Reles, Angela : MOLECULAR GENETIC ALTERATIONS IN OVARIAN CANCER The Role of the p53 Tumor Suppressor Gene and the mdm2 Oncogene



The p53 gene is a tumor suppressor gene which has been named the "guardian of the genome„ (Lane 1992). It is activated in response to cellular stresses and DNA damage and regulates cell cycle arrest and apoptosis. It has been found to be mutated in more than 50% of human carcinomas and the loss of its function through mutation is thought to be one of the major steps in carcinogenesis (Hollstein et al. 1994). The mdm2 (murine double minute 2) gene was initially described as a proto-oncogene and is amplified and overexpressed in human soft tissue sarcomas and gliomas (Oliner et al. 1992). Most importantly though, MDM2 was found to be activated by p53 and subsequently inhibit p53 in an autoregulatory feedback-loop and promote its nuclear export and rapid degradation (Momand et al. 1992, Haupt et al. 1997, Kubbutat et al. 1997, Roth et al. 1998). Thereby it allows the cell to re-enter the cell cycle after repair of DNA damage.

In this investigation we analyzed frozen tumor tissue of a series of 178 ovarian carcinoma patients with long term follow-up data for alterations of the p53 and mdm2 gene and their impact on response to chemotherapy and survival. p53 mutations and polymorphisms were characterized by screening the entire coding region of the gene (exon 2-11) with PCR/SSCP (Single Strand Conformation Polymorphism) and DNA sequence analysis. Overexpression of p53 was analyzed by immunohistochemistry in frozen tissue.

Since MDM2 promotes the rapid degradation of p53 protein, alterations of MDM2 are expected to cause p53 stabilization and accumulation, as it is seen in a number of ovarian carcinomas with wildtype p53 sequence. We therefore analyzed mdm2 for amplification and overexpression by Southern and Northern hybridization and for RNA alternative splicing by RT-PCR and cDNA sequencing. The frequency and the type of alternative mdm2 RNA splice variants were also analyzed in ovarian tumors of borderline malignancy, benign cystadenomas, and normal ovarian tissue to identify patterns of splicing which may be involved in carcinogenesis.

Results of p53 and mdm2 alterations were statistically evaluated for correlations to clinical and histopathological parameters, response to platinum-based chemotherapy, time to relapse or progression of disease, and overall survival. Furthermore p53 mutation and overexpression were analyzed in correlation to mdm2 alterations to clarify whether loss of the p53 binding domain in mdm2 splice variants would cause stabilization and accumulation of p53 protein in the absence of mutations.

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