6-Thioguanine-induced DNA damage as a determinant of cytotoxicity in cultured Chinese hamster ovary cells.

The mechanism of action of 6-thioguanine (TG) has been examined in cultured Chinese hamster ovary cells by direct measurement of the incorporation of the compound into DNA and by analysis of the resulting DNA damage. The predominant lesions as monitored by alkaline elution were DNA strand breaks. Very few, if any, interstrand or DNA-protein cross-links could be definitively observed. The cytotoxicity of TG as measured by colony-forming ability appeared closely related with its incorporation into DNA and the DNA strand scission events. As TG concentrations were increased, cytotoxicity, DNA incorporation, and strand scission reached a plateau; this result is consistent with earlier reports that TG produces a reversible block of DNA synthesis. Strand breaks appeared to be related to the incorporation of TG into DNA, since the addition of 1 microM cycloheximide during a 24-hr treatment with 3 microM TG prevented the cytotoxicity, prevented incorporation of TG into DNA, and eliminated the strand breaks. Alkali-labile sites were detected in the DNA of TG-treated cells by alkaline elution at pH 12.8, suggesting that depurination of TG residues by a glycosylase mechanism may occur. It is also postulated that TG residues are recognizable by the long-patch repair system, since UV-sensitive cells deficient for long-patch repair were more sensitive to TG than were wildtype cells. Furthermore, caffeine (1 mM) was shown to enhance the lethality of TG (3 microM), as monitored by colony formation, without altering levels of TG incorporation into DNA or the strand scission as measured immediately after treatment. This result, coupled with the known delayed cytotoxic response of TG, suggests that gaps may occur in newly synthesized DNA opposite TG residues and that the repair of these gaps by a postreplication repair mechanism is inhibited by caffeine.