Cisplatin-induced apoptosis andp53 gene status in a cisplatin-resistant human ovarian carcinoma cell line

Abstract

Cisplatin-induced apoptosis and pS3 gene status were analyzed in human ovarian carcinoma usin a parental IGR-OVI line and a derived cisplatin-resistant I&-OVI /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-OVI/ 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 pS3 gene in sensitive and resistant cells revealed the presence of the same heterozygous mutation in exon 5. A 2-fold increase in pS3 mRNA and protein amounts was observed in resistant cells as assessed by Northern and Western blots, respectively. lmmuncqtochemical staining revealed a higher percentage of p53 stained nuclei in resistant cells. RT-PCR analysis of pS3 transcripts showed that both wild-type and mutated alleles were transcribed in sensitive as well as in resistant cells. However, mutated transcripts were I .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.o 1996 Wiley-Liss, Inc.Platinum-based chemotherapy is a standard treatment for ovarian canccr. Howevcr, the initial positive responses to therapy are often limited by thc development of drug rcsistance. Resistance to anti-tumor treatments may be due to impaired drug availability, altered host metabolism or the induction or selection of resistant cells. DNA is the critical intracellular target for platinum compounds, and cytotoxicity is thought to be mediatcd by thc inhibition of DNA synthesis after formation of platinum-DNA cross-links. Various mcchanisms responsiblc for the development of cellular resistance to platinum complexes have bcen reported and include altered membrane transport, inactivation of the drug by cellular thiols such as glutathione as well as mctallothioneins and incrcascd tolerance of DNA damage o r incrcascd repair (Andrews and Howell, 1990).However, there is cvidence that drug-target interaction is not the sole determinant of cellular sensitivity to chemothcrapcutic agents. Although they intcract with different targets, most of the cytotoxic ant...

Gamma-rays-induced death of human cells carrying mutations of BRCA1 or BRCA2

“…It was cultured in DMEM medium supplemented with 20% FBS. IGROV1 is an ovarian tumor cell line heterozygote for a mutant BRCA1 allele (BRCA1 +/7 ) (Fajac et al, 1996). The constitutional BRCA1 or BRCA2 genotype of the woman from whom the tumor was collected is not known.…”

Growth inhibition and apoptosis induction in ovarian cancer cells

“…Alterations in the p53 gene in ovarian carcinoma cell lines are rarely described (22,23,38). We found that both cell lines expressed high levels of p53 indicating a stabilization and inactivation of the protein.…”

“…This might be explained by the observation that cisplatin resistant tumor cells show an elevated level of the p53 protein. Sequence analysis revealed that this p53 could either be mutant or wild-type (22,23). Moreover, these tumor cells also exhibited a less reduced inhibition of DNA synthesis after Á-irradiation.…”

“…It is known that cisplatin induced apoptosis in human ovarian cancer cells (22). However, many other tumors are intrinsically resistant against cisplatin.…”

Temperature‐sensitive Ovarian Carcinoma Cell Line (OvBH‐1)

“…2,[13][14][15][16][17] Some authors reported point mutations in the highly conserved DNA binding domain encoded by exons 5-8 of the gene, resulting in single amino acid changes. 2,13,15,18) However, no data are available concerning the p53 status in ovarian clear cell carcinoma cell lines. Recently, we established a new ovarian clear cell carcinoma cell line designated OvBH-1.…”