Theses - Department of Molecular Biology and Genetics

Permanent URI for this community

https://hdl.handle.net/11693/13819

Faculty of Science

Browse

Now showing 1 - 20 of 255

Open Access
A-To-I RNA editing events, potential biomarkers for prognosis and chemosensitivity in gastric cancer
(Bilkent University, 2022-09) Çela, Isli; Güre, Ali Osmay
Gastric cancer (GC) is one of the leading causes of cancer mortality, and it frequently presents in advanced stages with a poor prognosis and response to treatment. Although extensive research has identified many potential biomarkers in GC, the heterogeneity of the disease is an impediment to validation, so only a small number find limited application in clinics. RNA editing is an epigenetic modification that results in nucleotide changes in the RNA sequence. Adenosine to Inosine (A-to-I) substitutions are the most common editing events in humans, and they are mediated by Adenosine deaminases acting on RNA (ADAR) enzymes. Inosine (I) mimics Guanosine (G) and creates pairs with Cytidine (C), resulting in changes in RNA structure and stability, amino acid substitutions, alternative splicing, or gene expression regulation via miRNA target site modifications.RNA editing dysregulations have been found in breast, lung, kidney, brain, and gastric cancers, but the utility of specific editing events as biomarkers is largely unexplored. In this study we investigate the potential of A-to-I editing events as chemosensitivity and prognostic biomarkers in GC. Across multiple datasets, our analysis shows that RNA editing events at 305 unique positions correlate with drug sensitivity measures of 17 approved chemotherapeutics in GC cell lines.The most significant editing event-drug sensitivity correlations indicate that higher editing levels are associated with higher chemosensitivity. Interestingly, the expression levels of genes with identified editing events have a weaker or no correlation with drug sensitivity, implying that editing events are biomarkers independent of transcript levels. We show that, while ADAR enzymes mediate editing events, ADAR expression levels are not interchangeable with editing frequencies as chemosensitivity biomarkers in GC. We discovered a non-synonymous editing event in the C11orf80 coding sequence, resulting in an amino acid substitution (S.p133G). Also, we identified an editing event in the 3'UTR of SOGA1 that correlates with increased SOGA1 expression. The presence of this editing site in a putative target site of miR-9-5p suggests that gene expression might be regulated by miRNA target site modifications. In the TCGA and Singapore cohorts, the prognostic role of editing events in GC was investigated. Overall, higher levels of editing are associated with better survival in GC patients. In both cohorts, we found an editing event in the CLPX gene at the position 65442098 to be an independent good prognostic factor. We chose editing events that would best categorize our patients into "High" and "Low" edited groups using the Log Rank Multiple Cut-off (LRMC) plot distribution. In each dataset, we propose two editing events, one good and one bad prognostic factor that independently correlate with survival in GC patients. In the Singapore Cohort, high editing levels in ZNF587 are associated with a good prognosis, while those in DCAF16 are associated with a poor prognosis.High editing levels in CTSB correlate with better overall survival (OS) in the TCGA cohort, while those in NUP43 correlate with worse OS. Because transcript levels do not correlate with survival, the prognostic effects of these editing events are unaffected by gene expression levels.We believe that editing levels at specific positions can be used as prognostic biomarkers in a significant way, providing a more cost-effective and applicable alternative to prognostic editing signature models. Our findings suggest that editing events could be used as independent biomarkers for chemosensitivity and prognosis in gastric cancer, however more investigation is required to elucidate the mechanisms underlying the observed relationships.
Open Access
The ability to generate differentiated and senescent progeny is a major determinant of breast cancer heterogeneity
(Bilkent University, 2009) Mumcuoğlu, Mine; Öztürk, Mehmet
Breast cancer displays distinct subtypes, such as luminal A, luminal B, and basallike. The prognosis and therapeutic response of each subtype is different. The mechanisms involved in the generation of these tumor types are poorly understood. Our aim was to test whether the ability to generate senescent progeny contributes to breast cancer heterogeneity. A panel of 12 breast cancer cell lines, 31 isogenic clones, and 12 breast tumors were used. We classified breast cancer cell lines into senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All ER+ cell lines tested and some ER-positive (ER+) breast tumors displayed senescence. Acute loss and tamoxifen-mediated inactivation of ER triggered a robust senescence response in SCP type T47D cell line. In contrast, ER-overexpression, estrogen treatment and p21Cip1 knockdown inhibited senescence. Neutralization of reactive oxygen species also abolished senescence. Breast cancer cell subtypes displayed divergent ability to produce differentiated progeny. The SCP subtype cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some cell lines generated only CD44+/CD24-/ ER-/ASMA- progenitor/stem-like cells, and others only CD24+/ERluminal-like, but not ASMA+ myoepithelial-like cells. SCP cell lines were less tumorigenic, and they clustered with luminal A/normal like tumors. In contrast, ICP subtypes were more tumorigenic, and they clustered together with basal/luminal B tumors. Our results show that breast cancer cell lines clustering with luminal A/normal-like and basal/luminal B tumors respectively, differ from each other by the ability to generate differentiated and senescence-arrested progeny.
Open Access
Acquired expression of transcriptionally active p73 in hepatocellular carcinoma cells
(Bilkent University, 2002) Sayan, A. Emre; Öztürk, Mehmet
P53 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.
Open Access
Acquired tolerance of hepatocellular carcinoma cells to selenium-deficiency : a selective survival mechanism
(Bilkent University, 2003) Irmak, Meliha Burcu; Çetin-Atalay, Rengül
Selenium-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.
Open Access
Adenosine regulation of danger signaling
(Bilkent University, 2017-07) Akdemir, İmran; Çekiç, Çağlar
Metabolic and immune related activities converge as main triggers of adenosine accumulation in extracellular space. Adenosine by engaging adenosine A2A and A2B receptors strongly suppresses innate and adaptive immune responses. Although adenosine receptors are being targeted in preclinical and clinical studies, how different danger signals are regulated by adenosine is poorly understood. Here we showed that adenosine receptor stimulation strongly inhibited inflammatory responses while sparing Type-I interferon responses downstream of different danger signals in dendritic cells and macrophages. Mechanistically, danger signals associated with MyD88-dependent inflammatory pathways such as LPS and CpG but not the danger signals associated with IRF3/Type-I interferon pathways such as pA:U and cGAMP increase the expression of adenosine A2A and A2B receptors. Expression of anti-inflammatory NR4A1 was increased after adenosine receptor stimulation in the presence of TLR ligands known to activate MyD88 pathway but not in the presence of cGAMP and pA:U. Overall these results indicate that there is a differential modulation of danger signaling by adenosine rather than overall suppression. Our results have important implications for developing combinatorial approaches to target adenosine and danger signaling pathways to cure immune-related diseases.
Open Access
Analyses and web interfaces for protein subcellular localization and gene expression data
(Bilkent University, 2007) Bilen, Biter; Çetin-Atalay, Rengül
In order to benefit maximally from large scale molecular biology data generated by recent developments, it is important to proceed in an organized manner by developing databases, interfaces, data visualization and data interpretation tools. Protein subcellular localization and microarray gene expression are two of such fields that require immense computational effort before being used as a roadmap for the experimental biologist. Protein subcellular localization is important for elucidating protein function. We developed an automatically updated searchable and downloadable system called model organisms proteome subcellular localization database (MEP2SL) that hosts predicted localizations and known experimental localizations for nine eukaryotes. MEP2SL localizations highly correlated with high throughput localization experiments in yeast and were shown to have superior accuracies when compared with four other localization prediction tools based on two different datasets. Hence, MEP2SL system may serve as a reference source for protein subcellular localization information with its interface that provides various search and download options together with links and utilities for further annotations. Microarray gene expression technology enables monitoring of whole genome simultaneously. We developed an online installable searchable open source system called differentially expressed genes (DEG) that includes analysis and retrieval interfaces for Affymetrix HG-U133 Plus 2.0 arrays. DEG provides permanent data storage capabilities with its integration into a database and being an installable online tool and is valuable for groups who are not willing to submit their data on public servers.
Open Access
Analysis of 45S rDNA promoter methylation and expression of rRNA transcripts in breast cancer
(Bilkent University, 2015-06) Karahan, Gurbet; Yulug, I. G.
Ribosome biogenesis has a central role in cell growth and proliferation that is usually disrupted in tumor cells by the inactivation of tumor suppressor genes and activation of oncogenes. Ribosomal RNA (rRNA) gene expression is one of the most important factors regulating ribosome production, which is controlled by CG rich 45S ribosomal DNA (rDNA) promoter. The effect of DNA methylation at 45S rDNA promoter on rRNA gene expression is a subject of controversy in the literature. In this thesis, a 434 bp region (-380 bp to +54 bp) spanning both upstream control element (UCE) and core promoter located in 45S rDNA promoter containing 54 CpGs was analysed in breast cancer. We also analysed the related rRNA expression levels in the same samples in order to clarify the role of 45S rDNA promoter methylation on rRNA gene expression. 45S rDNA promoter region was highly methylated (74%-96%) in all cell lines including non-tumorigenic breast cell line (MCF10A). Even though 45S rDNA promoter region of breast cancer cell lines are extensively methylated, rRNAs (18S, 28S, 5.8S and 45S ETS) were expressed independent of the heavy methylation. Expression levels of rRNAs are assessed either using housekeeping genes (ACTB, TBP, ACTB&TBP) or geometric mean of rRNAs (GM-rRNAs). We propose GM-rRNA normalization as a new method to identify relative expression differences between rRNA transcripts. Epigenetic drugs 5-Aza-2'-deoxycytidine (5-AZA) and Trichostatin A (TSA) were used to determine the effect of DNA methylation and histone acetylation on rRNA expression. Demethylation with 5-AZA resulted in an unexpected decrease in the expression of all rRNA. TSA treatment did not lead to any significant expression difference in cell lines. To better evaluate the effect of DNA methylation on the expression of rRNA transcripts we analysed the methylation status of 19 breast tumor and matched normal frozen tissue samples. The results showed that majority of the tumors (13/19) have significantly higher methylation levels than their normal pairs. Using the GM-rRNA as reference helped us to determine significant differences in the proportionate expression of rRNAs in these tissue samples. The 5.8S rRNA ratio was significantly lower whereas the 18S rRNA ratio was significantly higher in breast tumor samples. Furthermore, the 45S rDNA promoter methylation levels in normal breast tissue samples were negatively correlated with the18S rRNA ratio but this correlation was disrupted in breast tumors. Similarly, rRNA transcript levels were significantly correlated with each other in normal samples, were lost in tumor samples. It is clear that, there is a dysregulation both in rDNA methylation levels and spliced rRNA transcripts specific to breast tumor samples, which was not observed in normal breast tissues. rRNA gene expression is controlled by mechanisms other than promoter DNA methylation. Tumorigenesis may cause disruption of many control mechanisms that are required for proper rRNA expression, splicing and maturation, resulting in a dysregulation of the correlation between spliced rRNA expression levels, which should be investigated further.
Open Access
Analysis of BRCA1 and BRCA2 genes in Turkish breast cancer patients
(Bilkent University, 2000) Özdağ, Hilal; Özçelik, Tayfun
Breast 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.
Open Access
Analysis of candidate molecular targets in adult (CML) and childhood (AML, ALL) Leukemias
(Bilkent University, 2004) Boylu, Cemaliye Akyerli; Özçelik, Tayfun
Candidate 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.
Open Access
Analysis of CHRNA5 expression in breast cancer cell lines in response to serum starvation and estrogen treatment
(Bilkent University, 2013) Açıkgöz, Azer Aylin; Konu, Özlen
Breast cancer is a complex disease that can be classified into distinct molecular subtypes including Basal, Luminal A, Luminal B and HER2 positive. These molecular subtypes mainly differ in their hormone receptor expression and response to treatment. This makes the discovery of new molecular markers for further classification important. Cholinergic nicotinic acetylcholine receptors are ion channels involved in smoking behavior, neurodegenerative diseases and cancer. Cholinergic nicotinic receptor alpha 5 (CHRNA5) has been associated with nicotine addiction and recently with lung cancer yet its importance in breast cancer remains relatively unexplored. In the present study, a panel of 10 breast cancer cell lines were used for quantification of isoform-specific CHRNA5 expression using qPCR. Changes in CHRNA5 expression in response to serum starvation and estrogen treatment were assessed. qPCR showed that CHRNA5 was alternatively spliced, with at least five different isoforms in breast cancer cell lines. qPCR analysis for CHRNA5 expression in serum treated and serum starved cells were analyzed after outlier detection and exclusion; and statistical tests included ANCOVA using geometric mean of TPT1 and SDHA, as reference genes. Our results demonstrated that, CHRNA5 expression differed between different subtypes of breast cancer cell lines. CHRNA5 expression significantly responded to serum starvation in ZR75-1 and MDA-MB-157 cell lines, isoform specifically. Isoform expression of CHRNA5 exhibited significant alterations upon estrogen treatment in a dose and time-dependent manner. Expression of 1000bp variant, isoform2 and isoform3 of CHRNA5 significantly increased upon E2 treatment and total CHRNA5 and isoform2 CHRNA5 increased in expression at 24 hours when compared with 12 hours of treatment. Our findings show that CHRNA5 has multiple isoforms in breast cancer, with potential to be modulated by serum starvation and estrogen treatment in a cell-specific manner.
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 Gür; Yuluğ, Işık G.
The 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.
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 Altay; Özçelik, Tayfun
The 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.
Open Access
Analysis of hMLH1 germline mutations in three Turkish hereditary nonpolyposis colorectal cancer kindreds
(Bilkent University, 1998) Akyerli, Cemaliye; Ricciardone, Marie D.
Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common genetic diseases in Western world. It is a clinical syndrome characterized by an inherited predisposition to early onset colorectal and an increased incidence of other cancers. The disease is caused by a germline defect in one of five human DNA mismatch repair genes, hMLHl, hMSH2, hPMSl, hPMS2, and hMSH6. Defects in hMLHl and hMSH2 account for the majority of mutations found in HNPCC families. In this study, a variety of mutation detection methods were used to identify hMLHl germline mutations in three Turkish HNPCC kindreds. Restriction enzyme analysis of genomic DNA was used to analyze five members of an HNPCC family with a previously described G884C mutation. The genotypes of all five individuals were determined by Dde\ digestion and the results were confirmed by DNA sequence analysis. Hph\ restriction enzyme analysis was used to analyze twenty-nine members of an unrelated HNPCC family for a previously identified A1652C mutation. Genotypes were determined for all individuals and the results were confirmed by DNA sequence analysis. Both of these restriction enzyme analyses are reliable, cost-effective methods that can be used in mutation screening programs for family members who request genetic counseling. Single strand conformation polymorphism analysis (SSCP) was used to screen for unknown germline mutations. Nine DN A samples with defined mutations in the hMLHl gene were analyzed using several gel formulations and electrophoretic conditions to determine the most sensitive protocols. These protocols were then used for routine mutation detection. In a third HNPCC family, for whom no mutation has yet been defined, the complete coding sequence of the hMLHl gene was screened by SSCP. Two exons, 7 and 15, showed an altered mobility compared to control sequences. The nucleotide sequence of these two exons was determined by automated fluorescence DNA sequence analysis. The differential mobility observed for exon 15 appears to be due to an intonic polymorphism in the control sample. Preliminary results for exon 7 show no difference between proband and control nucleotide sequences. Thus, the DNA mismatch repair defect in this kindred appears not to be in hMLHl. Further studies will focus on the analysis of hMSH2.
Open Access
Analysis of LSAMP gene as a tumor suppressor in neuroblastoma
(Bilkent University, 2009) Saydere, Atıl Çağdaş; Yakıcıer, M. Cengiz
Neuroblastoma constitutes approximately 10 % of all childhood tumors with a worldwide considerable morbidity and mortality. Frequently, in children under the age of 1, it can spontaneously regress and transform into a benign tumor. However, in children older than age of 1 the disease often behaves aggressively and metastasizes to other organs. This unpredictable behavior of unknown origin makes therapeutic applications ineffective. Limbic system associated membrane protein gene (LSAMP) functions in neurite growth, axonal guidance and acts as a cell adhesion and recognition molecule. Recent studies revealed its association to several cancer types and proposed a potential tumor suppressor role. Markers in the LSAMP gene region were also shown to be homozygously deleted in neuroblastoma. In the framework of this study, we investigated LSAMP gene in respect of its potential tumor suppressor role in neuroblastoma. 6 clinical patient samples and 2 neuroblastoma cell lines were studied via PCR methodology to detect any loss in LSAMP gene. Immunohistochemistry (IHC) was applied to 6 neuroblastoma tissue sections to determine protein level changes of LSAMP. Moreover, expression analysis in a set of brain tumors was performed. As a result of these efforts, one possible LOH and one homozygous deletion in two different patients were observed. Low levels of LSAMP protein in all of the tumor samples compared to controls were recorded.Downregulation of LSAMP in brain tumors was detected. Based on these results, LSAMP is suggested as a candidate tumor suppressor in neuroblastoma and in broader aspect for nervous system tumors.
Open Access
Analysis of MECP2 gene mutations in Rett syndrome patients
(Bilkent University, 2001) Sayı, Ayça; Özçelik, Tayfun
Rett Syndrome (RTT) is a progressive X-linked dominant childhood neurodevelopmental disorder, affecting 1/10,000-15,000 girls. The disease-causing gene was identified as MECP2 on chromosome Xq28, and mutations have been found in approximately 80% of patients diagnosed with RTT. We screened for eight recurrent MECP2 mutations (R106W, P152R, T158M, R306C, R168X), one rare mutation (F155S) and one polymorphism (E397K) in 63 RTT patients divided into four groups as classic-RTT (n=43), variant-RTT (n=14), male-RTT (n=4), and familial-RTT (n=2). We identified the recurrent mutations in 18 cases. These are three R106W, two P152R, five T158M, five R306C, and three R270X mutations. R168X and F155S were not detected in our patients. Only one patient had the E397K polymorphism who also had the R306C mutation. All these mutations were confirmed via sequencing analysis. In exon 4 of MECP2, several deletion types of mutations are known. By PCR analysis, two patients were found to have an approximately 44 bp deletion in exon 4. Also, a novel mutation – T197M– was identified in one of the patients. We identified a boy affected by RTT who is mosaic for the R270X mutation, and had a normal male karyotype. This result show that a recurrent MECP2 mutation could lead to a similar phenotype in females and males, if the male is a mosaic for the mutation in his somatic cells. MECP2 mutation frequency for the four groups is as follows: 37.2% for the classic-RTT, 28.57% for the variantRTT, and 25% for the male-RTT groups. No mutation was found in the familial group. We could not find a consistent correlation between the clinical symptoms and the type of mutations or the X chromosome inactivation patterns of the patients.
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 Ender; Yağcı, Tamer
Hepatocellular 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.
Open Access
Analysis of p73 gene in hepatocellular carcinoma
(Bilkent University, 1998) Fındıklı, Necati; Öztürk, Mehmet
Hepatocellular carcinoma (HCC) is the eighth most frequent cancer worldwide, iipidemiologically-studied risk factors include hepatitis B virus (greater than 80%), hepatitis C virus and aflatoxins. Molecular mechanisms of hepatocarcinogenesis are poorly understood. The only gene known to be consistently involved in these tumors is the p53 tumor suppressor gene. However, this gene was found to be mutated or ifiactivated in about 30% of HCC. There is a need to study additional genes in order to fully understand hepatocellular carcinogenesis. p73 has been identified recently as a p5i -homolog gene. In this study, we analyzed the possible involvement of this gene in HCC. We investigated both the expression and structure of p73 gene in HCC for possible alterations.We first developed a novel method to analyze the expression of alternatively spliced transcripts of p73 (p73a and p73p) simultaneously. This technique, based on RT-PCR, allows the analysis of p73 transcripts semi-quantitatively. We found that p73a was expressed ubiquituously in 8 cell lines derived from normal liver or HCC tumors. Interestingly, p73|3 was present only in 5 differentiated but not in 3 undifferentiated cell lines. The differentiation status of these cell lines were tested by the analysis of albumin and a-fetoprotein transcripts by RT-PCR. These transcripts were present in 3/5 differentiated but not in 3 undifferentiated cell lines. Next, we screened 25 HCC samples for possible mutations of p73 gene at selected exons with non-radioactive heteroduplex test, radioactive SSCP analysis, restriction enzyme analysis and DNA sequencing. No alterations were found in exons homologous to those of p53 known to harbor mutational hotspots.From these observations, we conclude that i) /3 gene is not mutated in HCC, but it may play a critical role in hepatocellular differentiation. As p73p was found in differentiated cell lines, this form may be involved in transcriptional regulation of liver-specific genes. Additional studues are needed to confirm this hypothesis.
Open Access
Analysis of the ATAD2 gene effect on the genes involved in epithelial mesenchymal transition in estrogen positive and negative breast cancers
(Bilkent University, 2021-02) Özerk, Zeynep Ilgım; Yuluğ, Işık
ATAD2 is overexpressed in many distinct cancer types including breast cancer. Its elevated expression is an indicator of poor prognosis. High ATAD2 expression correlates with short overall survival, disease-free survival as well as shorter recurrence-free survival. Moreover, ATAD2 is associated with migration and invasion in some cancer types such as hepatocellular carcinoma and cervical cancer. In breast cancer, ATAD2 expression is higher in invasive tumors. ATAD2 is coactivator of steroid hormone receptor ERα. It directly interacts with ERα and enhances its target genes expressions. ERα is also a regulator of EMT in breast carcinomas. Based on our current knowledge it is proposed that ATAD2 may have a role in EMT and migration capacity of breast cancer cells and this role may be ER dependent as both ATAD2 and EMT are associated with ERα. Bioinformatics analysis revealed that siATAD2 silencing decreased mesenchymal gene expressions significantly in MCF7 and T47D cells. To investigate the possible mesenchymal inducing role of ATAD2, ATAD2 was silenced with shRNA transfection in ER+ MCF7 and T47D cells and ER- mda-mb-231 and sk-br-3 cells. ATAD2 silencing decreased mesenchymal markers expression at both the mRNA and protein level in ER- cells. In ER+ cells, no change in EMT marker proteins and mRNAs were observed with ATAD2 silencing. ER was silenced in ER+ cells and ER silencing introduced a mesenchymal phenotype to them. In this case, ATAD2 silencing reduced this mesenchymal phenotype introduced with ER loss. The EMT effect of the ATAD2 silencing on migration capacity of breast cancer cells was assessed with a scratch assay. Consistent with changes in the epithelial and mesenchymal markers, ATAD2 silencing reduced the migration capacity of mda-mb-231 cells. On the other hand, sk-br-3 migration did not change significantly. In ER+ cells ATAD2 silencing alone had no influence on migratory capacity. ER silencing increased their migration significantly while ATAD2 downregulation in ER-silenced cells suppressed this migration. Over all, this study suggests a possible involvement of ATAD2 in EMT and migration regulation in ER- cells. Targeting ATAD2 in ER- mesenchymal breast cancer cells could be a strategy to reduce their migration capacity.
Open Access
Analysis of the effect of Erbin on the epithelial to mesenchymal transition related genes in breast cancer
(Bilkent University, 2022-09) Şahin, Filiz; Yuluğ, Işık
Breast cancer is a heterogeneous disease and has complex mechanisms, which brings the need to come up with different approaches for its various types. Erbin is a member of the LAP family and directly interacts with ErbB2 (HER2), which is a crucial component for breast cancer sub-typing and treatment. Research on the relationship between Erbin and breast cancer showed different results since Erbin was seen to be able to act as both a tumor suppressor and a tumor promoter. There are multiple findings that Erbin has a role in the epithelial to mesenchymal transition in different cancer types. The role of Erbin in EMT is said to be multi-faceted, it affects multiple pathways that act on EMT progress. However, like Erbin’s contradictory role in breast cancer, there are different findings that support the conflicting claims on whether Erbin is an EMT promoter or not. These findings in the literature lead us to analyze how Erbin affects EMT in breast cancer. To investigate if there is a role of Erbin in EMT regulation, we have overexpressed Erbin in MCF7 and silenced Erbin in MDA-MB-231 with MDA-66 cell lines. With this experimental setup, we aimed to see how Erbin plays a role in EMT in both epithelial and mesenchymal cells as well as Luminal A, TNBC, and ER positive breast cancer types. Our results showed that in TGFB induced EMT, Erbin levels are decreased in epithelial cells. Erbin overexpressed MCF7 cells show significant changes in the EMT markers compared to the control group. Experiments conducted with Erbin silenced MDA-MB-231 and MDA-66 cells also demonstrated significant changes in EMT marker levels. Our results suggest that Erbin has a role in EMT in breast cancer, and this role is not limited to one pathway and not streamlined, but possibly a big orchestration of different effects.
Open Access
Analysis of the function of the nuclear matrix-associated protein C1D
(Bilkent University, 1999) Bilican, Bilada; Yavuzer, Uğur
DNA Double-strand breaks (DSBs) are generated as intermediate stnictures during V(D)J recombination, as a consequence of oxidative metabolism, or can be induced by exogenous factors such as gamma irradiation and radiomimetic drugs. Mutational studies identified the serine/threonine kinase, DNA-PK, as an essential component of DNA DSB repair machinery. The activation of the multi-component DNA-PK complex requires either free DNA ends or an association with the nuclear-matrix associated protein CID, which facilitates the activation of DNA-PK in a DNA end-independent fashion. The activation of DNA-PK through its interaction with CID, joins an increasing body of evidence which suggests a role for higher order nuclear organisation in the orchestration of complex cellular processes such as transcription, RNA splicing, nucleotide excision repair, replication and double-strand DNA break repair. In this study, the yeast two hybrid system was employed to screen a B-cell cDNA library to identify the interacting proteins with CID, and the interactions determined were further characterised. It was found that, CID interacts specifically with the recombinational hotspot binding protein Translin and Translin associated factor X, TRAX, both in vitro and in vivo, providing evidence that C 1D may play a critical role in DNA repair and recombination. Interestingly, an interaction between TRAX and DNAPKcs has also been identified under in vivo conditions. Tlie interaction of TRAX with DNA-PKcs and C ID indicates a connection between DNA double-strand break repair, recombination, and dynamic nuclear architecture.