Cross-link formation and chemopotentiation of EMT-6/Ro cells exposed to MISO after CCNU treatment in vitro.

Experiments were designed to measure cross-link formation following combined treatment of EMT-6/Ro tumor cells with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and misonidazole (MISO) in vitro. To avoid MISO-induced glutathione (GSH) depletion, which might contribute to enhanced monoadduct formation by reducing the protective GSH pools, a post-incubation (i.e. treatment with CCNU for one hour in air followed by MISO treatment in hypoxia) protocol was adopted. Utilizing this treatment scheme, it was possible to significantly enhance CCNU toxicity by post-treating with MISO immediately after exposure to CCNU. Enhanced cross-link formation detected by alkaline elution, at this time, correlated well with the magnitude of cell-kill enhancement, thereby implicating enhanced cross-link formation in the mechanism of potentiation. However, if the cells were allowed to incubate for various intervals between CCNU and MISO treatments, the magnitude of potentiation progressively diminished. Beyond approximately 8-10 hours (corresponding to the time required for maximal cross-link formation after CCNU treatment), treatment with MISO was ineffective at potentiating CCNU cytotoxicity. These experiments suggest that chemopotentiation can be produced by treating with MISO after treatment with CCNU (post-incubation) and that enhanced cross-link formation is involved in the mechanism of MISO chemopotentiation of CCNU activity. The kinetic studies, using the post-incubation protocol, further suggest that the chemopotentiating effect of MISO is exerted subsequent to monoadduct formation and probably does not involve inhibition of DNA-DNA cross-link repair.