Studies on the mechanism of the cytotoxic action of the mitomycin antibiotics in hypoxic and oxygenated EMT6 cells.

The mitomycin antibiotics, because of their preferential toxicities for hypoxic cells, have significant potential as adjuncts to ionizing radiation in the treatment of solid tumors. To gain information on the mechanism by which these agents exert their cytotoxicities to hypoxic and aerobic cells, the effects of MC, POR and several of their analogs were studied in EMT6 mammary carcinoma cells. The rate of uptake of POR by these cells was directly correlated with the cytotoxicity produced by this agent under both hypoxia and aeration. At equivalent concentrations, uptake of POR into hypoxic cells was more rapid than into aerobic cells. Hypoxic cells also accumulated the antibiotic in concentrations well in excess of that present in the extracellular medium, presumably as a result of reductive activation and covalent binding of POR to cellular structures. Such activation and binding occur to a much lesser degree in aerated cells, resulting in the rapid efflux of POR from these cells when the antibiotic is removed from the extracellular environment. To gain information on the reaction of POR with DNA, mono- and bis-adducts formed in EMT6 cells exposed to this agent were measured. Three major adducts were formed. Two were mono-adducts consisting of deoxyguanosine linked at its N2-position to the C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct in which POR was cross-linked to two deoxyguanosines at their N2-positions. More adducts were formed in hypoxia than in air, and more bis-adducts were present in hypoxic cells. Simultaneous exposure of cells to both POR and DIC reduced the total adduct level and a new unknown adduct was formed, primarily under hypoxia. Several mitomycins were evaluated for their capacity to kill EMT6 cells and to produce DNA cross-links in both hypoxia and aeration. The number of cross-links required to produce a given amount of cell kill was similar, regardless of the mitomycin employed or the degree of oxygenation. The findings support the concept that DNA is a critical target in the action of the mitomycins and that cross-linking of the DNA creates an important lesion for cytodestruction.