Tumor suppressor functions of p 53 gene
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- Tez No: 101269
- Danışmanlar: PROF.DR. MEHMET ÖZTÜRK
- Tez Türü: Doktora
- Konular: Biyoloji, Biology
- Anahtar Kelimeler: Genler, Genler-P53, Neoplazmlar, p53, plö1^48, hepatocellular carcinoma, temperature sensitivity, DNA-binding domain. (V, Genes, Genes-P53, Neoplasms
- Yıl: 2000
- Dil: İngilizce
- Üniversite: İhsan Doğramacı Bilkent Üniversitesi
- Enstitü: Mühendislik ve Fen Bilimleri Enstitüsü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: Belirtilmemiş.
Özet
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Özet (Çeviri)
ABSTRACT TUMOR SUPPRESSOR FUNCTIONS OF p53 GENE Kezban Unsal Ph.D. in Molecular Biology and Genetics Supervisor: Prof. Dr. Mehmet Ozturk May 2000 The subject of this Ph D thesis work is the study of the tumor suppressor functions of p53 gene. Our first aim is to test whether wild-type p53 can exert its growth suppressive activity in the absence of retinoblastoma gene. This hypothesis was tested on the model of hepatocellular carcinoma because these tumors display both p53 and retinoblastoma gene mutations. Our second aim was to understand the mechanism of temperature-dependent activity of p53 protein. A novel experimental approach based on the exchange of functional domains of p53 protein from different species was developed and applied for comparative study of human andXenopus p53 protein domains. Both p53 and plö“^4* are known to inhibit cell growth by modulating retinobastoma protein phosphorylation by different mechanisms. In human hepatocellular carcinomas, the loss of function of p53 and plö11^48 as well as retinoblastoma genes by different mechanisms has been largely documented, but their hepatocellular effects are poorly known. In the first part of this study, we compared the growth inhibitory effects of p53 and plö1^48 proteins by transfecting the pRb protein-deficient hepatocellular carcinoma cells with inducible plö0*4”and p53 expression vectors. Stable clones were analyzed for transgene expression by western blotting and immunoperoxidase staining. Effects on cell growth were analyzed by in vitro growth assay, thymidine incorporation and flow cytometry. Biochemical effects of p53 were tested by northern blotting of p21c,pl, mdm-2, bax, cyclin-dependent kinase 2 and cyclin E proteins. Retinoblastoma protein was studied by western blotting and immunoprecipitation assays. The induction of plö1^48 protein expression did not affect in vitro growth of cells. In contrast, p53 protein in its wild- type conformation provoked a growth arrest accompanied by transactivation of p21.. » IIIand BTG-2 genes and accumulation of p21, bax and mdm-2 proteins. p53-induced growth arrest was due to a cell cycle arrest at the Gl/S transition, probably mediated by p21 protein, which inhibits cyclin-dependent kinase 2/cyclin E complexes. The lack of detectable retinoblastoma protein and resistance of cells to plo1^4“ strongly suggest that p53 is able to arrest the growth of hepatoma cells by a mechanism independent of ”p53-retinoblastoma pathway". In the second part of this study, we have originated a novel experimental model in order to provide better information about the possible functions of domains of p53, and generated hybrid proteins with human and Xenopus p53 gene products. We show by an immunological technique that the Xenopus p53 forms specific complex with mammalian hsp72/73 only at temperature (37°C) well above the optimal growth temperature for Xenopus. It seems that at 37°C this protein is altered in conformation, while at 32°C it exhibits wild-type behavior. To investigate this thermal sensitivity of p53, we have specifically exchanged the DNA-binding domains of Xenopus and human p53 with each other and tested their transcativation ability by luciferase assay at permissive and non-permissive temperatures. When the DNA- binding domain of Xenopus p53 is substituted by that of human, this hybrid protein behaves like a human wild-type p53; it is able to activate a reporter gene at 37°C. The effects of temperature-dependent change in the conformation also reflected in the growth suppression ability of p53. This is the most compelling evidence to date for the involvement of DNA-binding domain of p53 in thermal sensitivity. Our results indicate that p53 protein is highly flexible and that its temperature-dependent change plays a key role in the regulation of its biological activity.
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