Cell cycle dependency of cisplatin cytotoxicity on ovarian cancer cells.

The cytotoxicity of Cisplatin (CDDP) is traditionally believed to be cell cycle independent. This general belief is not in agreement with CDDP-induced apoptosis. Therefore, cell cycle dependency of cisplatin was investigated using human ovarian carcinoma OV 2008 cell line synchronized at the different phases of the cell cycle (i.e. subpopulations). Clonogenic assay, flow cytometry, fluorescence microscopy, electron microscopy (EM), atomic absorption spectroscopy (AA), and various biochemical techniques were used in this project. OV 2008 cell subpopulations at the different phases of G1, G1/S, S and G2/M were exposed to 1 m g/ml of CDDP for one hour (n = 3). CDDP resulted in 66 +/- 6(SE)%, 50 +/- 6%, 42 +/- 5%, and 41 +/- 5% survival for cells in G2/M, S, G1/S boundary and G1 of the cell cycle, respectively. Flow cytometry confirmed a general pattern of arrest in S and G2/M for all subpopulations, in spite of the different percentages of death. A cell cycle delay in arrest was noted when cells were exposed to CDDP in late S or G2/M, but not for the other two phases. EM revealed both apoptotic and necrotic cell death. For all subpopulations, the appearance of apoptotic and necrotic cells were 70% and 30% in about 12 hours, with 25% and 75% in 30 hours, respectively. All subpopulations of cells had accumulated the same amount of intracellular elemental platinum after exposure to CDDP. However, different amounts of DNA adducts were formed for cells in each subpopulation. In general, the rank order of CDDP cytotoxicity on the different phases of OV 2008 cell cycle is as follows; G1>G1/S>S>G2/M. This rank order is found in good agreement with the rank order of CDDP-DNA binding, as well as intracellular acidity (pHi) of OV 2008 cells; being at the lowest pH in G1 and highest in G2/M. Control experiments have shown the conversion of CDDP to its active metabolite of aquated CDDP (AP) in acidic media. pH dependent metabolism of CDDP to AP, which will then bind to DNA was confirmed. We have concluded that OV 2008 cells have different sensitivity to CDDP at the different phases of the cell cycle, which is related to the variations in pHi, and consequently the intracellular metabolism of CDDP to AP