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Association between Cisplatin Resistance and Mutation of p53 Gene and Reduced Bax Expression in Ovarian Carcinoma Cell Systems¹

Divisions of Experimental Oncology B [P. P., S. R., R. S., C. C., F. Z.] and Experimental Oncology A [D. D., M. G., M. A. P.], Istituto Nazionale Tumori, Via Venezian I, 20133 Milan, Italy, and La Jolla Cancer Research Foundation, La Jolla, California 92037 [T. M., J. C. R]

p53 status may be a determinant of chemosensitivity of tumor cells; however, its involvement in cellular resistance to cisplatin remains uncertain. To investigate the relationships between p53 and the development of resistance to cisplatin, the p53 gene status was studied in ovarian carcinoma cell systems which included two cisplatin-resistant variants (IGROV-1/Pt 0.5 and IGROV-1/Pt 1) selected in vitro after prolonged drug exposure of the cisplatin-sensitive parental IGROV-1 cell line. IGROV-1/Pt 0.5 and IGROV-1/Pt 1 cell lines exhibited a degree of resistance of approximately 6 and 14, respectively, following 96-h exposure to the drug and were cross-resistant to other DNA-damaging agents (ionizing radiation and melphalan). Resistance to cisplatin paralleled a reduced cell susceptibility to cisplatin-induced apoptosis. DNA single-strand conformation polymorphism analysis of exons 5–9 demonstrated the presence of two mutant alleles at exon 8 in the two resistant cell lines, in contrast to the parental IGROV-1 cell line which exhibited the wild-type p53 gene. Direct DNA sequencing revealed that the mutations consist of two nucleotide changes in the DNA-binding domain at codons 270 (T/A) and 282 (C/T). The constitutive levels of p53 protein were lower in IGROV-1 than in IGROV-1/Pt cells. Following exposure to ionizing radiation or cisplatin, accumulation of the p53 protein was markedly enhanced only in the sensitive cells. Concomitantly, the expression of WAF-1 protein was strongly induced in the parental IGROV-1 cells, whereas WAF-1 protein remained undetectable in the IGROV-1/Pt 1 subline after DNA-damaging treatment. Consistent with this finding is the observation that ionizing radiation caused a different pattern of cell cycle perturbation in sensitive and resistant cells. Northern blot analysis demonstrated a marked reduction in bax mRNA levels in IGROV-1/Pt 1 cisplatin-resistant cells. Co-transfection assays with wild-type or mutant p53 expression plasmids and a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase were consistent with the role of p53 in regulation of bax expression in these cells. Taken together, these observations support a role for mutations of the p53 gene in the development of cisplatin resistance in ovarian cancer as a consequence of loss of the ability of p53 to transactivate bax, an apoptosis-inducing gene.

¹ This work was supported in part by grants from the Consiglio Nazionale delle Ricerche (Finalized Project Applicazioni Cliniche della Ricerca Oncologica), Associazione Italiana per la Ricerca sul Cancro, Ministero della Sanitá, NIH (CA-60181), and American Cancer Society (DHP-32C).

² To whom requests for reprints should be addressed. Phone: 39-2-2390267; Fax: 39-2-2390764.

Received 3/22/95. Accepted 11/20/95.

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