Browsing by Subject "Genes, p53."

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Open Access
Acquired expression of transcriptionally active p73 in hepatocellular carcinoma cells
(2002) Sayan, A. Emre
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
Establishment of an experimental system to study p53 effects in Saccharomyces cerevisiae
(1997) Dinçer, Tuba
The aim of this vv^ork was to establish an experimental model to study human wild type and mutant p53 protein effects in yeast Saccharomyces cerevisiae cells. Wild type p53 was previously shown to be a DNA damage response gene that controls the genome stability in mammalian cells. We are interested in using yeast cells to study human p53-mediated cellular events after DNA damage. In this study we established an experimental model as an initial step. For construction of this system pAK31 plasmid expressing a human mutant p53-248W as well as control plasmid pL3 were used to obtain two different yeast cell populations, one expressing the mutant p53 248W protein and the other without p53 expression. Wild type p53 expression vector was avoided because of its known growth inhibitory effects in yeast cells. In this experimental system initially p53 expression at pAK31 transformed yeast cells was shown. Then, the effect of mutant p53-248W expression to growth rate of yeast cells was analysed and no growth rate difference was detected between the cells expressing and non-expressing mutant p53-248W protein. To test the participation of mutant p53-248W protein in DNA damage response in yeast cells, cells were exposed to DNA damaging agents; UVC and cisplatin that were reported to induce wild type p53 protein in mammalian cells. Codon 248 is a common site of ‘hot spot’mutation and the arginine residue that corresponds to codon 248 encoded by the wild type p53 sequence is in the DNA interacting face of the p53 protein. Mutant p53-248 (arg->trp) is defective in specific DNA binding and it has lost the ability to act as a transcription factor. Although there are not many reports about mutant p53-248W response to DNA damaging agents, it was shown that mutant p53- 248W exhibit decreased repair of active genes upon UV radiation. In this study cell survival was analysed in mutant p53-248W protein expressing yeast cells in parallel to mutant p53 protein levels following to DNA damage and no effect of mutant p53 248W expression on cell survival upon DNA damage was detected. Also no difference was detected in mutant p53 protein levels following DNA damage.
Open Access
Tumor suppressor functions of p53 gene
(2000) Ünsal, Keziban