Light and electron microscopy, flow cytometry and DNA gel electrophoresis have been used to study the induction of apoptosis in mammalian cells by platinum-based drugs. In particular, they have been used to observe the effects of the chemotherapeutic drug cisplatin on three human ovarian carcinoma cell lines-one sensitive to the drug (CH1), one with acquired resistance (CHlcisR) and one with intrinsic resistance (SKOV-3).
The 50% inhibitory concentrations after a 2 h exposure to drug were: CH1 -2.5 laM; CHlcisR -7.5 DM; SKOV-3 -33 DM. Despite the variation in sensitivity, the amount of platinum bound to DNA and the subsequent rate of platinum removal was similar for the three lines.
After treatment with cisplatin, the cells detached from the culture dish in a time-and dose-dependent fashion. The detached cells morphologically were quite distinct from the attached cells and showed changes in their chromatin structure indicative of apoptosis. However their DNA had not been degraded into oligonucleosomal fragments (200 bp and multiples thereof) but had been cut into larger fragments (30 kbp) of a size associated with chromatin domains (chromatin loops). At equitoxic doses of drug, the number of cells undergoing apoptosis was similar for the three cell lines. Flow cytometry showed that the most prominent effect on cell cycle kinetics was a stow down in S phase transit. Subsequently there was a G 2 block. Although apoptosis could be induced in G~, the cells appeared to undergo apoptosis predominantly from S phase.
We conclude that: (1) The sensitivity of these cell lines to cisplatin was governed by their ability to handle damage caused by platination of the DNA; (2) the mechanism of cisplatin-induced cell death in all three cell lines was the induction of apoptosis; (3) apoptotic cell death does not require degradation of DNA into nucleosomal sized fragments; (4) the results provide further evidence in support of the hypothesis that endonucleolytic degradation of DNA at the linkers between the chromatin domains is an important early event in apoptosis.