Browsing by Subject "Pathology, Molecular."
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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 Development of a non-radioactive diagnostic test for the detection of microsatellite instability in colorectal cancer(Bilkent University, 1997) Vata, KorkutStepwise accumulation o f mutations in the human genome is the initial step in carcinogenesis. Microsatellites are the regions which are first hit by the mutations resulting from mismatch repair deficiency. Until today microsatellite alterations have been shown mainly in hereditary non-polyposis colon cancer (HNPCC) and several other cancer types. Recent advances indicate that microsatellite alterations can also be detected in DNA samples (such as blood, urine, etc.), which are shed fiOm tumors. This is an important finding for the early diagnosis o f cancer since malignant cells can be detected in tissues other than the primary tumor. Therefore microsatellite analysis, when coupled with an easy, powerful screening technique could have a high diagnostic value for cancer types other than colorectal cancer. Despite its drawbacks the most common microsatellite screening method is based on the use of radioisotopes and autoradiography. However in the clinical setting non-radioactive detection methods are preferred. The aim o f this thesis is development o f a non-radioactive diagnostic test for the detection o f microsatellite instability in genomic DNA which can be used for the early detection o f some forms o f cancer. Therefore we have optimized the PCR conditions for eight microsatellite markers which are: i. mononucleotide repeats BAT25 and intragenic repeat region o f BAX gene, ii. dinucleotide repeats D5S105, D6S291, D11S904, D13S175, D17S855, and in. tetranucleotide repeat FGA. In addition, we have analyzed the mononucleotide repeat markers in blood, paraffin embedded and fi"esh tumor samples o f six colorectal cancer patients with polyacrylamide gel electrophoresis and silver stainingItem Open Access Genetic analysis of Smad4 gene in TGF-Beta signalling pathway in human liver cancer(Bilkent University, 1998) Irmak, Meliha BurcuHCC is a multistep genetic disease in which many genomic changes occur as a result of uncontrolled proliferation of hepatocytes. Molecular events leading to HCC is still unclear. Until now, neither an oncogene nor a tumor suppressor gene has been shown to be prefentially altered in HCC. Genetic alterations other than p53, pl6, BRCA2 (Breast Carcinoma Associated Protein), M6P/IGFIIR (lyiannose 6 Phosphate/ Insulin Like Growth Factor II Receptor), Rb (Retinoblastoma), PRLTS (Platelet Derived Growth Factor Receptor-|3-Like Tumor Suppressor Gene), and Tg737 (Candidate polycyctic kidney disease gene) genes remain unknown. TGF-P is a strong inhibitor of hepatocyte proliferation. In HCC and cirrhosis increased levels of TGF-P is observed, so this shows that the presence of high levels of TGF-3 does not avoid hepatocyte proliferation. Thus, there may be a disruption in the signalling pathway of TGF-p. The common mediator Smad4 gene, which is among the genes located in TGF-P signalling pathway, is found to be mutated in many cancer types. We decided to do the mutational analysis of Smad4 gene, which is located in the signalling pathway of the hepatocyte antiproliferative factor, TGF-p. Exons 8, 9, 10, and 11 which are in MH2 region, and exon 2 which is in MHl region of Smad4 is mutationally analysed by SSCP for 35 HCC cases. In the 35 HCC tumors, 5 alterations were observed (14%), 3 of them being in exon 8, one of them being in exon 9a, and the last one being in exon 10 of Smad4 gene. In the samples we tested, no big deletions were observed, but the alterations observed are probably single base changes. Also HCC cell lines namely, HepG2, Hep3B, Huh-7, FOCUS, Mahlavu, and PLC/PRF/5 were checked for their mutations and cell lines other then PLC/PRF/5 were analysed for their mRNA transcription. There were no big deletions or alterations in Nand C- terminals of the cell lines and we have shown mRNA transcription for all cell lines except Hep3B in which PCR has revealed very weak amplification. Our results suggest that Smad4 might be involved in at least a part of primary HCC tumor development.