Characterization of the DNA-DNA cross-linking activity of 3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin.

3'-(3-Cyano-4-morpholinyl)-3'-deaminoadriamycin (CMA) is a highly potent analogue of the antitumor agent, Adriamycin (ADR), being up to 1500 times more cytotoxic both in vivo and in vitro. In contrast to ADR, CMA, and 5-imino-3'-(3-cyano-4-morpholinyl)-3'-deaminoadriamycin (ICMA) have been shown to possess alkylating activity, as seen by their ability to produce DNA-DNA cross-links in human and murine tumor cells and in isolated lambda-phage DNA. We have compared the pharmacological activities of CMA, ICMA, and the alkylating agent, chlorambucil (CHL), in order to determine the roles of intercalation, the quinone ring, and DNA base composition, in cross-linking by CMA. CMA was 27-and 1000-fold more active than ICMA and CHL, respectively, in cross-linking DNA in L5178Y cells. In addition, the maximum level of cross-linking in L5178Y cells was reached more rapidly with CMA than with CHL, and the CMA cross-links were removed faster and more efficiently by these cells. CMA was 26- and 450-fold more active than ICMA and CHL, respectively, in producing DNA cross-links in isolated lambda-phage DNA. In contrast, the alkylating activity of CMA was only 6-fold greater than CHL, as measured by the ability of the drugs to bind to the nucleophile, p-nitrobenzyl pyridine. CMA was a better DNA intercalator than ICMA, whereas CHL did not intercalate. In addition, the intercalating agent, ethidium bromide, inhibited the cross-linking activity of CMA, but not that of CHL, suggesting that intercalation contributed to the cross-linking activity of CMA. CMA produced an increasing level of cross-linking, but showed no difference in intercalation, with isolated DNA of increasing G-C content, suggesting a preference for alkylating G-C bases. Both the cross-linking and intercalating, but not the alkylating, activities of CMA and ICMA were decreased by the reducing agent, sodium borohydride, providing additional evidence that the intercalative interaction of the ADR analogues with DNA contributes to their DNA cross-linking activity. Thus, alterations to the quinone group may effect the intercalating activity of these analogues and may contribute to the difference in cross-linking activity between CMA and ICMA.

Increased sensitivity of quinone resistant cells to mitomycin C.

L5178Y cells resistant to the model quinone antitumor agent, hydrolyzed benzoquinone mustard, were four-fold more sensitive to mitomycin C compared to parental cells. Mitomycin C also produced increased DNA-DNA crosslinking in these cells compared to parental L5178Y cells, but did not induce DNA double strand breaks in either cell line. The resistant cells have a 24-fold increased level of DT-diaphorase activity, an enzyme that produces two electron reduction of quinone groups. Dicoumarol, an inhibitor of DT-diaphorase, significantly inhibited crosslinking and cytotoxicity by mitomycin C in the quinone resistant cells. These findings suggest that DNA-DNA cross-linking may be a major contributor to mitomycin C cytotoxic activity in L5178Y cells, and that the hydroquinone of mitomycin C may play a major role in the crosslinking activity of this agent.

Role of NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase) in activation of mitomycin C under hypoxia.

The role of the two-electron reducing enzyme DT-diaphorase in the activation of mitomycin C under hypoxic conditions was investigated. Mitomycin C activity was compared in L5178Y murine lymphoblasts, which have low levels of DT-diaphorase activity, and L5178Y/HBM10 cells, which have elevated levels of enzyme activity. The cytotoxic and DNA cross-linking activities of mitomycin C were greater in L5178Y/HBM10 cells than in L5178Y cells. In L5178Y/HBM10 cells, dicoumarol, an inhibitor of DT-diaphorase, decreased cell kill and DNA cross-linking by mitomycin C in air but had no significant effect on these activities under hypoxia. By comparison, in L5178Y cells, dicoumarol had no effect on drug activity under either aerobic or hypoxic conditions. A model for the activation of mitomycin C by both one-electron and two-electron reduction is proposed. Our findings suggest that two-electron reduction by DT-diaphorase has only a limited role in the activation of mitomycin C under hypoxic conditions, although this enzyme appears to be an important contributor to drug activation under aerobic conditions.