Emergence of p53 mutant cisplatin-resistant ovarian carcinoma cells following drug exposure: Spontaneously mutant selection

We have previously shown that p53 mutations are associated with cisplatin r esistance in ovarian carcinoma IGROV-1/Pt 1 cells. The relationship between p53 status and the development of resistance has not been completely eluci dated; in particular, the biological mechanisms behind the acquired drug-re sistant p53-mutant phenotype were not clearly explained. Thus, in this stud y, we investigated whether the p53 mutations found in IGROV-1/Pt 1 cells (2 70 and 282 codons) resulted from selection, under the selective pressure of the cytotoxic treatment, of a spontaneously mutant cell population preexis tent in the cisplatin-sensitive parental cell line (IGROV-1) or were induce d by drug (genotoxic) treatment. For this purpose, an allele-specific PCR a pproach was used. Primers carrying the desired mutations (T-->A codon 270, C-->T codon 282) in the 3' terminus, and the corresponding wild-type primer s were used to amplify genomic DNA from the original IGROV-1 cell line used to select the mutant IGROV-1/Pt 1, To increase sensitivity, we hybridized blots of the PCRs with the radiolabeled PCR fragment from IGROV-1/Pt 1, Amp lification was obtained for IGROV-1 DNA with the mutated allele-specific pr imers, indicating the preexistence of a mutated population in the IGROV-1 c ell line. Titration experiments suggested that the frequency of the mutated alleles was <0.1%, Single-strand conformation polymorphism and allele-spec ific PCR analysis of the IGROV-1/Pt 0.1 cells, which are less resistant to cisplatin than IGROV-1/Pt 1 cells and which carry both mutant and wild-type p53 alleles with a wildtype predominance, suggested a progressive selectio n of the mutant population by cisplatin treatment. This is the first observ ation that indicates that a subpopulation of p53 mutant cells can occasiona lly be selected by cisplatin treatment. Thus, considering the susceptibilit y to spontaneous mutations of the p53 gene in advanced ovarian carcinoma, t he selection process resulting in emergence of p53 mutant tumors is a possi ble origin of resistance of ovarian carcinoma to DNA-damaging agents, The s urvival advantage of p53 mutant cells in the presence of genotoxic agents c ould be related to a loss of susceptibility to p53-dependent apoptosis and to defects in checkpoints pathways, resulting in genomic instability.