoma.

Our approach was to knock down expression of DEK or E2F3 in retinoblastoma cell

lines and examine the effect that this knockdown has on the proliferation rate of retinoblastoma

cell lines. Two retinoblastoma cell lines were chosen for this study, Y79 and RB247c. Status of

6p and DEK and E2F3 loci in these two cell lines is well established, by karyotype studies

[43], spectral karyotyping (Chapter IV), mBand analysis of chromosome 6 (Chapter IV), CGH

[51], locus specific FISH for DEK and E2F3 (Chapter IV), and gene-specific QM-PCR [54].

6p gain is not present in Y79, and Y79 has two copies of both E2F3 and DEK loci. RB247c

has 4 copies of 6p and four copies of both DEK and E2F3 loci. Expression of DEK and E2F3

in these two cell lines was examined at the mRNA level by real-time RT-PCR, and at the

protein level by immunoblotting. Both cell lines overexpress E2F3, while only RB247c

overexpresses DEK [118].

72

Knocking down DEK gene expression in Y79 with four different constructs had no

effect on the growth rate or the proliferation index determined by BrdU incorporation and

staining for proliferation marker Ki67, when compared with empty vectors expressing

selection markers only. Similarly, knocking down E2F3 with two different shRNA constructs

had no effect on the growth of Y79 or proliferation index, when compared with the control

vector.

RB247c, however, was affected by both DEK and E2F3 knockdown. Treatment with

any of the two anti-DEK shRNA constructs resulted in the negative growth rate. Number of

live cells was decreasing over time, and increased level of cell death was evident from both

staining for the apoptosis marker activated Caspase 3, and from trypan blue staining that was

used to quantify live cells during growth assay (data not shown). This is in accordance with the

suggested role of DEK as an apoptosis inhibitor [121]. Since proliferation index determined by

both BrdU incorporation and staining for the proliferation marker Ki67, which labels all cells

except cells in G0, is evidently decreased in comparison to the cells infected with the control

vector, this suggests that a portion of cells exit the cell cycle when DEK is knocked down.

Although cellular senescence upon DEK knockdown has been demonstrated, we did not detect

senescence-related -galactosidase activity in RB247c upon DEK depletion (data not shown).

E2F3 knockdown with a less potent vector, PLKO.1_PURO_E2F3_2879, had no effect

on the growth rate or proliferation index of RB247c, suggesting that the remaining level of

E2F3 was enough for RB247c to continue to proliferate. Knockdown by the more potent

vector, PLKO.1_PURO_E2F3_1214, however, significantly slowed down the proliferation of

RB247c. In the same time, no significant change in the proliferation index by staining for Ki67

or BrdU incorporation was evident, suggesting that the cells did not exit the cell cycle.

However, expression of Cyclin B1, marker of G2 phase and G2/M transition [206] was

increased several fold compared to all the other treatments in both cell lines, suggesting that

73

RB247c cells accumulate in G2 or at G2/M transition when they are depleted of E2F3. G2/M

arrest is a documented consequence of E2f3 inactivation in mouse fibroblasts [182], which can

be explained by the finding that E2f3 loss results in disruption of centrosome duplication cycle

and, consequently, mitotic spindle defects [158]. It was shown that accumulation of cells in

G2, characterized by cyclin B1 overexpression can result in shifting back to G1 and

endoreduplication [207], which would explain the fact that no large reduction in BrdU

incorporation is seen in RB247c upon PLKO.1_PURO_E2F3_1214 treatment. Positive

association between cyclin B1 overexpression and sensitivity to apoptosis is suggested but not

clearly established [208], and increased apoptosis in RB247c infected with

PLKO.1_PURO_E2F3_1214 is evident by staining for activated Caspase 3, and was evident

from trypan blue staining used for quantification of live cells during growth assay (data not

shown).

Although E2F3 was implicated in the positive regulation of DEK expression [81, 82],

we did not detect change in DEK levels upon E2F3 knockdown in Y79 or RB247c, which

could be explained by the tissue-specific regulation of gene expression.

We conclude that both DEK and E2F3 exhibit oncogenic potential in retinoblastoma

when 6p22 genomic gain is present, with RB247c being dependent on DEK overexpression.

Since DEK and E2F3 are frequently gained together and they both have a functional role in

retinoblastoma, it is plausible that they perform complementary roles. E2F3 overexpression

could be the proliferation hit needed for indefinite cycling of RB1-/- cells [209], while DEK

overexpression would enable transformed cells to escape apoptosis.

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CHAPTER IV:

Novel 6p rearrangements and recurrent translocation breakpoints in

retinoblastoma identified by SKY and mBand analyses

This chapter has been submitted to Cancer Genetics and Cytogenetics as follows:

Paderova J.*, Orlic-Milacic M.*, Yoshimoto M., DaCunha Santos G., Gallie, B.L. and

Squire J.A. Novel 6p rearrangements and recurrent translocation breakpoints in

retinoblastoma identified by SKY and mBand analyses.

*Authors made equal contribution

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1. ABSTRACT

Gain of short arm of chromosome 6, usually through isochromosome 6p formation, is

present in approximately 50% of retinoblastoma tumors. The minimal region of gain maps to

chromosome band 6p22. Two genes, DEK and E2F3, are implicated as candidate oncogenes.

However, chromosomal translocations have been overlooked as a potential mechanism of

activation of oncogenes at 6p22 in retinoblastoma. Here, we report combined spectral

karyotyping (SKY), DAPI-banding, mBand and locus-specific fluorescence in situ

hybridization (FISH) analyses of four retinoblastoma cell lines, RB1021, RB247c, RB383, and

Y79. The first three lines show genomic gain of 6p, with formation of isochromosome 6p and

four copies of both E2F3 and DEK. In RB1021 and RB247c, 6p undergoes additional

structural rearrangements involving a common translocation breakpoint at 6p22. In addition to

6p22 and 6p10, other recurrent translocation breakpoints identified in this study are 4p16,

11p15, 17q21.3 and 20q13. Common minimal regions of gain map to chromosomal arms 1q,

2p, 6p, 17q and 21q. In Y79, which does not possess 6p gain, two different translocations of

chromosome 6 are present. Collectively these data imply that 6p translocations may represent

another mechanism of activation of 6p oncogene(s) in a subset of retinoblastomas, in addition

to copy number increase.

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2. INTRODUCTION

Retinoblastoma is a rare childhood eye cancer initiated by the loss of function of both

alleles of the RB1 tumor suppressor gene, mapping to 13q14 chromosomal band [16, 20, 210,

211]. Mutations 1 and 2 (M1 and M2) that target RB1 are rate-limiting events in tumor

formation, in accordance with the Knudson’s two-hit hypothesis [1], and may be sufficient for

the development of the benign retinal tumor, retinoma (Dimaras et al., manuscript in

preparation). Retinoblastoma tumorigenesis is thought to involve mutations in additional

genes, named M3-Mn events [45].

As shown by karyotype analyses [43], chromosomal and array-based comparative

genomic hybridization [49-53, 201], the two most frequently gained chromosomal regions in

retinoblastoma are 1q and 6p, both thought to harbor oncogenes. The most frequently lost

region, hypothesized to harbor a tumor suppressor gene, is 16q.

By quantitative multiplex PCR (QM-PCR) analyses of genomic copy numbers of

UniSTS markers within minimal regions of gain at 1q [55, 212] and 6p [65, 66] and subsequent

expression analyses, KIF14 was identified as the candidate oncogene at 1q [55], and E2F3 and

DEK were identified as candidate onocogenes at 6p [66, 118]. CDH11 was identified as the

candidate tumor suppressor gene at 16q [57].

Gain of 6p in retinoblastoma is usually present as an isochromosome i(6p) [59], and

tumors with this rearrangement possess four copies of the entire 6p genomic region [59]. The

candidate 6p oncogenes DEK and E2F3 are 2.1 Mb apart at band 6p22.3 and are frequently

gained together in tumors with gain of 6p [54]. Both genes are overexpressed in tumors with 6p

gain [66, 118] and DEK shows statistically significant correlation between genomic copy

number of 6p and mRNA level in retinoblastoma, while E2F3 is overexpressed in all

retinoblastoma tumors, with expression levels being higher if 6p genomic gain is present [118].

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There is no data concerning recurrent chromosomal translocations or other types of genomic

alterations within the 6p region.

In this study we utilized SKY analysis and precise mBand evaluation of chromosome 6,

to generate detailed karyotypes of four retinoblastoma cell lines. We identify two different

translocations of chromosomes 6 within cytoband 6p22 close to the DEK and E2F3 genes, as

determined by dual colour locus-specific FISH.

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3. MATERIALS AND METHODS

3.1. Cell lines and metaphase preparation

Retinoblastoma cell lines RB247c, RB383 and RB1021 were grown in Iscove’s

modified Dulbecco’s Medium, supplemented with 15% Fetal Clone Serum III, 10 g/ml

insulin, 55 M -mercaptoethanol and 100 U/ml penicillin-streptomycin. Retinoblastoma cell

line Y79 was grown in RPMI 1640 medium supplemented with 15% Fetal Calf Serum and

2mM Glutamine. Cell lines were incubated at 37C and 5% CO2. For metaphase preparation,

cell lines were treated with 0.1 g/ml KaryoMAX Colcemid Solution (Gibco, Invitrogen,

Carlsbad, California, USA) for 4 hours, followed by 20 minute incubation in 0.075M KCl

hypotonic solution. Cells were fixed in acetic acid-methanol fixative and cell suspensions were

dropped on microscope slides.

3.2. FISH

FISH probe for DEK was prepared using the PAC clone RP1-298J15 (obtained from

The Centre for Applied Genomics at the Hospital for Sick Children, Toronto, Ontario,

Canada). FISH probe for E2F3 was prepared using the BAC clone CTD-2347D10 (obtained

from Invitrogen, Carlsbad, California, USA). Plasmid DNA was labeled with Spectrum Green

dUTPs (DEK) or Spectrum Red dUTPs (E2F3) by Nick Translation Kit (Vysis, Downers

Grove, Illinois, USA). Labeled probes were hybridized to metaphase spreads from all four cell

lines. Hybridization and post-hybridization washes were carried out according to labeling kit

manufacturer’s instruction. Number and localization of signals was analyzed. Metaphase

images were acquired by ZEISS Axioskop 2 plus microscope (ZEISS, Oberkochen, Germany)

using Isis imaging and analysis software (MetaSystems, Altlussheim, Germany).

79

3.3. SKY

The human SkyPaint probe (Applied Spectral Imaging (ASI), Migdal Ha’emek, Israel)

was hybridized to metaphase spreads obtained from the cell lines. Hybridization and post-

hybridization washes were carried out according to manufacturer's instructions. Spectral

images were acquired and analyzed with SD 200 Spectral Bio-Imaging System (ASI, Migdal

Ha’emek, Israel) attached to ZEISS Axioplan 2 microscope (ZEISS, Oberkochen, Germany)

and with SKY View 1.5 software (ASI, Migdal Ha’emek, Israel). 10 metaphase spreads were

analyzed according to spectral and inverted DAPI data. Resulting karyotypes are described

according to the ISCN 2005 guidelines [213].

3.4. mBand analysis

mBand analysis of chromosome 6 was performed using the human mBand XCyte 6

probe (MetaSystems, Altlussheim, Germany). Procedure was carried out according to

manufacturer’s instructions. Metaphase images were acquired by ZEISS Axioskop 2 plus

microscope (ZEISS, Oberkochen, Germany) using Isis imaging and analysis software

(MetaSystems, Altlussheim, Germany). 10 metaphase spreads were analyzed and evaluated in

comparison to mBand pattern of normal chromosome 6.

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4. RESULTS

4.1. Chromosome 6 rearrangements

Overall SKY karyotype analysis (described in section 4.2) and chromosome 6 mBAND

analysis permitted a complete identification of all chromosomal aberrations in each cell line

(classified banding pattern of normal chromosome 6 is presented in Figure 4.1.c.I). Dual-

colour locus-specific FISH with the DEK and E2F3 gene probes was used to determine the

position of DEK and E2F3 relative to the translocation breakpoints.

4.1.1. Retinoblastoma cell line RB1021

RB1021 cell line was derived from a tumor of a female patient with bilateral

retinoblastoma. The germline RB1 mutation is CGA-TGA substitution in exon 10 (data not

shown). RB1021 lacks a normal chromosome 6. SKY, mBand and FISH showed the presence

of i(6p) chromosome (Figure 4.1.a.III). A reciprocal translocation between p and q arms of two

chromosomes 6 resulted in one derivative chromosome 6 harboring two copies of DEK as well

as E2F3 (Figure 4.1.a.II) and another derivative chromosome 6 carrying neither DEK nor E2F3

(Figure 4.1.a.I). mBand and FISH data suggest that the breakpoints of this reciprocal

translocation are at 6p22 and 6q22.3.

4.1.2. Retinoblastoma cell line RB247c

The cell line was derived from an irradiated tumor of a male patient with bilateral

retinoblastoma. The germline RB1 mutation is an 8 bp deletion within exon 1 (data not shown).

RB247c exhibits one normal chromosome 6 with one DEK and one E2F3 signal (Figure 1.b.I)

and two derivative chromosomes 6: der(6)t(6;6)(qter;p22) (Figure 4.1.b.II) and der(6)

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(6pter6p10::6p106p22::6q23.3 ~6q27::17q21.317qter) (Figure 4.1.b.III). While the

precise order of events leading to this complex chromosomal alteration is not known, it seems

probable that two derivative chromosomes 6 resulted from a non-reciprocal balanced

translocation between 6qter of one normal chromosome 6 (Figure 4.1.b.II) and i(6p)

chromosome at the translocation breakpoint 6p22. Both the SKY and mBAND chromosome 6

analyses suggested that additional translocation events occurred at the affected p arm of i(6p)

chromosome (Figure 4.1.b.III) including rearrangement of material from the 6q region and

translocation of material from chromosome 17.

4.1.3. Retinoblastoma cell line RB383

The cell line was derived from a tumor of a male patient with unilateral retinoblastoma,

with no germline mutation (data not shown). RB383 possesses three chromosomes carrying

genomic material of chromosome 6 (Figure 4.1.c). Two of these chromosomes are normal

chromosomes 6, according to SKY, FISH, DAPI banding and mBand analyses (Figures 4.1.c.I

and 4.1.c.II), and possess single unrearranged E2F3 and DEK loci at 6p22.3. The third

chromosome 6 is an isochromosome, with deletion of terminal 6p25 band on one of the arms,

resulting in der(6)i(p10)del(p25) (Figure 4.1.c.III). The isochromosome possesses E2F3 and

DEK loci on each of the 6p arms, increasing the total number of copies of E2F3 and DEK to

four, as in RB1021 and RB247c.

4.1.4. Retinoblastoma cell line Y79

Retinoblastoma cell line Y79 was derived from a tumor of a female patient with

unilateral retinoblastoma, with a germline G-T substitution at the 3’ end of exon 12 of the RB1

gene (data not shown). Y79 does not possess an isochromosome 6p and showed no gain of

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DEK and E2F3 loci (Figure 4.1.d). However, SKY analysis and mBAND findings revealed

abnormalities of chromosome 6. One chromosome 6 is involved in translocation with

chromosome 21, resulting in the formation of der(6)t(6;21)(q24;q21) (Figure 4.1.d.I ). The

other chromosome 6 is involved in translocation with chromosome 7, resulting in the formation

of der(6)t(6;7)(7?q317?q36::6pter6qter) (Figure 4.1.d.II).

4.1.5. Overall pattern of chromosome 6 rearrangements

Collectively these data show that 3 out of 4 investigated cell lines have 4 copies of

DEK and E2F3 genes (and majority of genes located on 6p) in near-diploid genome due to

initial formation of i(6p) chromosome. i(6p) chromosome frequently participates in further

rearrangements (in 2 out of 3 cell lines with i(6p)). Interestingly, 2 of the 4 cell lines, RB1021

and RB247c, also have translocations between different regions of chromosomes 6, involving

cytoband 6p22.

4.2. Spectral karyotype analysis

Representative karyotype tables of all four retinoblastoma cell lines based on SKY

analysis are shown in Figure 4.2, and detailed karyotype descriptions are provided in Table 4.1.

All cell lines exhibit near-diploid karyotypes with multiple structural changes, predominantly

non-reciprocal translocations. Two common breakpoints were evident on chromosome 6 close

to E2F3 and DEK: at 6p22 in 2 cell lines and at 6p10 in 3 cell lines. Other recurrent

breakpoints present in at least two cell lines were 4p16, 11p15, 17q21.3 and 20q13 (Figure

4.3). Regions of minimal genomic gain were determined as 1q23-1q42 (all 4 lines), 2p (3 lines,

plus amplification of a smaller region of 2p in Y79), 6p10-6pter (3 lines), 17q21.3-17qter (3

lines), 21q21-21qter (all 4 lines), shown in Figure 4.4. In addition, 2 cell lines showed gain of

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the whole chromosome 5 and region 13q13-13qter x2-3. Both cell lines that were derived from

tumors of female patients (RB1021 and Y79) exhibited loss of one X chromosome.

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5. DISCUSSION

Increase in the copy number of chromosomal arm 6p is a frequent event in

retinoblastoma, and isochromosome 6p is considered to be a hallmark of retinoblastoma [42,

59, 214]. Several mechanisms of isochromosome formation have been proposed. The most

widely accepted mechanism is the transverse division of the centromere [63], first suggested by

Darlington in 1939 [62]. Alternative mechanisms involve translocation between two

homologous chromosomes, and either intra-or interchromosomal mitotic chromatid exchange

[63]. By examining the relative dosage of polymorphic 6p and 6q alleles in retinoblastoma

tumors with i(6p), Horsthemke et al. showed that mitotic nondisjunction leading to trisomy 6p

precedes isochromosome 6p formation, as suggested by Squire et al. [59], and that the

transverse division of the centromere or intrachromosomal chromatid exchange are the most

likely mechanisms of the subsequent i(6p) formation [64].

Most retinoblastoma tumors with 6p gain are tetrasomic for all 6p genes, but trisomic

tumors with no i(6p) were also identified in the study by Squire et al., leading the authors to

suggest that increase in dosage of 6p genes, and not isochromosome formation per se was the

critical event in tumor development [59]. In several CGH studies of retinoblastoma tumors, it

was confirmed that, although tetrasomy for the entire short arm is most frequently observed

with 6p gain, a proportion of tumors possess gain of smaller portions of 6p [49, 51-53, 201].

This allowed mapping of the minimal region of 6p gain to band 6p22 [51], an area that

partially overlaps with the minimal region of gain in bladder cancer [116]. Two candidate

oncogenes in this region, DEK and E2F3, are overexpressed in tumors with 6p gain at both

mRNA and protein level [66, 118].

In the present study, four retinoblastoma cell lines were characterized in detail by SKY,

chromosome 6 mBAND, and by E2F3 and DEK locus specific FISH. Three retinoblastoma

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cell lines, RB1021, RB247c and RB383, possess the isochromosome 6p, increasing the

genomic copy number of DEK and E2F3 to four, in a near-diploid genome. Two of these cell

lines, RB247c and RB1021, have translocations at 6p, with a breakpoint at 6p22 in the vicinity

of the E2F3 and DEK loci. These rearrangements may be considered as potential oncogene

activation events because of their close proximity. In addition, this rearrangement emphasizes

the involvement of 6p22 chromosomal band in retinoblastoma etiology.

The role of E2F3 gene in RB1 pathway of cell cycle control is well established [140,

142, 203]. Levels of E2F3 increase in late G1 phase in rat fibroblast cells [140], promoting the

transcription of genes needed for G1/S phase transition [143, 149]. The biological role of DEK

has not yet been fully elucidated. DEK is an abundant nuclear phosphoprotein, implicated in

chromatin architecture, transcription, and mRNA splicing [67, 85, 93, 94, 97, 130]. DEK levels

do not change during cell cycle, but the phosphorylation of DEK is cell cycle dependent,

reaching its peak at G1 [70]. E2F3 and DEK have both been implicated in cancer. E2F3 is

amplified and overexpressed in bladder cancer [187-190], and its expression level is an

independent prognostic indicator in prostate cancer [192]. DEK is translocated in acute

myeloid leukemia to nucleoporin CAN locus on chromosome 9, resulting in the production of

the fusion DEK-CAN protein with an unknown function [101]. DEK is overexpressed in a

number of different malignancies [66, 104, 110, 115, 118]. Recently, DEK was implicated as

the inhibitor of RB1-dependent cellular senescence [80, 120] and p53-dependent apoptosis

[121]. In in vitro studies, both E2F3 and DEK behave as oncogenes, with their overexpression

resulting in the promotion of anchorage independent growth [80, 172]. E2F3 is also weakly

oncogenic in vivo, when overexpressed from a keratin 5 promoter in transgenic mice [183]. It

is plausible that E2F3 and DEK could both be involved in retinoblastoma progression, by

performing complementary functions. In this model, E2F3 would promote proliferation of

RB1-/- retinal cells, which would otherwise stop dividing after a defined number of cell cycles

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[209]. DEK overexpression would inhibit RB1-/- cells from undergoing senescence as a

mechanism to stall the uncontrolled proliferation.

Retinoblastoma cell line Y79 does not possess gain of 6p, and has no i(6p). The

rearrangements involving chromosome 6 were, nonetheless, present. One chromosome 6 was

involved in interchromosomal translocation with chromosome 21, with the translocation

breakpoint mapping to the chromosomal band 6q24. The other chromosome 6 was involved in

the interchromosomal translocation with chromosome 7, at 6pter. Our SKY, DAPI-banding

and mBand analyses did not reveal the t(4;6)(p15;p21.1) described by Imbert et al. [215],

although other aberrations characteristic of Y79 were present. It is well known that

continuously cultured cell lines may exhibit minor variation in chromosomal content with time

and cytogenetic discrepancies between different clones of Y79 have been reported by Gilbert et

al. [9]

This study represents the first karyotype analysis of the retinoblastoma cell line

RB1021. Besides the gain of 1q, chromosome 2, 6p and a part of 14q, which were previously

detected by CGH [51], there is a gain of chromosome 21, as well as duplication of 7q.

Genomic losses involve 16q and one X chromosome.

Karyotypes of retinoblastoma cell lines RB247c and RB383 based on G-banding

analysis have been published [43]. The total number of chromosomes in RB247c has not

changed over the twenty years that this cell line has been in culture. SKY analysis resolved a

number of translocations present in this cell line. In RB383, number of chromosomes has

increased compared with the original karyotype published, with gain of chromosomes 7, 15,

17, 18, 21 and 22.

Overall, common minimal regions of gain in the examined cell lines were 1q23-1q42 (in all

examined cell lines), 2p (in RB1021, RB247c and RB383, plus the documented MYCN

amplification at 2p24 in Y79 [44, 216]), 6p (in RB1021, RB247c and RB383), 17q21.3-qter (in

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Y79, RB247 and RB383) and 21q21-21qter (all four cell lines; none of the patients had

constitutional chromosome 21 trisomy). Both cell lines derived from female patients (RB1021

and Y79) exhibited the loss of one X chromosome. MYCN gene, at 2p24, is implicated as the

target of 2p gain in retinoblastoma [43, 44]. The gain of 2p is present in ~30% retinoblastoma

tumors [51, 56, 217], with MYCN amplification (>10 copies) reported in 5-10% of tumors [43,

56]. The MYCN gene is frequently coamplified with DDX1 gene [218, 219], which is also

overexpressed in retinoblastoma [220]. In Y79, the hsr contains coamplified MYCN and

DDX1(both residing at 2p24) and ATP5A (residing at 18q12~21) as published previously

[219]. Genomic gain of 21q is found in several cancer types [195, 221-228], but is well

documented only in leukemia [229-234] with RUNX1 (AML1) being a target oncogene [104,

235-238]. Gain of 21q has not been studied in retinoblastoma.

Common translocation breakpoints map to 4p16 (in RB1021, RB247c and Y79), 11p15

(in RB247c and Y79), 17q21.3 (in RB1021, RB247c and Y79) and 20q13 (in RB247c, RB383

and Y79). A 4p16 translocation breakpoint common in multiple myeloma [239] leads to the

oncogenic activation of the fibroblast growth factor receptor 3 (FGFR3) gene. FGFR3 is an

activator of MAPK pathway and is frequently mutated in bladder cancer [240]. Its expression

in retinoblastoma has not been examined. Chromosomal band 4p16 is also listed as one of the

chromosomal fragile sites [241], hence, the observed translocation breakpoint could just be

coincidental to decreased chromosomal stability of tumor cell lines. 11p15 translocation

breakpoint is frequently found in hematopoietic malignancies and results in fusion of the

nucleoporin 98 (NUP98) gene with a number of different partners, resulting in the expression

of various fusion proteins [242]. Expression of NUP98 in retinoblastoma has not been

examined. A candidate tumor suppressor gene, p75NTR, maps to the chromosomal band

17q21.3. Expression of p75NTR is frequently lost or decreased in retinoblastoma [204], but no

changes in the genomic copy number of p75NTR are detected [54]. It is possible that a

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translocation breakpoint at 17q21.3 affects the expression of the p75NTR locus. Chromosomal

rearrangements involving 20q have been reported in osteosarcoma [54], and the gain of

chromosomal band 20q13 is a frequent event in solid tumors [243]. The candidate 20q13

oncogene in breast cancer is the transcription factor ZNF217 [30, 55, 244]. Expression of

ZNF217 or other 20q13 genes in retinoblastoma has not been studied.

Chromosomal translocations have been overlooked as a mechanism of M3-Mn events

in retinoblastoma. While classic karyotype studies had technical limitations, recent studies of

retinoblastoma M3-Mn events have mainly relied on CGH and quantitative multiplex PCR,

thereby focusing on recurrent regions of chromosomal gain and loss. By using spectral

karyotype analysis, DAPI banding and mBand analysis we have identified several common

translocation breakpoints in retinoblastoma. Precise localization of the recurrent translocation

breakpoint within chromosomal band 6p22 will provide more insight into the identity of the

6p22 oncogene in retinoblastoma and mechanisms of activation besides copy number increase.

In addition, pursuing other recurrent translocation breakpoints at 4p16, 11p15, 17q21.3 (along

with the simultaneous genomic gain of 17q21.3-qter) and 20q13 has potential to identify

additional M3-Mn target genes involved in retinoblastoma development and progression.

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ACKNOWLEDGMENTS

This work has been supported by the National Cancer Institute of Canada (NCIC) with

funds from the Canadian Cancer Society and Terry Fox Foundation.

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CHAPTER V:

Discussion

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CHAPTER OUTLINE:

1. DEK and E2F3 as targets of 6p chromosomal gain in retinoblastoma

(Chapter II)

2. Evidence that DEK and E2F3 overexpression in retinoblastoma is not the consequence of the

loss of function of RB1

(Chapter II)

3. Decrease of DEK or E2F3 level negatively affects the growth of retinoblastoma when 6p

genomic gain is present.

(Chapter III)

4. Recurrent translocation breakpoint at 6p in retinoblastoma as a mechanism of oncogene

activation

(Chapter IV)

5. Future directions :

5.1. Function of DEK and E2F3 in retinoblastoma

5.2. Identification of 6p translocation breakpoint

5.3. Mutational analysis of DEK and E2F3 in retinoblastoma

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1. DEK AND E2F3 AS TARGETS OF 6p CHROMOSOMAL GAIN IN

RETINOBLASTOMA

Formation of the isochromosome 6p, a retinoblastoma hallmark [42, 59], results in the

increased dosage of 6p genes in over 50% of retinoblastoma tumors. Chromosomal regions that

are recurrently gained in cancer were shown to harbor oncogenes. Increased dosage of a proto-

oncogene may be sufficient for it to display its oncogenic role. In this scenario, increased

dosage results in increased proto-oncogene product level, and the increased product level gives

cancer cells selective advantage [30-33].

Over 800 genes map to chromosomal arm 6p. To identify the genes that are relevant for

retinoblastoma, two approaches are crucial. First approach involves narrowing down the region

of gain. Tumors that have the gain of smaller portions of 6p provide more precise information

on the location of 6p oncogene(s). The second approach involves expression analysis of 6p

genes. Increased gene dosage will not result in increased expression of all 6p genes, due to

different mechanisms of gene regulation. Genes that are overexpressed when the genomic gain

is present are more likely to be involved in tumor development. By combining the two

described approaches, it is possible to limit the search to few oncogene candidates.

Comparative genomic hybridization is commonly used to map recurrent regions of

chromosomal gain and loss to particular chromosomal bands [48]. The overlapping minimal

region of 6p gain in four independent CGH studies of retinoblastoma is the chromosomal band

6p22 [50-53]. This chromosomal band spreads across ~15 Mb and is gene-rich, containing over

200 genes. Two groups attempted to further narrow down this region of gain using the

quantitative multiplex PCR to determine the copy numbers of UniSTS markers spanning 6p22

band, and identify the markers that were most frequently gained. In the first QM-PCR study,

published by Chen et al. in 2002, UniSTS marker SHGC-103950, that overlaps with KIF13A

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gene, was identified as the hotspot marker of gain, and the minimal region of gain was

narrowed to 0.6 Mb, delineated with DEK and NUP153 loci (markers X64229 and WI-19208,

respectively) [65]. The major disadvantage of this study was that the Human Genome

Sequencing Project was not completed, therefore the number of UniSTS markers used was

limited. Only five UniSTS markers were used in the first round of QM-PCR, which defined a 5

Mb minimal region of gain (delineated by UniSTS markers SHGC-130608 and WI-22629,

which overlaps with E2F3 locus), and additional four UniSTS markers were used to further

narrow down the MRG to 0.6 Mb. Only one UniSTS marker was used per gene locus. After the

completion of the Human Genome Sequencing Project, it became evident that one of the

markers used, WI-22629, which overlaps with E2F3 locus, is not unique for chromosome 6,

but also maps to chromosome 17, making it unsuitable for the QM-PCR study. The second

QM-PCR study was published by Grassman et al. in 2005. In this study a total of 15 UniSTS

markers were used, 13 covering the chromosomal band 6p22 and two markers mapping to

either 6p21 or 6p23 chromosomal band. Some of the gene loci were covered by two UniSTS

markers. The hotspot marker of gain was identified to be X64229, overlapping with the DEK

locus (the same hotspot marker was identified in the study of 6p22 gain in bladder cancer

[116]) and the minimal region of gain was narrowed down to 2.5 Mb, delineated by DEK and

E2F3 genes [66]. Therefore, two independently identified 6p22 minimal regions of gain

partially overlap on the DEK locus.

Expression analysis of 6p22 MRG genes in retinoblastoma, presented in Chapter II,

showed that, of the six genes within 0.6 Mb MRG, only DEK was overexpressed at both

mRNA and protein level in retinoblastoma tumors with genomic gain of 6p. The expression

analysis was based on real-time RT-PCR and western blotting of retinoblastoma tumors and

healthy human retinas, and on immunofluorescent staining of eyes enucleated to treat advanced

retinoblastoma. Furthermore, DEK mRNA level correlated with 6p genomic copy number.

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E2F3 gene was included in the study because, as discussed in Chapter II, it is a plausible

oncogene candidate that was in the initial 5 Mb minimal region of gain defined by QM-PCR, it

is involved in RB1 pathway of cell cycle control [138, 139, 145], and there was accumulated

evidence for the oncogenic involvement of E2F3 in bladder and prostate cancer [188, 189,

192]. Similarly to DEK, E2F3 was overexpressed at both mRNA and protein level in

retinoblastoma tumors with 6p gain, with all three described expression analysis techniques.

Overall, E2F3 mRNA level was higher in tumors with 6p genomic gain. However, it was

overexpressed in all retinoblastoma tumors examined.

Findings of the study presented in Chapter II were corroborated by the study conducted

by Grasemann et al., which was published at the same time that our manuscript was accepted

for publication. Grasemann et al. used a different approach for expression analysis, RNA

expression microarray, testing many more 6p22 genes than our study. The only two candidate

oncogenes identified by Grasemann et al., based on the integration of genomic copy number

analysis and differential expression analysis between tumors with and without genomic 6p

gain, were E2F3 and DEK [66].

The combined findings of four CGH studies, two QM-PCR studies and two expression

analysis studies, provide strong evidence that 6p22 is the region containing an oncogene(s) in

retinoblastoma, and that DEK and E2F3 are the two most likely 6p22 oncogene candidates,

minimizing the probability that another important 6p22 proto-oncogene exists.

DEK and E2F3 have been proposed as oncogenes in other cancers. DEK is translocated

to nucleoporin CAN in 1% of AML patients [101, 103], and overexpressed in AML [104, 105],

liver cancer [110, 111], glioma [112], melanoma [82, 113], colon cancer [82], larynx cancer

[82], prostate cancer [114], bladder cancer [82, 115] and neuroblastoma [117]. E2F3 is

amplified and overexpressed in bladder carcinomas with 6p22 gain [187-189], Wilm’s tumor

[184], prostate cancer [192], ovarian cancer [185] and lung cancer [186]. The oncogenic

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potential of DEK and E2F3 was demonstrated in cultured cells [80, 172, 190, 191], and E2F3

was shown to be oncogenic in vivo [183].

The 0.6 Mb MRG at 6p22 in retinoblastoma is syntenic to a 0.4 Mb region on mouse

chromosome 13. E2F3 is, however, much more distant from this region on mouse chromosome

13 (17 Mb, compared to only 2 Mb on human chromosome 6). mRNA expression analysis of

mouse retinoblastoma tumors, presented in chapter II, shows that Dek and E2f3 are both

overexpressed in mouse TAg-induced retinoblastoma, compared to adult and P7 mouse retina,

with the higher relative increase in Dek mRNA levels. Immunofluorescent analysis of Dek

expression in TAg-RB mice, presented in Chapter II, shows that the cell of origin of TAg-

induced mouse retinoblastoma expresses Dek and that Dek levels are increased in a portion of

TAg-induced retinoblastoma tumors, further supporting the oncogenic role of DEK in

retinoblastoma.

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2. EVIDENCE THAT DEK AND E2F3 OVEREXPRESSION IN

RETINOBLASTOMA IS NOT DIRECTLY DUE TO THE LOSS OF

pRB

Transcriptional activity of cell cycle promoting E2Fs, including E2F3, is repressed by pRB

[23]. In addition, transcription of E2F loci is also regulated by pRB/E2F complexes. The E2F3

promoter contains binding sites for E2F/pRB and in G0/early G1 phase transcription of E2F3

is repressed [139, 142]. Therefore, it is expected that the loss of function of RB1 will result in

the increased expression of E2F3, promoting the cell cycle. During G1/S transition, E2F3

transcription levels rise 15-fold in rat fibroblasts [140]. Indeed, many retinoblastoma cell lines

with no genomic gain of 6p exhibit ~10-fold increase in E2F3 mRNA level. Tumors with 6p

gain show E2F3 mRNA levels that are on average higher than those attributed to the release of

the pRB transcriptional block characteristic for dividing cells. This, however, does not prove

that E2F3 overexpression is the consequence, directly or indirectly, of M3-Mn events that

occur after the loss of function of RB1.

Transcriptional regulation of DEK has not been thoroughly established. DEK was

shown to be regulated by cancer-implicated transcription factors YY1 and NF-Y [72]. It also

possesses pRB/E2F binding sites in its promoter [82], and YY1 was also shown to function in

the complex with E2F2 or E2F3 [161]. It has recently been published that an increase in E2F

levels increases the transcriptional activity of the DEK locus [82]. Therefore, it is highly likely

that DEK transcription is pRB dependent.

Dimaras et al. show that retinoma, a benign retinal tumor that precedes retinoblastoma

in ~15% of eyes removed to treat retinoblastoma, contains identical RB1 gene mutations as the

adjacent retinoblastoma (manuscript in preparation). Therefore, M1 and M2 events may be

97

insufficient for uncontrolled cell-of-origin proliferation, rather giving rise to the benign tumor,

retinoma. If M3-Mn events do not ensue, retinoblastoma does not develop.

Analysis of DEK and E2F3 expression in retinoblastoma, adjacent retinoma and

healthy retina of the same eye by immunofluorescent staining, presented in Chapter II,

indicates that although DEK is expressed in retinoma, its expression is much higher in 2/4

examined adjacent retinoblastomas; E2F3 protein is almost non-detectable by

immunofluorescent staining in healthy retina and retinoma, and is overexpressed in all

examined adjacent retinoblastoma cases.

Thus overexpression of DEK and E2F3 cannot be attributed to M1 and M2 events that

result in the loss of function of RB1, but rather is the consequence, either directly or indirectly,

of additional M3-Mn mutational changes.

The biological relevance of E2F3 overexpression in retinoblastoma can be speculated based on two studies. Bremner et al. showed that retinal cells that have lost Rb1 do proliferate excessively, but after a defined number of cell cycles they arrest and become quiescent. Hence, it is plausible that another hit in a proliferation-promoting gene in the pRB pathway of cell cycle control is the by which a RB1-/- retinal cell avoids cell cycle arrest to proliferate indefinitely [209]. Olsson et al. showed that E2F3 overexpression gives selective growth advantage only to pRB depleted prostate cancer cell lines [191], supporting the hypothesis stated by Bremner et al. [209]. Check 1.Wenzel P. L., Wu L., de Bruin A., Chong J. L., Chen W. Y., Dureska G., Sites E., Pan T., Sharma A., Huang K., Ridgway R., Mosaliganti K., Sharp R., Machiraju R., Saltz J., Yamamoto H., Cross J. C., Robinson M. L., Leone G. Rb is critical in a mammalian tissue stem cell population. Genes & development. 2007 Jan 1;21(1):85-97.

DEK was shown to inhibit senescence in cell lines in which pRB has been inactivated

by expression of the pRB binding protein E7 of the human papilloma viruses [80]. DEK is also

proposed as an inhibitor of p53-dependent apoptosis [121]. As shown by Dimaras et al. (in

preparation) , retinoma cells exhibit senescence-like properties, and it is plausible that DEK

overexpression is one way for RB1-/- retinal cells to avoid senescence and proliferate. However,

data presented in Chapter III of this thesis is more supportive of the role of DEK as an

98

apoptosis inhibitor, since the level of apoptosis of the retinoblastoma cell line RB247c was

increased upon DEK knockdown.

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3. DECREASE OF DEK OR E2F3 EXPRESSION NEGATIVELY

AFFECTS THE GROWTH OF RETINOBLASTOMA WHEN 6p

GENOMIC GAIN IS PRESENT

In Chapter III of this thesis the oncogenic potentials of DEK and E2F3 in retinoblastoma

were tested by shRNA-mediated knockdown of gene expression in retinoblastoma cell lines.

Two cell lines were studied that differ with respect to chromosome 6p changes, Y79 and

RB247c. Y79 does not show genomic gain of 6p, has two copies of both DEK and E2F3 loci,

and overexpresses E2F3 but not DEK. Retinoblastoma cell line RB247c has four copies of 6p,

four copies of DEK and E2F3, and overexpresses both genes.

We show that knocking down either DEK or E2F3 had no effect on the growth rate of Y79,

while the knockdown of any of the two genes affected the growth of RB247c.

In the case of E2F3 knockdown in RB247c, slower growth was observed, with an increased

number of cells expressing activated caspase 3, an apoptosis marker. Also, approximately 40%

of cells expressed cyclin B1, suggestive of their accumulation at G2/M transition and inability

to undergo mitosis. This is corroborated by the finding that no decrease in the rate of BrdU

incorporation was detected, and cells unable to progress through mitosis were found to bounce

back to G1 and undergo endoreduplication [207].

DEK knockdown in RB247c resulted in a negative growth rate. There was a reduction in

the proliferation index, measured by both Ki67 proliferation marker or BrdU incorporation,

and ~20% of cells with relatively intact nuclei were expressing activated caspase 3, suggesting

that they were en route to programmed cell death. No senescent cells were detected.

Combined, these data show that both E2F3 and DEK overexpression are important for

growth of retinoblastoma cell line RB247c, with the viability of RB247c being dependent on

the level of DEK. In addition, since RB247c possesses genomic gain of 6p, while Y79 does

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not, these experiments suggest that DEK overexpression related to the genomic gain of 6p

drives the oncogenic process, while E2F3 may be a consequence of oncogenesis.

A similar approach was used to test the oncogenic potential of E2F3 in bladder cancer

[190] [191]. The 6p22 amplicon in bladder cancer cell lines usually contains two co-amplified

genes: E2F3 and CDKAL1. Both genes are overexpressed when the amplification is present.

These genes were separately knocked down by siRNA in two bladder cancer cell lines, CRL-

7930, which does not have 6p22 amplification, and HTB-5, which has amplification of both

E2F3 and CDKAL1. Only E2F3 knockdown had an effect on HTB-5, leading the authors to

conclude that E2F3 is the gene that drives 6p22 amplification.

4. RECURRENT TRANSLOCATION BREAKPOINT AT 6p IN

RETINOBLASTOMA MAY ACTIVATE ONCOGENE(S)

Analysis of four retinoblastoma cell lines by spectral karyotyping, DAPI banding, mBAND

for chromosome 6 and locus-specific FISH for DEK and E2F3 genes, presented in Chapter IV,

revealed that two retinoblastoma cell lines with genomic gain of 6p, RB247c and RB1021,

exhibit a translocation involving 6p and 6q chromosomal arms, with a common translocation

breakpoint at 6p22.

Chromosomal translocations are a well established mechanism of oncogene activation [25].

Translocations may involve coding regions of two different genes, resulting in the formation of

a fusion gene, that gives rise to a fusion protein product. The fusion protein may perform a

novel role in the cell such as promoting cancer cell growth, i.e. exhibiting gain of function.

This is the case with the chromosomal translocation between chromosomes 9 and 22 that

results in the formation of the Philadelphia chromosome in chronic myelogenous leukemia,

which leads to the production of the BCR/ABL fusion protein [36]. Translocations may also

involve the coding region of one gene and the regulatory region of another gene, resulting in

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aberrant expression of either a full length or a truncated protein that functions as an oncogene.

This is the case with MYC translocation to promoters of immunoglobulin genes in Burkitt’s

lymphoma [34].

DEK was discovered as the gene involved in translocation to the nucleoporin

CAN(NUP214) locus on chromosome 9, resulting in the expression of a fusion DEK-CAN

protein in 1% of acute myeloid leukemia patients [101, 103]. The role of DEK-CAN protein

has not yet been established. E2F3 translocations have not been reported in cancer.

In retinoblastoma cell lines, the translocation breakpoint at 6p22 lies in the proximity of

the DEK and E2F3 loci. Both DEK and E2F3 show only the protein products of expected size

by immunoblotting, excluding the possibility of fusion protein formation or expression of a

truncated protein product. Since the DEK locus is telomeric to E2F3 and the breakpoint is

centromeric to E2F3, E2F3 is more likely to be affected by the translocation. If the recurrent

translocation breakpoint at 6p22 affects the E2F3 locus, it likely acts to deregulated gene

expression.

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5. FUTURE DIRECTIONS

5.1. Function of DEK and E2F3 in retinoblastoma

In Chapter III of this thesis it is shown that both DEK and E2F3 perform oncogenic

functions in retinoblastoma. However, the mechanism of their action and the relationship

between pathways involving DEK and pathways involving E2F3 remain to be examined and

elucidated.

Acute loss of RB1 in primary quiescent cells is sufficient for cell cycle re-entry [245].

However, most RB1-/- cells will go through a defined number of cell cycles before they become

growth arrested [209]. The mechanism of this arrest is not known. Aberrant expression of pRB

repressed genes, such as E2F3, upon RB1 loss, may represent an oncogenic signal that

activates p14ARF expression [168]. p14ARF in turn was shown to induce degradation of E2F3

[178], which would relieve the inhibition of the p53-p21 tumor suppressor axis and halt the cell

cycle [182]. p53 mutations have not been detected in retinoblastoma [246-249]. Therefore,

retinoblastoma tumors likely have the means of keeping this important tumor suppressor

pathway in check. The missing link could be p14ARF. It is plausible that DEK is involved in

negative regulation of p14ARF (figure..….). It has been shown that DEK inhibits the activation

of p53 pathway by an unknown mechanism [121]. Knocking down DEK in HeLa cells induces

both p53 expression and expression of downstream targets of p53, such as p21Cip1 and BAX

[121]. The level of DEK remaining in the cell is proposed as the switch that will decide

between p53-mediated senescence and p53-mediated apoptosis [121].

Based on the regulatory sites present in the promoter region of DEK, DEK is probably

transcriptionally repressed by pRB [72, 82]. However, slight increase in DEK transcription

levels upon RB1 loss may be insufficient for DEK to perform its oncogenic role. Therefore,

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6p22 gain resulting in DEK overexpression would enable DEK to inhibit p14ARF function in

E2F3 degradation, which would enable E2F3 to perform its cell cycle promoting role, and

allow RB1-/- cells to avoid p53 pathway activation. In addition, levels of E2F3 would also be

boosted by genomic gain of E2F3 locus (Figure 5.1). In support to DEK’s role in apoptosis, it

was found that DEK protein undergoes specific post-translational modifications in apoptosing

cells, represented by the reduction in phosphorylation levels [250]. The umerous post-

translational modifications of DEK have not yet been taken into account when examining DEK

protein-protein interactions and different processes in which it is implicated.

Future experiments should therefore focus on examining the connection between DEK and

E2F3 overexpression and p53-mediated pathways of cell cycle arrest and apoptosis. This could

be done by examining the effect of DEK/E2F3 knockdown on the expression of p53 regulated

genes, such as p21Cip1 and BAX, and the expression of p14ARF, in retinoblastoma. Also, it could

be determined if knock down of any p53 downstream genes or p14ARF, simultaneously with

DEK/E2F3 knockdown, rescues the growth properties of retinoblastoma cells.

The importance of DEK and E2F3 could be examined in the large T antigen (TAgRB)

transgenic mouse retinoblastoma model, by crossing the mice with Dek-/- or E2f3-/- mice and

measuring tumor formation. Alternatively, treatment of developed retinoblastoma in TAg-RB

mice can be attempted by injecting the eyes with anti-Dek or anti-E2f3 viruses or anti-Dek/anti-

E2f3 siRNAs.

5.2. Identification of 6p translocation breakpoint

The recurrent translocation breakpoint at 6p22 is potentially another clue into the identity

of the 6p22 oncogene in retinoblastoma and the mechanism of its activation. To precisely

determine the breakpoint, two approaches may be attempted.

104

The first approach would involve comparison of restriction fragments in RB1021 and

RB247 and in healthy retina, or in the white blood cells of patients from which RB1021 and

RB247 developed. Specific probes for the E2F3 gene could be used, since E2F3 is more likely

affected by translocation than DEK, based on its position relative to the breakpoint. If the

breakpoint is not found within E2F3, than probes for other 6p22 genes centromeric to E2F3

should be used.

Alternatively, genomic libraries of RB1021 and RB247c should be made, and all the clones

that contain portions of 6p22 sequenced in order to identify the breakpoint(s). This approach is

potentially more costly, but is straightforward and will yield the answer within a defined

timeframe.

5.3. Mutational analysis of DEK and E2F3 in retinoblastoma

Mutations in the DEK gene have been identified in acute myeloid leukemia, as the

consequence of DEK translocation to CAN(NUP214) locus on chromosome 9 [101]. Other

mutations in the DEK gene have not been reported.

Both DEK and E2F3 function as regulators of transcription. DEK is subject to numerous

post-translational modifications, and the E2F3 protein product is known to contain several

protein-protein interaction domains important in the control of its function. It is very important

to examine the existence of mutations in the regions that are known to be or could be involved

in the negative regulation of DEK/E2F3, since these mutations could represent the activators of

the oncogenic function of DEK/E2F3. The mutational analysis should involve tumors with and

without gain of DEK and E2F3.

105

E2F3 mRNA possesses a long 3’ UTR, which is implicated in the negative regulation of

E2F3 by microRNAs [171]. Thorough mutational analysis of this noncoding region of E2F3

cDNA is therefore necessary.

106

APPENDICES

107

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