Maria Tomasz

Mitomycin C: small, fast and deadly (but very selective).

Mitomycin C, an important antitumor drug and antibiotic, has an extraordinary ability to crosslink DNA with high efficiency and absolute specificity for the sequence CpG. Recent results have shown how mitomycin C crosslinks DNA, and why the sequence specificity is so complete. This new understanding may allow the design of agents that mimic mitomycin Cs economy of structure and can crosslink other sequences.

The mitomycin bioreductive antitumor agents: Cross-linking and alkylation of DNA as the molecular basis of their activity

This review focuses on the chemical and enzymatic aspects of the reductive activation of mitomycin C, its disulfide analogs KW-2149 and BMS-181174, and, in less detail, FR66979 and FR900482, newly discovered antitumor antibiotics related to mitomycins. Furthermore, structural aspects of DNA damage induced by these drugs in vitro and in vivo are described, including the chemical and conformational characteristics of DNA interstrand and intrastrand cross-links and monofunctional alkylation products, with emphasis on DNA adducts of mitomycin C. The DNA sequence specificity of the damage and its mechanism is reviewed. The relationship between the chemical and structural properties of the DNA damage on the one hand, and the antitumor and other biological activities of the mitomycins on the other, is discussed.

Recruitment of Fanconi Anemia and Breast Cancer Proteins to DNA Damage Sites Is Differentially Governed by Replication

Fanconi anemia (FA) is characterized by cellular hypersensitivity to DNA crosslinking agents, but how the Fanconi pathway protects cells from DNA crosslinks and whether FA proteins act directly on crosslinks remain unclear. We developed a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. Interdependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Consistent with the distinct recruitment mechanism, recombination-independent crosslink repair was inversely affected in cells deficient of FANC-core versus BRCA-related FA proteins. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status.

H2O2 generation during the redox cycle of mitomycin C and dna-bound mitomycin C.

Reduction of mitomycin C by NaBH4 or by NADPH in the presence of a cell extract followed by exposure to air results in the generation of H2O2. This phenomenon occurs not only with free mitomycin but also with mitomycin irreversibly bound to DNA. In view of these findings, the antibiotic activity of mitomycin was tested in two bacterial systems: a facultative aerobic bacterium grown in the presence or absence of oxygen and obligate anaerobic bacterium. No oxygen effect could be demonstrated in either case in the growth-inhibitory and bactericidal activity of the drug. Nevertheless, the H202 generating capacity of mitomycin-DNA complexes inside the nucleus may play a role in the drug-induced biological damage to the genetic material of cells.

Extreme lability of the C-8 proton: A consequence of 7-methylation of guanine residues in model compounds and in DNA and its analytical application

Abstract 1. The C-8 proton of 7-methylguanosine rapidly exchanges with the solvent. The half-life of deuterium exchange is 5.5 min at pH 4.1, 28°, while at pH 7 the exchange is too fast to be measured by NMR spectroscopy. 1,7-Dimethylguanosine and 7-methylinosine behave analogously. The mechanism of this exchange seems to involve acidic dissociation of the C-8 proton. Two mechanistic alternatives, namely, tautomerism and reversible hydrolytic opening of the imidazole ring can be ruled out. This behaviour of 7-methylguanosine is analogous to that of compounds related to thiamine. 2. 7-Methylation of guanine residues in DNA results in a similar rapid isotope exchange at C-8. This was shown by methylating [8-3H]guanine-labeled DNA with dimethylsulfate. The amount of tritium released from the DNA as tritiated water corresponded to the amount of 7-methylguanine. 3. This observation provides a simple and selective method for the determination of the extent of 7-methylation of guanine residues in alkylated nucleic acids.

DIFFERENTIAL ACTIVATION OF P53 BY THE VARIOUS ADDUCTS OF MITOMYCIN C

Mitomycin C (MC) is a cytotoxic chemotherapeutic agent that causes DNA damage in the form of DNA cross-links 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.

Cellular pharmacology of quinone bioreductive alkylating agents

The cellular pharmacology of the mitomycin bioreductive alkylating agents is complex. This reflects in part the chemical characteristics of these quinones, which have multiple sites of reactivity and the capacity to produce a large number of different lesions of biological importance. Moreover, at least six different enzymes are capable of activating these compounds; the nature of the active species and the resultant biological lesions can vary with the activating enzyme. The relative activities of these reductases vary in different cell lines and can be modulated by pH and oxygenation. The effects of a quinone bioreductive alkylating agent therefore depend upon both the cell line and the microenvironment. DNA damage appears to be critical to the cytotoxic effects of these compounds. Both monoadducts and bis-adducts (forming interstrand and intrastrand crosslinks) have been identified in DNA from drug-treated cells. The pattern of adduct formation varies with the compound and the environment. Alkaline elution studies suggest a correlation between DNA cross-linking and cytotoxicity, both in air and in hypoxia. The rate of production of oxygen radicals and the importance of radical reactions in producing cytotoxic damage vary for different quinones and for different environments. While the potency of the bioreductive quinones varies with their redox potential, the direction and magnitude of the oxic/hypoxic differential cannot yet be predicted from the structures.

Novel assay of 7-alkylation of guanine residues in DNA: Application to nitrogen mustard, triethylenemelamine and mitomycin C

Abstract Release of tritium from DNA labeled at the 8-position of guanine residues was used as a test for the interaction of DNA with the bifunctional nitrogen mustard, methyl-(bis-β-chloroethyl)amine, the synthetic mutagen triethylenemelamine and the antibiotic mitomycin C. This method seems to offer a diagnostic and quantitative assay of 7-alkylation of guanine residues since alkylation experiments with nitrogen mustard showed a stoichiometric correspondence between the amount of guanine alkylated and the amount of tritium released into the medium. Triethylenemelamine caused substantial release of tritium. There was no detectable tritium release with mitomycin C under the optimal conditions for covalent binding of the drug to DNA in vitro . This finding indicates that, contrary to earlier suggestion, mitomycin C does not alkylate the N-7 position of guanine residues in DNA.

Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model

Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5′ incision on 1 strand and a 3′ incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA + UvrB and UvrA + UvrB + UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB + UvrC–DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations.

Absence of Strand Breaks in Deoxyribonucleic Acid Treated with Metronidazole

The deoxyribonucleic acid (DNA)-degrading potential of metronidazole was evaluated in vitro by three techniques: determination of melting curve, measurement of viscosity, and centrifugation in neutral or alkaline sucrose gradients. Studies were performed on calf thymus DNA and on 3H-labeled or unlabeled pneumococcal and T7 phage DNA after treatment with metronidazole alone or metronidazole reduced by sodium dithionite in the presence of DNA. This latter process is known to elicit covalent binding of metronidazole to DNA. Reduced or unreduced metronidazole had no effect on the melting properties, viscosity, or sedimentation velocity of the nucleic acids studied. Sodium dithionite alone, however, caused a 25% decrease in the intrinsic viscosity of pneumococcal DNA, and decreased the sedimentation velocity of pneumococcal and T7 phage DNA in both neutral and alkaline sucrose gradients. These data suggest that degradation of DNA is not important in the interaction of metronidazole with nucleic acids, an interaction assumed relevant to the cytotoxic, radiosensitizing, and mutagenic activities of this compound.