Reaction of DNA with chemically or enzymatically activated mitomycin C: isolation and structure of the major covalent adduct.

Abstract

The antitumor antibiotic mitomycin C is shown to form a covalent complex with calf thymus DNA under anaerobic conditions in the presence of either NADPH cytochrome c reductase/NADPH, xanthine oxidase/NADH, or the chemical reducing system H2/PtO2. Digestion of the complex with DNase I/snake venom diesterase/alkaline phosphatase yields a single mitomycin deoxyguanosine adduct as the major DNA alkylation product, identified as N2-(2",3,7"-diaminomitosen-l"a-yl) 2'-deoxyguanosine (Structure 2). Two minor adducts, 2-5% each of the total adduct pool, are isolated and identified as the 1"j8 stereoisomer of 2 (Structure 3), and 10"-decarbamoyl-2 (Structure 7). The same results were obtained with M13 DNA and poly(dG-dC) poly(dG-dC); however, in the latter case, a minor adduct apparently possessing two deoxyguanosine and one mitomycin unit is isolated. Digestion of the covalent mitomycin-calf thymus DNA complex with nuclease P1 yields four dinucleotide adducts, all of which consist of 2 linked at its 3' end to each of the four possible 5' nucleotides (A, T, G, and C). Upon treatment of each dinucleotide adduct with snake venom diesterase/alkaline phosphatase, 2 is released along with the corresponding free nucleoside. In apparent conflict with the present results, previous reports from another laboratory have indicated that modification of calf thymus DNA by mitomycin C under conditions identical to those described here result in the isolation of three mitomycin C mononucleotide adducts possessing linkages of the drug to N2 and 06 of guanine and N6 of adenine. Evidence is shown suggesting that the latter adducts are actually three of the above four dinucleotide derivatives of 2 obtained independently by us and, thus, all of them in fact possess an identical N2-mitosenylguanine adduct moiety. Model-building studies indicate an excellent fit of the guanine N2-linked drug molecule inside the minor groove of B-DNA with no appreciable distortion of the DNA structure.Mitomycin C (MC; Structure 1), a potent antibiotic and clinically used antitumor agent, is known to interact covalently with DNA in vivo and in vitro. This is manifested by the reversible melting behavior of MC-exposed DNA, attributed to formation of covalent crosslinks between the complementary strands (1) and by covalent association of the ultraviolet chromophore of MC to DNA (2). These processes require reduction of MC into a transiently activated form, which is thought to occur in cells and can be mimicked easily in vitro chemically or enzymatically (1). The cytotoxicity of MC is most likely a direct result of DNA alkylation, as indicated by the parallels in biological activity of MC with a number of known "DNA damaging agents": selective inhibition of DNA replication (1) and sister chromatid exchange (4), and cross-resistance or cross-hypersensitivity of bacterial (1, 5) and mammalian (6) cells to UV light and MC. The molecular nature of the covalent interactions between MC and DNA has remained elusive, mainly because of the difficulty of isolating low...

DNA sequence‐specific adducts of adriamycin and mitomycin C

“…The transcriptional blockages observed following reductive activation of mitomycin C confirm the well-documented observation of' DNA adducts with this compound [16][17][18]. The fact that these blockages are independent of elongation time is consistent with such covalent adducts.…”

“…If these adducts occur to a significant degree in vivo, then it is expected that they would result in termination of transcription, and may therefore comprise an important mode of action of these drugs, particularly in the light that tumour ceils contain a more reducing environment than normal cells [15]. Similar adducts with mitomycin C have been well documented [16][17][18]. We present here an in vitro transcription assay which exhibits blockage of transcription by E. coli RNA polymerase at specific DNA sites when mitomycin C is reduced by xanthine oxidase/NADH, or when adriamycin is present with Fe 2÷ or Fe 3+.…”

Mode of Action for Mitomycin C Inactivating Factors ofPseudomonas convexa4-87

“…Recently, Tomasz et al also proposed a structure for the covalent adducts. 9 ) The formation of these metabolites supports the hypothesis that the reduction of the heterocyclic quinone of MMC led to the cleavage of the aziridine ring and generated a reaction center at the C-1 position.…”

32P-postlabeling assay for carcinogen-DNA adducts: nuclease P1-mediated enhancement of its sensitivity and applications.

“…In the intensification procedure, apparent RAL values (<RAL>) were divided by adduct intensification factors to obtain actual RAL values (21,29 (22,32). In in vitro, chemically or enzymatically reduced MMC has been shown to react with DNA, forming monofunctional and bifunctional adducts in ratios, which vary depending on the reductive conditions (33,34); however, little information is available to date on in vivo adduction of tissue DNAs by MMC (23), probably because of unavailability of the drug in radiolabeled form. Using the 32P-postlabeling assay (Fig.…”