Emergence of p53 Mutant Cisplatin-resistant Ovarian Carcinoma Cells following Drug Exposure: Spontaneously Mutant Selection 1 Sabina C. Righetti, Paola Perego, Elisabetta Corna, Marco A. Pierotti, and Franco Zunino 2 Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy Abstract We have previously shown that p53 mutations are associated with cisplatin resistance in ovarian carcinoma IGROV-1/Pt 1 cells. The relationship between p53 status and the development of resistance has not been completely elucidated; in particular, the biological mechanisms behind the acquired drug-resistant p53- mutant phenotype were not clearly explained. Thus, in this study, we investigated whether the p53 mutations found in IGROV-1/Pt 1 cells (270 and 282 codons) resulted from selection, under the selective pressure of the cytotoxic treatment, of a spontaneously mutant cell population preexistent in the cisplatin-sensitive parental cell line (IGROV-1) or were induced by drug (genotoxic) treatment. For this purpose, an allele-specific PCR approach was used. Primers carrying the desired mutations (T3A codon 270, C3T codon 282) in the 3 terminus, and the corresponding wild-type primers 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. Amplification was obtained for IGROV-1 DNA with the mutated allele-specific primers, indicating the preexistence of a mutated population in the IGROV-1 cell line. Titration experiments suggested that the frequency of the mutated alleles was <0.1%. Single-strand conformation polymorphism and allele-specific 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 wild- type predominance, suggested a progressive selection of the mutant population by cisplatin treatment. This is the first observation that indicates that a subpopulation of p53 mutant cells can occasionally be selected by cisplatin treatment. Thus, considering the susceptibility to spontaneous mutations of the p53 gene in advanced ovarian carcinoma, the selection process resulting in emergence of p53 mutant tumors is a possible origin of resistance of ovarian carcinoma to DNA-damaging agents. The survival advantage of p53 mutant cells in the presence of genotoxic agents could be related to a loss of susceptibility to p53-dependent apoptosis and to defects in checkpoints pathways, resulting in genomic instability. Introduction Cisplatin is among the most effective agents that are clini- cally available for the treatment of a variety of solid tumors, including ovarian carcinoma. However, the development of drug resistance is a common problem for the efficacy of the pharmacological treatment (1, 2). Different mechanisms may contribute to define the resistant phenotype, including alter- ations in drug-target interactions, expression of defense and/or detoxification mechanisms, and cellular responses to DNA damage (3–5). In particular, it has been proposed that reduction of the apoptotic response is a critical determinant of cisplatin efficacy (6, 7). Wild-type p53 is an important component of the pathway leading from DNA damage to apoptosis because p53 protein is implicated in multiple functions that include control of cell cycle, DNA repair, cell senescence, genomic stability, and stress responses (8, 9). Mutations of the p53 gene are common alterations found in a variety of human tumors (10). Although loss of normal p53 function can confer resistance to DNA-dam- aging agents as a consequence of a reduced cell suscep- tibility to apoptosis, the relevance of p53 mutations in chemosensitivity remains controversial (11–13). Several studies indicate that p53 can be inactivated in cisplatin- resistant cell systems (14, 15). Clinical studies support a correlation between missense mutations and resistance to platinum drug therapy (16). On the basis of these observations, the aim of this study was to clarify whether, in an ovarian carcinoma p53 mutant cisplatin-resistant variant, IGROV-1/Pt 1, the presence of p53 mutations was a consequence of the genotoxic treat- ment or resulted from a selection process. For our purpose, we used allelic-specific gene amplification by PCR. These results support that the p53 allele carrying a mutation at codon 270 preexisted in the IGROV-1 parental cell line with a frequency 0.1% and that the cell population exhibiting the mutation can occasionally be selected by cisplatin treat- ment. Results Cellular Sensitivity to Cisplatin. Fig. 1 shows cellular sen- sitivity to cisplatin of IGROV-1 cells and cisplatin-resistant Received 2/4/99; revised 4/29/99; accepted 5/24/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- cate this fact. 1 This work was partially supported by the Consiglio Nazionale delle Ricerche, Finalized Project ACRO, (Rome), by the Associazione Italiana per la Ricerca sul Cancro (Milan) and by the Ministero della Sanita ` (Rome). 2 To whom requests for reprints should be addressed, at Istituto Nazio- nale Tumori, Via Venezian 1, 20133 Milan, Italy. Phone: 39-02-2390267; Fax: 39-02-2390692; E-mail: zunino@istitutotumori.mi.it. 473 Vol. 10, 473– 478, July 1999 Cell Growth & Differentiation