Cisplatin-induced apoptosis and p53 gene status in a cisplatin-resistant human ovarian carcinoma cell line.
Cisplatin-induced apoptosis and p53 gene status were analyzed in human ovarian carcinoma using a parental IGR-OV1 line and a derived cisplatin-resistant IGR-OV1/DDP subline. Compared with parental cells, cisplatin-resistant cells exhibited a 5-fold higher resistance index and a 2-fold longer doubling time. Cisplatin induced apoptosis in both cell lines, as assessed by cell morphology and the presence of a DNA ladder. However, high concentrations were necessary to induce apoptosis in resistant cells. These cells elicited a 5-fold decrease in the number of platinum atoms bound per nucleotide. IGR-OV1/DDP cells also exhibited enhanced drug efflux and a higher glutathione content. Our data suggest that the levels of cisplatin-DNA lesions are critical for drug sensitivity and apoptosis induction in this in vitro ovarian carcinoma model. Comparative analysis of the p53 gene in sensitive and resistant cells revealed the presence of the same heterozygous mutation in exon 5. A 2-fold increase in p53 mRNA and protein amounts was observed in resistant cells as assessed by Northern and Western blots, respectively. Immunocytochemical staining revealed a higher percentage of p53 stained nuclei in resistant cells. RT-PCR analysis of p53 transcripts showed that both wild-type and mutated alleles were transcribed in sensitive as well as in resistant cells. However, mutated transcripts were 1.5-fold more abundant than wild-type transcripts in sensitive cells, whereas they were 2-fold higher in resistant cells. In addition, mdm-2 protein was over-expressed in resistant cells. Our results address the question of the functionality of p53 protein and its possible role in apoptosis induction in this model. In resistant cells, p53 protein might be inactivated by 2 mechanisms: mutation and complexation with mdm-2 protein. Therefore, the presence of non-functional p53 in resistant cells might be involved in the relative failure of cisplatin-induced apoptosis in these cells.
Laboratory of Clinical and Molecular Pharmacology, Institut G. Roussy, Villejuif, France.
A
A
Fajac
J
J
Da Silva
J C
JC
Ahomadegbe
J G
JG
Rateau
J F
JF
Bernaudin
G
G
Riou
J
J
Bénard
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology*; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D002945:Cisplatin / Q000378:metabolism / Q000494:pharmacology*; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D005260:Female; D016158:Genes, p53 / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D012333:RNA, Messenger; D005982:Glutathione Transferase; D012194:RNA-Directed DNA Polymerase; D005978:Glutathione; D002945:Cisplatin