Induction of apoptosis and stress response in ovarian carcinoma cell lines treated with ST1926, an atypical retinoid.

To understand the molecular mechanisms mediating apoptosis induction by a novel atypical retinoid, ST1926, the cellular response to drug treatment was investigated in IGROV-1 ovarian carcinoma cells carrying wild-type p53 and a cisplatin-resistant p53 mutant subline (IGROV-1/Pt1). Despite a similar extent of drug-induced DNA strand breaks, the level of apoptosis was substantially higher in p53 wild-type cells. p53 activation and early upregulation of p53-target genes were consistent with p53-dependent apoptosis in IGROV-1 cells. Stress-activated protein kinases were activated in both cell lines in response to ST1926. This event and activation of AP-1 were more pronounced in IGROV-1/Pt1 cells, in which the modulation of DNA repair-associated genes suggests an increased ability to repair DNA damage. Inhibition of JNK or p38 stimulated ST1926-induced apoptosis only in IGROV-1 cells, whereas inhibition of ERKs enhanced apoptosis in both the cell lines. Such a pattern of cellular response and modulation of genes implicated in DNA damage response supports that the genotoxic stress is a critical event mediating drug-induced apoptosis. The results are consistent with apoptosis induction through p53-dependent and -independent pathways, regulated by MAP kinases, which likely play a protective role.

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells.

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.

Topoisomerase I activity and cellular response to radiation in Chinese hamster cells.

The aim of the present study was to investigate the synergistic potential of the combination of camptothecin, a specific inhibitor of topoisomerase I, and radiation in the the induction of chromosome aberrations and cell cycle delay in actively proliferating mammalian cells. Synergistic effects of the combined treatments were obtained for induced frequencies of aberrations in exponentially growing Chinese hamster ovary cells. The potentiating effects were more pronounced for aberrations of the exchange type, suggesting that interaction of unrepaired radiation- and camptothecin-induced lesions during replication may be involved in the observed drug-radiation synergism. Cytofluorimetric analysis of cell cycle progression in cells receiving the combined treatments displayed enhanced responses of CHO cells to S- and G2 phase delay induced by the single treatments. To investigate the determinants of the synergistic response, the influence of radiation exposure on the catalytic activity of topoisomerase I was assayed. A decreased plasmid supercoiled DNA relaxation capacity of crude extracts derived from irradiated CHO cells was found which suggests a decrease in the topoisomerase I catalytic activity following irradiation. In addition, a lower sensitivity of the enzyme from irradiated cells to inhibition of topoisomerase I activity by camptothecin was also observed using the same DNA relaxation test.