Isolation and characterization of a major adduct between mitomycin C and DNA

Tomasz, Maria; Lipman, Roselyn; McGuinness, Brian; Nakanishi, Kōji

doi:10.1021/ja00225a048

Cited by 68 publications

(58 citation statements)

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“…For example, the frequencies of total adduct in calf thymus DNA were 1.3 × 10 -3 and 29 × 10 -3 with DMC and MC, respectively (from data from Figure S2, panels a and c, Supporting Information). Similar findings in various cell-free systems were reported previously (6,32,33). What could be the mechanism for the much higher efficiency of alkylation of DNA by DMC than by MC in EMT6 cells?…”

Section: Methodssupporting

confidence: 86%

Relative Toxicities of DNA Cross-Links and Monoadducts: New Insights from Studies of Decarbamoyl Mitomycin C and Mitomycin C

Palom

Kumar

Tang

et al. 2002

Chem. Res. Toxicol.

139

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Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs. Paradoxically, DMC is slightly more toxic than MC to hypoxic EMT6 mouse mammary tumor cells as well as to CHO cells. To resolve this paradox, EMT6 cells were treated with MC or DMC under hypoxia at equimolar concentrations and the resulting DNA adducts were analyzed using HPLC and UV detection. MC treatment generated both intrastrand and interstrand cross-link adducts and four monoadducts, as shown previously. DMC generated two stereoisomeric monoadducts and two stereoisomeric ICL adducts, all of which were structurally characterized; one was identical with that formed with MC, the other was new and unique to DMC. Overall, adduct frequencies were strikingly higher (20-30-fold) with DMC than with MC. Although DMC monoadducts greatly exceeded DMC cross-link adducts ( approximately 10:1 ratio), the latter were equal or higher in number than the cross-link adducts from MC. DMC displayed a much higher monoadduct:cross-link ratio than MC. The similar cytotoxicities of the two drug show a correlation with their similar DNA cross-link adduct frequencies, but not with their total adduct or monoadduct frequencies. This provides specific experimental evidence that the ICLs rather than the monoadducts are critical factors in the cell death induced by MC. In vitro, overall alkylation of calf thymus DNA by DMC was much less efficient than by MC. Nevertheless, ICLs formed with DMC were clearly detectable. The chemical pathway of the cross-linking was shown to be analogous to that occurring with MC. These results also suggest that the differential sensitivity of Fanconi's Anemia cells to MC and DMC is related to factors other than a selective defect in cross-link repair.

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Section: Methodssupporting

confidence: 86%

Relative Toxicities of DNA Cross-Links and Monoadducts: New Insights from Studies of Decarbamoyl Mitomycin C and Mitomycin C

Palom

Kumar

Tang

et al. 2002

Chem. Res. Toxicol.

139

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“…The cytotoxicity of MC has been attributed mainly to the generation of lethal DNA cross-links. DMC was initially thought to be devoid of DNA cross-linking activity, based on chemical considerations and on tests carried out in cell-free systems that employed chemical reductive drug activation (13,31). Conse- quently, DMC has traditionally been regarded as the monofunctional counterpart of MC; however, this is not the case.…”

Section: Dna Adducts Do Not Always Activate the P53mentioning

confidence: 99%

Differential Activation of p53 by the Various Adducts of Mitomycin C

Abbas

Olivier

Lopez

et al. 2002

Journal of Biological Chemistry

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Mitomycin C (MC) is a cytotoxic chemotherapeutic agent that causes DNA damage in the form of DNA crosslinks as well as a variety of DNA monoadducts and is known to induce p53. The various DNA adducts formed upon treatment of mouse mammary tumor cells with MC as well as 10-decarbamoyl MC (DMC) and 2,7-diaminomitosene (2,7-DAM), the major MC metabolite, have been elucidated. The cytotoxicity of DMC parallels closely that of MC in a number of rodent cell lines tested, whereas 2,7-DAM is relatively noncytotoxic. In this study, we investigate the ability of MC, DMC, and 2,7-DAM to activate p53 at equidose concentrations by treating tissue culture cell lines with the three mitomycins. Whereas MC and DMC induced p53 protein levels and increased the levels of p21 and Gadd45 mRNA, 2,7-DAM did not. Furthermore, MC and DMC, but not 2,7-DAM, were able to induce apoptosis efficiently in ML-1 cells. Therefore the 2,7-DAM monoadducts were unable to activate the p53 pathway. Interestingly, DMC was able to initiate apoptosis via a p53-independent pathway whereas MC was not. This is the first finding that adducts of a multiadduct type DNA-damaging agent are differentially recognized by DNA damage sensor pathways.

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“…Alkylation of DNA by MC via either one-or two-electron reduction has been implicated in its ability to limit tumor cell growth (Iyer & Szybalski, 1964;Tomaszet al, 1974;Lownet al, 1976). MC reacts covalently to DNA both in monofunctional and bifunctional manners, generating, in the latter case, inter-or intrastrand DNA crosslinks that are chemically stable (Szybalski & Iyer, 1967;Tomasz et al, 1986Tomasz et al, ,a,b, 1987Tomasz et al, , 1988bBizanek et al, 1992a). 0006-2960/93/0432-4708%04.00/0…”

mentioning

confidence: 99%

Effect of site specifically located mitomycin C-DNA monoadducts on in vitro DNA synthesis by DNA polymerases

Basu¹,

Hanrahan²,

Malia³

et al. 1993

Biochemistry

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A series of site-specifically modified oligodeoxynucleotides were synthesized that contained either of the two known mitomycin C-DNA monoadducts. In vitro DNA synthesis was carried out on some of these templates using a modified bacteriophage T7 DNA polymerase (Sequenase), AMV reverse transcriptase, and two different varieties of Escherichia coli DNA polymerase I (Klenow fragment)--one that carries the normal 3'-->5' exonuclease activity and a mutant protein that lacks this enzymatic function. Regardless of the type of DNA polymerase being used, DNA synthesis was terminated nearly quantitatively at the nucleotide 3' to each of these two monoadduct sites, although primer extension to full length of the template was noted with the unmodified control template. Substitution of Mn2+ for Mg2+ at a high concentration of the deoxynucleotide triphosphates resulted in incorporation of nucleotides opposite the adduct in the incubations with Sequenase or the 3'-->5' exonuclease-free Klenow fragment; however, primer extension beyond the adduct site did not take place. These studies demonstrated that the mitomycin monoadducts are strong blocks of replication and are likely to be toxic lesions in vivo. Since previous molecular modeling studies and molecular mechanical calculations indicated that the mitomycin adduction does not induce severe distortions at the site of adduction, a lack of base-pairing ability of the modified base in the extended product is unlikely to be the reason for the inhibitory effect. Instead, energy-minimized structural models indicated that additional hydrogen-bonding interactions have been introduced by the mitomycin moiety, and perhaps this increased thermodynamic stabilization of a distorted structure of the replication fork, in turn, may block the replication bypass. Experimental evidence of increased thermodynamic stability was provided by thermal melting of a template/primer complex that presumably a polymerase encounters in a typical replication fork. Consistently higher Tm of the adducted "replication fork" was noted when compared to its unmodified counterpart.

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Isolation and characterization of a major adduct between mitomycin C and DNA

Cited by 68 publications

References 0 publications

Relative Toxicities of DNA Cross-Links and Monoadducts: New Insights from Studies of Decarbamoyl Mitomycin C and Mitomycin C

Relative Toxicities of DNA Cross-Links and Monoadducts: New Insights from Studies of Decarbamoyl Mitomycin C and Mitomycin C

Differential Activation of p53 by the Various Adducts of Mitomycin C

Effect of site specifically located mitomycin C-DNA monoadducts on in vitro DNA synthesis by DNA polymerases

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