Dept. of Molecular Biology and Genetics - Ph.D. / Sc.D.
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Item Open Access Analysis of BRCA1 and BRCA2 genes in Turkish breast cancer patients(Bilkent University, 2000) Özdağ, HilalBreast cancer is the most frequent cancer type and the second cause of death among women. It is estimated that 10 to 15% of breast cancer cases are hereditary. The majority of hereditary breast cancers can be attributed to germ-line mutations in ^Jgeast CAncer susceptibility genes BRCAl and BRCA2. In this study, germ-line BRCAl and/or BRCA2 gene mutations were screened in 50 Turkish breast and/or ovarian cancer patients divided into four groups of hereditary, familial, early onset, and male cancer by heteroduplex analysis and DNA sequencing. Two BRCA2 mutations, one novel (6880insG) and one previously reported (3034delAAAC), were found in the hereditary group. A novel BRCAl (1200insA) mutation was formd in the early onset group. All three mutations cause premature- termination codons. In addition, five BRCAl sequence variants have been identified in 23 patients. K654E (2080 A—>G), D693N (2196 G->A), P871L (2731 C—>T), and K1183R (3667 A—>G) result in a change of amino acids. 1013 T—^>C and 2201 C—>T are silent mutations. One patient in the early onset group was compound heterozygote for K654E and D693N. These results indicate that BRCAl and BRCA2 genes are involved in some but not all hereditary breast cancers in the Turkish population.Item Open Access Tumor suppressor functions of p53 gene(Bilkent University, 2000) Ünsal, KezibanItem Open Access Analysis of GSTM1, GSTT1, GSTP1, and TP53 polymorphisms as genetic risk factors for bladder cancer in the Turkish population(Bilkent University, 2001) Törüner, Gökçe AltayThe effect of the GSTM1 and GSTT1 null genotypes, the GSTP1 Ile105Val, and TP53 Arg72Pro polymorphism on bladder cancer susceptibility was investigated in a case control study of 121 bladder cancer patients, and 121 age-sex matched controls in the Turkish population. The adjusted odds ratio (for age, sex, and smoking status) for the GSTM1 null genotype is 1.94 (95% CI 1.15- 3.26) and for the GSTP1 105 Ile/Val or Val/Val genotypes is 1.75 (95% CI 1.03- 2.99). GSTT1, and TP53 loci was not shown to be associated with bladder cancer. Combination of the two high risk genotypes, GSTM1 null and GSTP1 105 Ile/Val or Val/Val, revealed that the risk increases by 3.91 times (95% CI 1.88-8.13) when compared with the combination of the low risk genotypes of these loci. In individuals with a combined risk of cigarette smoking and the GSTM1 null genotype, bladder cancer risk is 2.81 (95% CI 1.23-6.35) relative to persons who do not smoke and carry the GSTM1 present genotype. The same risk for the GSTP1 105 Ile/Val or Val/Val genotypes is 2.38 (95% CI 1.12-4.95). These findings support the role for the GSTM1 null and the GSTP1 105 Ile/Val or Val/Val genotypes in the development of bladder cancer. Furthermore, gene-gene (GSTM1- GSTP1) and gene-environment (GSTM1-smoking, GSTP1-smoking) interactions increase this risk substantially.Item Open Access Identification of the role of the nuclear matrix protein C1D in DNA repair and recombination(Bilkent University, 2002) Erdemir, TubaThe nuclear matrix protein C1D is an activator of the DNA dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks (DNA-DSBs). C1D is phosphorylated very efficiently by DNA-PK, and its mRNA and protein levels are induced upon γ-irradiation, suggesting that C1D may play a role in repair of DNA-DSBs in vivo. In an attempt to identify the possible biological functions of C1D and the nuclear matrix, two approaches were employed. One of the strategies was to apply yeast-two hybrid system to screen for polypeptides that interact with C1D. This screening revealed a number of cDNA clones that encode mainly proteins involved in recombination, DNA repair and transcription. Among the identified proteins, TRAX (Translin Associated protein X) was chosen for further analysis. Although, the biological function of TRAX remains unknown, its bipartite nuclear targeting sequences suggest a role for TRAX in the movement of associated proteins including Translin, into nucleus. After cloning the full-length TRAX ORF into bacterial and mammalian expression vectors, the specificity of the interaction between C1D and TRAX was confirmed both in vivo and in vitro conditions. Notably, it was shown that C1D and TRAX interact in mammalian cells only after γ- irradiation. In addition, it was demonstrated that the induced interaction of TRAX and C1D is not due to the alterations in their subcellular localizations but possibly through post-translational modifications in response to γ-irradiation. The second strategy was identification of the S. cerevisiae C1D homolog. Because of the high homology between yeast and mammalian C1D proteins, S. cerevisiae was used as a model organism to reveal the function of Yc1d. YC1D gene was knocked-out in yeast and the phenotypic and functional consequences of disruption of the YC1D gene was analyzed. It was found that yc1d mutant strain was slightly sensitive to γ-irradiation and was defective in DNA-DSB repair, thus, raising the possibility that yeast C1D and human C1D might be functional homologs.Item Open Access p53 mutations as a source of aberrant Beta-catenin accumulation in cancer cells(Bilkent University, 2002) Çağatay, T.β-catenin is involved in both cell-cell interactions and wnt pathway-dependent cell fate determination through its interactions with E-cadherin and TCF/LEF transcription factors, respectively. Cytoplasmic/nuclear levels of β-catenin are important in regulated transcriptional activation of TCF/LEF target genes. Normally, these levels are kept low by proteosomal degradation of â-catenin through Axin1- and APC-dependent phosphorylation by CKI and GSK-3β. Deregulation of β-catenin degradation results in its aberrant accumulation, often leading to cancer. Accordingly, aberrant accumulation of β-catenin is onberved at high frequency in many cancers. This accumulation correlates with either mutational activation of CTNNB1 (β-catenin) or mutational inactivation of APC and Axin1 genes in some tumors. However, there are many tumors that display β-catenin accumulation in the absence of a mutation in these genes. Thus, there must be additional sources for aberrant β-catenin accumulation in cancer cells. Here, we provide experimental evidence that wild-type β-catenin accumulates in hepatocellular carcinoma (HCC) cells in association with mutational inactivation of p53 gene. We also show that worldwide p53 and β-catenin mutation rates are inversely correlated in HCC. These data suggest that inactivation of p53 is an important cause of aberrant accumulation of β−catenin in cancer cells.Item Open Access Acquired expression of transcriptionally active p73 in hepatocellular carcinoma cells(Bilkent University, 2002) Sayan, A. EmreP53 gene is the most common mutated tumor suppressor gene during tumorigenesis. From its description till 1997, p53 gene was thought to stand alone in the human genome. In 1997, p73 gene and in 1998, p63 gene was identified which are encoding functional homologues of p53 protein. Unlike p53, the knock out mice for p73 and p63 genes did not yield a tumor prone phenotype and the mutation frequency of these genes is very low compared to p53 gene. There is also extensive alternative splicing and changes in the expression pattern of p73 and p63, unlike p53. Thus the new p53 homologues were considered as non-classical and non-Knudson type tumor suppressor genes. A codon specific, aflatoxin ingestion related p53 mutation was shown to be important in the ethiopathology of HCC so loss of p53 function is a major factor during HCC development. The rate of p53 functional inactivation was determined by lots of studies in HCC but the knowledge for new p53 homologues is scarce. We aimed to define the probable function of the new p53 homologue, p73 in HCC development. For this purpose, we have analyzed the 3’ alternative splicing and expression pattern of p73 in a series of HCC derived cell lines. Our results showed the alteration of splicing and expression in HCC cell lines compared to normal liver. After the completion of human genome project, the contig harboring the p73 gene was entered to the public database. With the hints of the presence of an alternative promoter in the p63 gene and the description of the alternative promoter in mouse p73 gene, we have made an in silico analysis to identify the probable promoter and exon within p73 gene. Our studies revealed the in vivo description of a new human p73 encoded transcript. The proposed protein product of the transcript was lacking the transactivation domain so it was named as Dominant Negative p73 (DN-p73) and the former p73 was renamed as Transactivating p73 (TA-p73). Since the promoters of these two transcripts are different and probably under the regulation of different transcription factors, we studied the expression pattern of them by semi quantitative RT-PCR method. We have shown the presence of only DN-p73 in normal in normal liver. HCC derived cell lines and primary HCC tumors also express DN-p73 together with the acquired expression of TA-p73 in most of the cell lines and some of the primary HCC tumors. The promoter of TA-p73 was shown contain E2F1 transcription factor binding sites. The Retinobastoma protein (pRb) is the most potent inhibitor of the E2F1 transcription factor and the dysrégulation of the Rb pathway components is a common event in HCC development (Rb gene mutations and proteolytic dysrégulation of pRb and mutational and epigenetic inactivation of pi6). We have shown the expression of TA-p73 in some of the HCC derived cell lines and primary HCC tumors so the acquired expression of TA-p73 in HCC cells might be the indicator and the effect of of Rb pathway dysrégulation. We tested this hypothesis by analyzing the expression of pRb and pi6, together with the endogenous E2F1 transcription factor targets such as cyclin E, pi4'^’^ and TA-p73. Our results showed a 75% inactivation of Rb pathway components and a partial correlation of TA-p73 expression in HCC cells. The acquired expression of TA-p73 in HCC cells is unfavorable during tumorigenesis since TA-p73 mimics the pro- apoptotic and cell cycle regulatory, function of wild type p53. Mutant p53 proteins were shown to inhibit the pro-apoptotic fliction of wild-type p53 and TA-p73. We have analyzed the p53 protein status of 15 HCC derived cell lines and defined the presence of mutant p53 or no functional p53 protein in 87% of the HCC derived cell lines. As a summary, we have identified the human homologue of mouse DN-p73 and defined the 3’ alternative splicing and 5’ differential promoter initiation products of p73 gene encoded products in normal liver versus a series of HCC derived cell lines and primary tumors. Moreover we have correlated the expression of TA-p73 with Rb pathway inactivation and expression of mutant p53 proteins.Item Open Access Identification of genes induced by BRCA1 in breast cancer cells(Bilkent University, 2002) Atalay, ArzuInherited mutations of the BRCA1 gene predispose to cancer of the breast, ovaries and other organs. The BRCA1 protein product is implicated in the maintenance of chromosomal integrity as BRCA1-deficient cells display gross chromosomal rearrangements. Chromosomal instability in BRCA1-deficient cells is related to inappropriate DNA double-strand break repair. The role of the BRCA1 gene in the maintenance of chromosomal integrity is linked to a number of biological properties of its protein product including transcriptional regulation. The aim of this study is to identify genes that are regulated by BRCA1. Initial attempts to overexpress BRCA1 in breast cancer cells with the tightly-regulated ecdysone inducible system did not result in the desired levels of BRCA1 protein and ectopic BRCA1 expression was therefore performed by using the constitutive expression vector. In this study, we have identified genes whose expression levels are upregulated as a result of BRCA1 overexpression in MCF7 breast carcinoma cells by using the suppression subtractive hybridisation (SSH) method. Differential screening, sequencing and homology search studies showed that BRCA1 overexpression in breast cancer cells leads to transcriptional upregulation of distinct classes of genes encoding proteins involved in cellular processes such as DNA repair, chromosome assembly and segregation, signal transduction, RNA surveillance, ubiquitin-mediated proteolysis, amino acid transport, RNA metabolism and glucose metabolism. This study is the first to report BRCA1- induced genes in breast carcinoma cells with the SSH technique. The identified genes in this study may provide new insights into the tumour suppressor functions of BRCA1.Item Open Access NAPO as a novel apoptosis marker(Bilkent University, 2002) Sayan, Berna S.Apoptosis or programmed cell death plays a pivotal role in embryonic development and maintenance of homeostasis. It is also involved in the etiology of pathophysiological conditions such as cancer, neurodegenerative, autoimmune, infectious and heart diseases. Consequently, the study of apoptosis is now at center of both basic and clinical research applications. Therefore sensitive and simple apoptosis detection techniques are required. This study involves identification and characterization of a monoclonal antibody-defined novel antigen, namely NAPO (negative in apoptosis), which is specifically lost during apoptosis. The anti-NAPO antibody recognizes two nuclear polypeptides of 60 kD and 70 kD. The antigen is maintained in quiescent and senescent cells, as well as in different phases of the cell cycle including mitosis. Thus, immunodetection of NAPO antigen provides a specific, sensitive and easy method for differential identification of apoptotic and non-apoptotic cells.Item Open Access Hepatocellular carcinoma viral etiology and molecular mechanisms(Bilkent University, 2003) Yıldız, EsraHepatocellular carcinoma (HCC) is one of the most frequent carcinomas throughout the world, being responsible for more than 1 million deaths annually and has a strong association with several etiological factors including aflatoxinB1, alcohol and Hepatitis virus B and C. Several studies suggested that HCV subtype 1b causes more severe liver diseases including HCC in a high manner and resistance to antiviral therapy. So, it is important to know genotype and some characteristics of HCV which are unique for the countries to develop better strategies regarding public health. By using direct sequencing information from 5`UTR and NS5B regions we identified subtype 1b as a predominant hepatitis C virus genome in Turkey. Next, the full genome sequence of a Turkish 1b isolate (HCV-TR1) was obtained by cloning of polypeptide-encoding region into 7 overlapping fragments. Although major structural and functional motifs of HCV proteins were maintained in HCV-TR1, it displayed amino acid substitutions in 6 out of 9 major cytotoxic T-cell epitopes. Several HCV proteins have been reported to contribute hepatocellular malignancy by interaction with critical cellular proteins involved in hepatocyte proliferation and survavil. Such studies often use HCC-derived cell lines as experimental models. As a prerequisite to future studies about the Turkish HCV 1b isolate in term of its contribution to HCC developments we investigated on phenotypic characterization of HCC cell lines. We provide experimental evidence that α-fetoprotein-producing HCC lines display in vitro liver stem cell-like properties with self-renewing capability and multi-lineage differentiation potential, even after single-cell cloning. However, their ability to generate fully differentiated normal progeny was disrupted, even if they modulate their differentiation program in response to external factors. These features qualify AFP-producing HCCs as “mis-specified” liver stem cell cancers whose cellular programs are deviated from repopulating liver to forming malignant tumors. Interestingly, stem-like cells described here have been used extensively to study the role HCV proteins. Our observations offer new opportunities for addressing the potential role of HCV in the misspecification of liver stem cells in relation with viral hepatocellular carcinogenesis.Item Open Access Acquired tolerance of hepatocellular carcinoma cells to selenium-deficiency : a selective survival mechanism(Bilkent University, 2003) Irmak, Meliha BurcuSelenium-deficiency causes liver necrosis. Selenium is protective against viral hepatitis and hepatocellular carcinoma (HCC). The underlying molecular mechanisms of selenium effects are ill-known. In this study in vitro response of hepatocellular carcinoma-derived cell lines to selenium-deficiency is examined alone or in conjunction with Vitamin E and Copper/Zinc. Here we show that in vitro selenium-deficiency in a subset HCC-derived ‘hepatocyte-like’ cell lines causes oxidative stress and apoptosis. The oxidative stress and consequent cell death induced by selenium-deficiency on these cells are reverted by the antioxidant effect of Vitamin E. However, ten among thirteen HCC cell lines are tolerant to selenium-deficiency and escape its deadly consequences. Nine of ten tolerant cell lines have integrated hepatitis B Virus (HBV) DNA in their genomes, and some display p53-249 mutation, indicating past exposure to HBV or aflatoxins, established factors for oxidative stress and cancer risk. Thus, as demonstrated by the gain of survival capacity of apoptosis sensitive cell lines with Vitamin E, such malignant cells have acquired a selective survival advantage that is prominent under selenium-deficient and oxidative stress conditions.Item Open Access Analysis of candidate molecular targets in adult (CML) and childhood (AML, ALL) Leukemias(Bilkent University, 2004) Boylu, Cemaliye AkyerliCandidate molecular targets were investigated in three different types of leukemias, chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). The first group of these molecular targets was identified through a cDNA based gene expression profile analysis in sixty-seven CML patients who were classified according to clinical parameters known as new prognostic score (NPS). CML patients can be divided into three groups of low-risk, intermediate-risk, and high-risk, based on NPS. Response of these risk groups to treatment is not uniform and the gene expression profiles associated with each risk group remain unknown. Seven genes were chosen from a cDNA microarray study in which two high versus two low-risk patients were analyzed. Semi-quantitative and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of these differentially expressed transcripts highly correlated with the microarray data. Expression levels of all genes, except PTGS1, were significantly different between the high (n=9) and low-risk (n=7) CML by semi-quantitative RT-PCR (IFITM1 and CXCL3 p=0.001; CCNH p=0.012; RAB1A p=0.01, PRKAR2B p=0.016; UCP2 p=0.04; and PTGS1 p=0.315). Real-time RT-PCR analysis showed similar results for IFITM1 expression in thirty-four low and eleven high-risk patients (p=9.7976 x 10-11). Higher IFITM1 or lower CXCL3 expression correlated with improved survival (p=0.01 and p=0.059 respectively). Gene expression profiling is a valuable tool to identify candidate risk group indicator genes for the development of a molecular classification system for CML, which may also predict survival. Although the connection between DNA-repair gene mutations and hematological malignancies are now well established, germ-line mutations in the base excision repair (BER) pathway was only recently documented in an inherited cancer syndrome in human homologue of E. coli mut Y (MYH). Interestingly, the cancer associated MYH missense mutations Tyr165Cys and Gly382Asp have been documented with a high frequency (1 percent) in a control group of the British population. Therefore, we screened the above mentioned missense variants in two different childhood leukemias, AML (n=45) and ALL (n=140). Neither mutation was present in any of the patient samples and controls, except for one patient diagnosed with AML/M3. Tyr165Cys mutation in the heterozygous state was present in the sample obtained at the time of initial diagnosis. Further sampling, at remission, and the analysis of parental DNA, showed only the normal allele. Therefore, the mutation was considered to be specific for the leukemic blasts. Based on these results, an association between childhood leukemias and the MYH missense variants Tyr165Cys and Gly382Asp was not observed. Also, these variants appear to be absent -if not at a very low frequency- in the Turkish population, contrary to the British population.Item Open Access WNT/(formula)-Catenin signaling pathway activation in epithelial cancers(Bilkent University, 2006) Benhaj, KhemaisWnt signaling is involved in a large set of cellular and developmental processes, and when mis-regulated can lead to both degenerative diseases and many types of cancer. The involvement of Wnt signaling was already well demonstrated in several types of human cancers such as colorectal cancer. However, in some others such as hepatocellular carcinoma (HCC) and breast cancer, the role of Wnt signaling is not fully understood. To study the role of Wnt pathway in liver cancer, we first classified human hepatoma cell lines into well-differentiated and poorly differentiated groups using hepatocyte-specific biomarkers. Wnt/β-catenin signaling activity was measured using TCF/LEF-dependent reporter assay. Canonical Wnt/β-catenin signaling was constitutively active in 80% of well differentiated and 14% of poorly differentiated cell lines, respectively. Furthermore, ectopic expression mutant of S33Y β-catenin resulted in strong canonical Wnt/β-catenin activity in well differentiated, but not in poorly differentiated HCC cells. Comprehensive analysis of major Wnt signaling components by a rapid RT-PCR assay showed redundant expression of many Wnt ligands, Frizzled receptors, co-receptors and TCF/LEF factors in HCC. In contrast, canonical signaling-inhibitory Wnt5A and Wnt5B ligands were selectively expressed in poorly differentiated HCC cell lines. Our observations indicate that canonical Wnt/β-catenin signaling is active in well differentiated, but repressed in poorly differentiated HCC cells. Thus, canonical Wnt/β-catenin signaling plays a dual role in HCC. To study the role of Wnt pathway in breast cancer, we performed a comprehensive expression analysis, by RT-PCR, of Wnt signaling molecules, including 19 Wnt ligands, ten Frizzled receptors, two LRP co-receptors and four Lef/TCF transcription factors in immortalized normal human mammary epithelial cells (HMECs), six breast cancer cell lines (BCCL) and 14 primary breast tumors (PBT). BCCL expressed/over-expressed all Frizzleds except FZD10, LRP5/6 and Lef/TCFs. They also overexpressed WNT4, WNT7B, WNT8B, WNT9A and WNT10B, but the expression of WNT1, WNT2B, WNT3, WNT5A, WNT5B and WNT16 was lost or decreased in most BCCL. Wnt expression correlated with nuclear β-catenin accumulation and cyclin D1 induction in BCCL, compared to HMECs, indicating a reactivation of the canonical Wnt signaling in malignant cells. Furthermore, the expression of FZD1, WNT-4, WNT7B, WNT8B, WNT9A and WNT10B, all implicated in canonical Wnt signaling, was upregulated in PBT, whereas the non-canonical WNT5A expression was down-regulated. Our study gave strong evidences for the differential involvement of Wnt pathway in liver and breast cancers. In liver cancer, Wnt pathway activity seems to be linked to the differentiation status of HCC cell lines. Furthermore, our data showed that the canonical Wnt pathway was active in well-differentiated HCC cell lines and repressed in poorly differentiated ones. In contrast, the study of Wnt pathway in breast cancer cell lines showed similarities rather than differences. Indeed, our study revealed a significant correlation between Wnt ligands mRNA expression profile and the induction of Cyclin D and nuclear β-catenin protein accumulation in all breast cancer cell lines studied. We concluded that, although involved in both types of cancers, Wnt signaling is acting differently in liver and breast cancers. More interestingly, in the same type of cancer such as HCC, Wnt signaling displayed differential activity depending on the cell differentiation status.Item Open Access Heterogeneity of hepatocellular malignant phenotype(Bilkent University, 2006) Öztürk, NuriHepatocellular carcinoma (HCC) is one of the most wide-spread carcinomas throughout the world - responsible for more than 600,000 deaths annually - and is strongly associated with several etiological factors; including aflatoxin B1, alcohol, and Hepatitis virus B and C. In HCC, many genes undergo somatic aberrations with a tendency to cluster at genes involved in cell cycle regulation, in the p53 and canonical Wnt signaling pathways, and in the TGF-β/IGF axis. Almost a third of HCCs display mutations affecting canonical Wnt signalling. However, the role of canonical Wnt signaling aberrations in HCC is not known in detail, since transgenic mice which express mutant β-catenin (an integral component of canonical Wnt signaling) do not develop liver tumours. To study the heterogeneity of hepatocellular malignancies, we have concentrated on canonical Wnt signaling in HCC cell lines. We have found that canonical Wnt signaling was active in 80% of well-differentiated, and 14% of poorly-differentiated cell lines respectively. Furthermore, ectopic expression of a mutant β-catenin resulted in strong canonical Wnt activity in well-differentiated, but not in poorly-differentiated HCC cells. Our findings suggested that heterogeneity in HCC exists even in the same pathway as exemplified by differential canonical Wnt signaling activity in well- and poorlydifferentiated HCC cell lines. During this study, we produced monoclonal antibodies against β-catenin to distinguish between the pools of nuclear/cytoplasmic and membraneassociated β-catenin in cells, since it is believed that the nuclear β-catenin pool is more potent in tumorigenesis. Monoclonal antibody (MAb), 4C9 recognised β-catenin out of adherens junctions, while another MAb, 9E10 recognised all β-catenin forms even though their epitopes were adjacent. Differential Wnt signaling activity in HCC cell lines prompted us to investigate the interactions between β-catenin and other molecules, which have important functions in hepatocytes and may affect β-catenin/TCF transcriptional activity. C/EBPα is a potent inhibitor of cell proliferation in HCC cell lines and is involved in liver-specific gene expression, and some somatic alterations of it have been observed in AML and HCC. We investigated the effect of C/EBPα on β-catenin signalling. We have found that C/EBPα inhibits mutant β-catenin-TCF transcriptional activity, and physically interacts with β- catenin in HCC cells. While we were analyzing some stably mock-transfected Huh7 clones to use as controls, we observed heterogeneity in their proliferation rates. Further analysis of these clones revealed that some clones ceased to proliferate when passaged extensively. One of these clones (C3) was not tumorigenic in immunodeficient mice. Based on these observations, we hypothesized that some cancer cells could produce senescencent progeny in cell culture. Indeed, we showed that breast- and liver-cancer-derived cells display senescencent phenotypes at variable ratios. By using our experimental system, we also showed that replicative senescence program may work independently of functional p53 and p16 pathways, and the SIP1 gene is partially responsible for replicative senescent phenotypes in our Huh7-derived senescent clone C3. Overexpression of mouse SIP1 in p53- and Rb-deficient Hep3B cells induced partial senescent phenotypes at early passages. However, stable Hep3B cells repressed mouse SIP1 expression by an unknown mechanism and escaped senescent arrest in late passages. Our results suggest that Wnt pathway may have a dual role in hepatocellular malignancy, as it is active/easily inducible in well-differentiated HCC cells and inactive/repressed in poorly-differentiated ones. The further study of β-catenin in tumor samples by using our monoclonal antibodies may reveal new aspects in β-catenin signaling. However, the mechanism of these phenomena and the inhibition of β-cateninTCF signaling by C/EBPα require more study to reach a more comprehensive conclusion. The study of reprogramming of replicative senescence in HCC-derived cells indicated that senescence program may work independent of p53 and p16 pathways and heterogeneity of hepatocellular malignancy exists even within the established HCC derived cell lines.Item Open Access Identification of novel genetic elements controlling transcriptional regulation of the human Na(formula)/I(formula) symporter (NIS) gene(Bilkent University, 2006) Alotaibi, HaniThe function of sodium iodide symporter (NIS) in mammary gland epithelial cells is essential for the accumulation of iodide in mother’s milk, which is the first source of iodide for the synthesis of thyroid hormones in the newborn. In addition to the lactating mammary gland, NIS expression has been also detected in breast tumors. Several hormones and ligands have been implicated in the functional expression of NIS in the mammary gland and breast cancer cell line models but the molecular determinants governing this expression are not yet identified. In this study we aimed to identify cis- and trans-acting elements regulating NIS expression in the breast cancer cell line MCF-7 in response to all-trans-retinoic acid (tRA), and to assess the possible role of 17-β-estradiol (E2) in regulating the expression of NIS. Using comparative bioinformatics, we have identified several regions that were conserved in human, mouse and rat in the sequences flanking and including the NIS gene. By using luciferase reporter assays, we have established that conserved clusters 3 and 4 respond to tRA in MCF-7. We have also shown that putative retinoic acid response elements controlling tRA-induced NIS expression in MCF-7 are located in the first intron of this gene. This tRA-responsive NIS expression was also correlated with the estrogen receptor status of mammary gland cell lines and we investigated roles of ERα in the regulation of NIS expression. We showed that the suppression of endogenous ERα by RNA interference resulted in down-regulation of both basal and tRA-induced NIS expression in MCF-7, furthermore, we have also shown that (E2) is capable of up-regulating NIS expression in MCF-7. In the ERα negative cell line MDA-MB-231, re-introduction of ERα resulted in NIS expression in a ligand independent manner. The role of ERα in the regulation of NIS expression was supported by the identification of an estrogen response element (ERE) in the promoter of NIS, this ERE was conserved in human, mouse and rat. We have also showed that this ERE could respond to E2 stimulation, and that ERα occupies the NIS promoter by binding to this novel element in vivo. These results indicate that E2 and ERα contribute to the regulation of NIS in the breast cancer cell line MCF-7.Item Open Access In silico identification of candidate MECP2 targets and quantitative analysis in rett syndrome(Bilkent University, 2006) Onat, Onur EmreRett syndrome (RTT) is an X-linked neuro-developmental disorder seen exclusively girls in the childhood. It is one of the most common causes of mental retardation with an incidence rate of 1/10,000-1/15,000. Mutations in MECP2 gene was described as a common cause of RTT. MECP2 is a transcriptional repressor that regulates gene expression. It is not fully understood which MECP2 targets are affected in RTT and therefore contribute to disease pathogenesis. Researchers approached the problem in two directions: a) Global expression profile analysis and b) Candidate gene analysis. Global expression profile analysis revealed which a limited number of genes including those on the X-chromosome are de-regulated. Candidate gene analysis studies showed that loss of imprinting as exemplified by DLX5 could also contribute to disease pathogenesis. We hypothesize that Xchromosome inactivation (XCI) is an important physiological epigenetic mechanism that could be involved in Rett pathogenesis. We predicted a MECP2 binding motif by a distinctive bioinformatic approach. Using this algorithm we searched for the candidate MECP2 target genes on the X-chromosome and whole genome. The genes FHL1 and MPP1, whose interaction with MECP2 were heuristically displayed were predicted by our algorithm. We identified more than 100 genes which are on the Xchromosome. 10 genes from the list were selected according to their MECP2 binding homology score and X-inactivation status. In order to test this hypothesis we analyzed these genes with quantitative RT-PCR .We expect to identify the key genes that potentially contribute to RTT pathogenesis via disturbances in X-chromosome inactivation.Item Open Access X chromosome inactivation in female predisposition to autoimmunity(Bilkent University, 2008) Uz, ElifThe high female preponderance is thought to be important in identifying the etiological factors. Sex hormones, pregnancy related microchimerism, and environmental factors are investigated as likely candidates. Disturbed Xchromosome inactivation (XCI) is another candidate, which may contribute to the break-down of self-tolerance. In this study, we tested the hypothesis that “loss of mosaicism” for X-linked gene expression may contribute to autoimmune disease etiology. Therefore, XCI status of healthy individuals and patients diagnosed with scleroderma (SSc), autoimmune thyroiditis (AITDs), Sjogren’s syndrome (SICCA), and juvenile idiopathic arthritis (JIA) in the Turkish population were analyzed by genotyping the methylation status of a CAG polymorphism in the androgen receptor (AR) gene. Extremely skewed XCI was observed in a significant proportion of SSc (OR: 38.9; P<0.0001), AITDs (OR: 9.6; P<0.0001), and JIA (OR: 4.4; P=0.0022). Further genotyping of AITDs in Tunisian and SSc in the US population supported the initial observations (OR: 3.8; P=0.0046; OR: 3.8; P<0.0001) respectively. Analysis of rheumatoid arthritis (RA) in the Tunisian population suggests that extremely skewed XCI (OR: 6.7; P<0.0001) could be involved in disease pathogenesis. Moreover, pre-eclampsia, a disease in which autoimmunity may be important, skewed XCI was observed (OR; 11.7; P=0.0005). However, in SICCA random patterns of XCI was observed suggesting that extreme skewing is not a common feature of all female prevalent autoimmune disorders. In conclusion, our results suggest that extremely skewed XCI may be important factor in autoimmune disease pathogenesis.Item Open Access Analysis of differentially expressed geExpression of notch signaling pathway recenes in breast cancer : BRCA1- induced gene expression profiles and meta-analysis gene signature(Bilkent University, 2009) Dedeoğlu, Bala GürThe aim of the first part of this study was to find out the expression profiles of the genes, which were selected from the former BRCA1-induced gene list (OVCA1, OVCA2, ERBIN, RAD21, XRN2, RENT2, SMG1 and MAC30) in normal-matched primary breast tumors and to correlate the gene expression profiles of selected candidate genes with BRCA1 and various pathology parameters. Among the target genes, the expression of ERBIN, SMG1 and RAD21 were found to be highly correlated with that of BRCA1 both in BRCA1 up- and down-regulated cells and this result was validated with qRT-PCR expression profiling of the eight genes in 32 normal-matched primary breast tumor samples. These genes were found to be discriminative between ER(-) and ER(+) tumors as well as grade 1 and grade 3 tumors. Target genes were also analyzed in independent microarray datasets to assess their predictive power for breast tumor grade, subtype and patient survival. ERBIN, SMG1 and RAD21 were found to have predictive roles in these datasets. The aim of the second part of the study was to found appropriate reference genes (RGs) for accurate quantification of target gene expressions in breast tumor tissues. The expression patterns of fifteen widely-used endogenous RGs and three candidate genes that were selected through analysis of two independent microarray datasets were determined in 23 primary breast tumors and their matched normal tissues using qRT-PCR. Additionally, 18S rRNA, ACTB, and SDHA were tested using randomly primed cDNAs from 13 breast tumor pairs to assess the rRNA/mRNA ratio. The tumors exhibited significantly lower rRNA/mRNA ratio when compared to their normals. Among the eighteen tested endogenous reference genes, ACTB and SDHA were identified as the most suitable reference genes for the normalization of qRTPCR data in the analysis of normal-matched tumor breast tissue pairs. The aim of the third part of this study was to develop a resampling-based metaanalysis strategy. Two independent microarray datasets that contain normal breast, invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC) samples were used for the meta-analysis. The resampling-based meta-analysis has led to the identification of a highly stable set of genes for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes. A subset of this meta-gene list was shown to predict well-established molecular tumor subtypes, e.g., basal vs luminal or ER+/ER-, with high accuracy and sensitivity based on class prediction analysis of existing breast cancer microarray datasets. Expression of selected genes, tested on 10 independent primary IDC samples and matched nontumor controls by real-time qRT-PCR, supported the meta-analysis results.Item Open Access Analysis of members of the SLIT-ROBO pathway as diagnostic and prognostic tools in hepatocellular carcinoma with special focus on ROBO2-associated cellular phenotype(Bilkent University, 2009) Avcı, Mehmet EnderHepatocellular carcinoma is the sixth most common cancer in the world, with an annual incidence exceeding half a million. It is associated mainly with hepatitis B and C viral infections; and is the main cause of death among cirrhotic patients. Aflatoxin B1 exposure, chronic alcohol consumption and virtually all cirrhosis-inducing conditions are of the other etiologies. For early diagnosis of HCC, surveillance of the risk groups is a crucial task requiring the development of novel markers for HCC with stronger sensitivity and specificity. In addition, description of biomarkers specific to hepatocellular carcinoma subtypes could identify novel targets for therapy. In this study, we analyzed members of the SLIT-ROBO gene families as novel diagnostic and prognostic markers in hepatocellular carcinoma. We defined an expression signature for members of the SLIT-ROBO gene families in HCC cell lines and tissues by real-time quantitative RT-PCR analysis. We showed that ROBO1 was overexpressed as stage and differentiation of the HCC proceeds. Furthermore, ROBO4 downregulation and SLIT2 overexpression marked late stage and poorly differentiated HCCs. Our results suggest that the expression of ROBO1 and ROBO4 can be used in early diagnosis of HCC. As another focus, we stably knockdowned ROBO2 expression in a model AFP positive cell line Huh7 and characterized the associated cellular phenotype. ROBO2 downregulation caused a significant decrease in proliferation rate whereas in wound-healing assay no significant difference in migration rate was observed. In addition, we performed a microarray experiment and found the differentially expressed genes between stable ROBO2 knockdown and negative clones. In this analysis, we found an overexpression of CK19, CD44, ABCG2/ BCRP1 hepatic progenitor cell markers and CD133 that is also a putative cancer stem cell marker of HCC, in stable ROBO2 knockdown clones. In addition KLF4 expression was augmented in these ROBO2 knockdown clones. We propose a genetic association between SLIT-ROBO pathway and CD133 at transcriptional level.Item Open Access Senescence and immortality genes as markers of hepatocellular carcinogenesis(Bilkent University, 2009) Arslan Ergül, AyçaCellular senescence is a tumor-suppression mechanism, and immortalization facilitates neoplastic transformation. Both mechanisms may be highly relevant to hepatocellular carcinoma (HCC) development. We worked on two major aspects of cellular senescence and immortality in HCC. First, we analyzed the role of ZEB2 (Smad-interacting protein SIP1, ZFXH1B) gene for a senescence-related role in HCC. Then, we extended our work on the identification and analysis of a senescence and immortality gene network (SIGN) in relation to hepatocellular carcinogenesis. ZEB2 is a transcriptional repressor of E-cadherin, and induces epithelial-mesenchymal transition (EMT), a key process involved in tumor metastasis and progression. However, ZEB2 is also a repressor of telomerase reverse transcriptase (TERT) gene, which encodes a key enzyme required for telomere maintenance and tumor cell immortality. We performed in-vivo, in-silico and in-vitro studies to explore potential implications of ZEB2 in hepatocellular carcinoma (HCC). Tissue expression of ZEB2 transcripts displayed stepwise decreases in HCC lesions, as compared to liver cirrhosis. This inverse correlation suggested that sustained ZEB2 expression is not compatible with HCC progression. Next, vekt¨or¨u ile transfekte edilen Hep3B h¨ucrelerinin, ya¸slanma ili¸skili !-galaktozidaz aktivitesi ile, kalıcı h¨ucre d¨ong¨us¨u hapsine girdiklerini g¨ord¨uk. ZEB2 ile ind¨uklenen ya¸slanma hapsi, TERT ifadesinin baskılanması ve e¸slik eden siklin-ba˘gımlı kinaz engelleyici p21Cip1’in ifadesindeki artı¸s ile ba˘gıntılı idi. ZEB2’nin ge¸cici ifadesi, p21Cip1 ifadesindeki artı¸sı ind¨uklemedi. Son olarak, ZEB2’yi a¸sırı ifade eden Hep3B ve A431 klonlarının, in vitro k¨ult¨urde dereceli olarak azalması ile ZEB2 a¸sırı ifadesinin, kanser h¨ucrelerinin in vitro ya¸samı ile uyumlu olmadı˘gı sonucuna varıldı. Bu g¨ozlemler, ZEB2 geninin, EMT’deki rol¨un¨un dı¸sında, HCC h¨ucre b¨uy¨umesi ve ya¸samasında negatif rol oynadı˘gını d¨u¸s¨und¨urd¨u. Di˘ger ¸calı¸smamızda SIGN imzasını olu¸sturmak i¸cin, ya¸slanmaya programlanmı ¸s ve ¨ol¨ums¨uz HCC h¨ucrelerinden gelen gen ifade datasını, siroz ve HCC dokularından gelen data ile birle¸stirdik. SIGN imzası normal karaci˘ger, siroz, displazi ve HCC lezyonlarını do˘grulukla sınıflandırdı ve ya¸slanmadan ¨ol¨ums¨uzl¨u˘ge d¨on¨u¸s¨um¨un, ilk olarak displaziden erken HCC’ye ge¸ci¸ste ger¸cekle¸sti˘gini belirledi. Bu d¨on¨u¸s¨um, t¨um¨or ilerlemesine de katkıda bulunur. Ya¸slanmadan ¨ol¨ums¨uzl¨u˘ge d¨on¨u¸s¨ume, hepatik geri ba¸skala¸sım ile ve h¨ucre ¸co˘galması, kromozom de˘gi¸simi ve DNA hasar yanıtı genlerindeki ifade artı¸sı e¸slik etti. Bu nedenle, HCC ¨ol¨ums¨uzl¨u˘g¨u, k¨ok h¨ucre/¨onc¨u h¨ucre benzeri ¨ozellikler ile yakından ili¸skilidir. Son olarak, DNA hasar kontrol noktası ve DNA tamir genlerindeki artı¸s ile, t¨um¨or ba¸slangıcı ve ilerlemesi arasında ili¸ski bulduk. Bu genler, HCC’nin engellenmesinde ve terapisinde potansiyel hedefler olabilirler.Item Open Access Functional identification of RASGEF1 family of exchange factors as activators of RAP2, and as interacting partners of CCDC124(Bilkent University, 2009) Yaman, ElifCoiled coil domain-124 gene is highly conserved among eukaryotes and the human counterpart encodes a protein with no domain similarities with any previously characterized eukaryotic proteins. In this study, we aimed to identify biological functions and interaction partners of human Ccdc-124. A yeast-two-hybrid analysis carried in this study has revealed that Ccdc-124 interacts with RasGEF1B which was predicted to be a member of Ras guanine exchange factors. The highly conserved RasGEF1 family of proteins contain C-terminal CDC25-homology domain (CDC25- HD) and an N-terminal RasGEF-N domain (Ras Exchange Motif, REM), and is of unknown function and specificity. In this thesis, the interaction of Ccdc-124 and RasGEF1 family of proteins was also established with co-immunoprecipitation and GST pull down assays. On the other hand, by using purified RasGEF1A and RasGEF1B proteins, as well as a large number of Ras family of G-proteins, we established that RasGEF1A and RasGEF1B function as very specific exchange factors for Rap2, a member of the Rap subfamily of Ras-like G-proteins. They do not act on Rap1 or other members of the Ras subfamily. On the other hand, Ccdc-124 protein did not change the stimulatory effect of RasGEF1 family of proteins on any of the tested G proteins in vitro. Furthermore, using reciprocal site-directed mutagenesis, we analyzed residues that allow RasGEF1 proteins to discriminate between Rap1 and Rap2, and we were able to identify Phe39 in the switch I region of Rap2 as a specificity residue. Mutation of the corresponding Ser39 in Rap1 changed the specificity and allowed the nucleotide exchange of Rap1(S39F) to be stimulated by RasGEF1B. This study describes for the first time GEFs that are uniquely specific for Rap2 among Rap family of G-proteins.