Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems.

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 mutants 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 consecutive 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. Cotransfection 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]  J. Bartek,et al.  Genetic and immunochemical analysis of mutant p53 in human breast cancer cell lines. , 1990, Oncogene.

[2]  John Calvin Reed,et al.  Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[3]  P. Workman.  To pop or not to pop: p53 as a critical modulator of tumour responsiveness to therapy in vivo? , 1995, Human & experimental toxicology.

[4]  A. Levine,et al.  Wild-type p53 mediates positive regulation of gene expression through a specific DNA sequence element. , 1992, Genes & development.

[5]  P. Burns,et al.  Increased accumulation of p53 protein in cisplatin‐resistant ovarian cell lines , 1993, International journal of cancer.

[6]  U. K. Laemmli,et al.  Cleavage of structural proteins during , 1970 .

[7]  C. Harris,et al.  Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. , 1994, Cancer research.

[8]  S. Korsmeyer,et al.  Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death , 1993, Cell.

[9]  P. May,et al.  Concomitant p53 gene mutation and increased radiosensitivity in rat lung embryo epithelial cells during neoplastic development. , 1994, Cancer research.

[10]  S. Howell,et al.  Cellular accumulation of the anticancer agent cisplatin: a review. , 1993, British Journal of Cancer.

[11]  D. Fisher.  Apoptosis in cancer therapy: Crossing the threshold , 1994, Cell.

[12]  R. Brown.  p53: a target for new anticancer drugs or a target for old drugs? , 1993, Annals of oncology : official journal of the European Society for Medical Oncology.

[13]  A. Eastman.  Activation of programmed cell death by anticancer agents: cisplatin as a model system. , 1990, Cancer cells.

[14]  A. Levine,et al.  Gain of function mutations in p53 , 1993, Nature Genetics.

[15]  M. Oren.  Relationship of p53 to the control of apoptotic cell death. , 1994, Seminars in cancer biology.

[16]  John Calvin Reed,et al.  Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. , 1994, Oncogene.

[17]  K. Kohn,et al.  p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents. , 1994, Cancer research.

[18]  M. Kashani-Sabet,et al.  Cisplatin resistance in human cancers. , 1991, Pharmacology & therapeutics.

[19]  J. Lehmann,et al.  Nuclear retinoic acid receptors: cloning, analysis, and function. , 1990, Methods in enzymology.

[20]  P. Shaw,et al.  Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[21]  D. Housman,et al.  p53 status and the efficacy of cancer therapy in vivo. , 1994, Science.

[22]  D. Lane,et al.  Mutant conformation of p53. Precise epitope mapping using a filamentous phage epitope library. , 1992, Journal of molecular biology.

[23]  A. Harris,et al.  A role for the human DNA repair enzyme HAP1 in cellular protection against DNA damaging agents and hypoxic stress. , 1994, Nucleic acids research.

[24]  M. Pierotti,et al.  Gene p53 mutations are restricted to poorly differentiated and undifferentiated carcinomas of the thyroid gland. , 1993, The Journal of clinical investigation.

[25]  M. Scheffner,et al.  Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression , 1993, Molecular and cellular biology.

[26]  M. Heinrich,et al.  Specific repression of granulocyte-macrophage and granulocyte colony-stimulating factor gene expression in interleukin-1-stimulated endothelial cells with antisense oligodeoxynucleotides. , 1992, Blood.

[27]  A. Pa,et al.  Cellular pharmacology of cisplatin: perspectives on mechanisms of acquired resistance. , 1990 .

[28]  S. Howell,et al.  Cellular pharmacology of cisplatin: perspectives on mechanisms of acquired resistance. , 1990, Cancer cells.

[29]  J. Blondal,et al.  The role of p53 in tumor progression. , 1994, Seminars in cancer biology.

[30]  R. Ozols,et al.  Platinum analogues in preclinical and clinical development , 1993, Current opinion in oncology.

[31]  J. Peacock,et al.  The role of apoptosis in cell killing by cisplatin: a flow cytometric study. , 1994, British Journal of Cancer.

[32]  John Calvin Reed,et al.  Identification of a p53-dependent negative response element in the bcl-2 gene. , 1994, Cancer research.

[33]  Bert Vogelstein,et al.  X-rays strike p53 again , 1994, Nature.

[34]  P. O'Connor,et al.  Induction of bax by genotoxic stress in human cells correlates with normal p53 status and apoptosis. , 1994, Oncogene.