Studies related to antitumor antibiotics. Part V. Reactions of mitomycin C with DNA examined by ethidium fluorescence assay

Abstract

The cytotoxic action of the antitumor antibiotic mitomycin C occurs primarily at the level of DNA. Using highly sensitive fluorescence assays which depend on the enhancement of ethidium fluorescence only when it intercalates duplex regions of DNA, 3 aspects of mitomycin C action on DNA were studied: cross-linking events; alkylation without necessarily cross-linking and strand breakage. Cross-linking of DNA was determined by the return of fluorescence after a heat denaturation step at alkaline pH''s. Under these conditions denatured DNA gave no fluorescence. The cross-linking was independently confirmed by S1-endonuclease (EC 3.1.4.-) digestion. At relatively high concentrations of mitomycin the suppression of ethidium fluorescence enhancement was not due to depurination but rather to alkylation, as a result of losses in potential intercalation sites. A linear relationship existed between binding ratio for mitomycin and loss of fluorescence. The proportional decrease in fluorescence with pH strongly suggests that the alkylation was due to the aziridine moiety of the antibiotic under these conditions. A parallel increase in the rate and overall efficiency of covalent cross-linking of DNA with lower pH suggested that the cross-linking event, to which the primary cytotoxic action was linked, occured sequentially with alkylation by aziridine and then by carbamate. Mitomycin C, reduced chemically, induced single strand cleavage as well as monoalkylation and covalent cross-linking in PM2 covalently closed circular DNA. The inhbition of this cleavage by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), and by free radical scavengers suggested that the degradation of DNA observed to accompany the cytotoxic action of mitomycin C was largely due to the free radical O2 -. In contrast to the behavior of the antibiotic streptonigrin, mitomycin C did not inactivate the protective enzymes superoxide dismutase or catalase. Lastly, mitomycin C was able to cross-link DNA in the absence of reduction at pH 4. This was consistent with the postulated cross-linking mechanisms.