Cell cycle-dependent cytotoxicity of alkylating agents: determination of nitrogen mustard-induced DNA cross-links and their repair in Chinese hamster ovary cells synchronized by centrifugal elutriation.

Chinese hamster ovary cells were synchronized into the different phases of the cell cycle by centrifugal elutriation and treated with nitrogen mustard (HN2) in order to investigate the role of DNA damage and repair processes in the cell cycle-dependent cytotoxicity of this alkylating antitumor agent. In agreement with previous studies, cell populations enriched in G1 were the most sensitive to HN2, and those enriched in late S phase-G2 were more resistant, as determined by clonogenic assay. Although the variation in surviving fraction through the cell cycle in response to a single dose (3 micrograms/ml; 1.0 h) of HN2 was as great as a factor of 10, complete dose-response curves generated for the most sensitive and most resistant elutriator fractions indicated that such changes could be accounted for by a ratio of D0 values of only 1.4 Cells synchronized by this same method were also analyzed for their relative levels of HN2-induced DNA cross-linking using the sensitive technique of alkaline elution. There was no significant difference in the levels of either DNA interstrand or DNA-protein cross-links induced in the two elutriator fractions described above immediately after the HN2 treatment. When the amount of DNA cross-linking in the two fractions was measured 6 h after treatment, considerable repair had occurred; however, there was no measurable difference in the rate of repair of either type of cross-link (i.e., DNA interstrand and DNA-protein) in the different phases. Differences in DNA damage and repair processes could not therefore be resolved within the confidence limits of available assays and probably cannot account for the differential cytotoxicity of HN2 towards cells in the different cell cycle phases.