Somatic Genetic Analysis of p53 Function and Cisplatin Resistance

p53 modulates a number of cellular responses to genotoxic stress including cell cycle arrest and apoptosis. Loss of p53 function either by mutation or otherwise may alter the sensitivity of cells to DNA damage. Introduction of a dominant negative p53 mutant (codon 143, Val to Ala) has been shown to confer cisplatin resistance in the chemosensitive human ovarian A2780 carcinoma cell line. Induced resistance of A2780 cells to cisplatin was used as the selection strategy to isolate genetic suppressor elements (GSEs) from retroviral libraries expressing random fragments of human or murine TP53 cDNA (Chapter 4). Six GSEs were identified, encoding either dominant negative mutant peptides or antisense RNA molecules which corresponded to various regions within the TPS3 gene. Both types of GSE induced cisplatin resistance when introduced individually into A2780 cells. Expression of antisense GSEs led to decreased intracellular levels of p53 protein. One sense GSE induced loss of p53-mediated activities such as DNA damage induced G1 arrest and apoptosis. A synthetic peptide, representing the predicted amino acid sequence of this GSE, conferred resistance to cisplatin when introduced into A2780 cells and inhibited the sequence specific DNA binding activity of p53 protein in vitro. Overall, these results directly indicate that inactivation of p53 function confers cisplatin resistance in these human ovarian tumour cells. We have identified short structural domains of p53 which are capable of independent functional interactions and highlighted the efficacy of this approach to discriminate biological active p53 GSEs from a random fragment library. This work may have use in the rational design of low molecular mass modifiers of the p53 response. Both G1 arrest and apoptosis induced by ionising radiation or cisplatin are dependent upon an intact p53-mediated DNA damage response pathway in the A2780 line. Evidence is presented for a defect in the p53-mediated DNA damage response pathway in cisplatin resistant derivatives of A2780 cells as determined by reduced DNA damage induced G1 arrest, apoptosis and transcription of certain target genes (Chapter 3). The basis of the p53 dysfunction in one of these resistant cell lines does not appear to be due mutation of the TP53 gene, overexpression of mdm2 protein, differences in the cell cycle related expression of p53 protein or gross changes in the pattern of p53 isoforms. Cisplatin resistant A2780 derivatives also acquire a microsatellite instability (RER+) phenotype, which is associated with defective mismatch repair (MMR) and loss of hMLH1 mRNA and protein expression. Defective MMR in these cisplatin resistant cells may lead to tolerance of DNA damage and reduced ability to engage both p53-dependent and -independent apoptotic pathways