Mitomycin C (MC) is a natural antitumor antibiotic used in the clinic for cancer chemotherapy. MC generates six different DNA adducts in tumor cells. One of these is a DNA interstrand cross-link, as a result of bifunctional alkylation of the complementary DNA strands by MC, while the others are adducts of the drug, linked to only one strand of DNA (monoadducts and an intrastrand crosslink). The broad, long-range objective of the present proposal is to elucidate the relationship of each of the six adducts to the cytotoxicity/antitumor activity of MC. In addition, the putative toxic role of reactive oxygen species (ROS), generated by redox cycling of MC, will be examined using Fanconi's Anemia (FA) cells.
The first aim of the proposal is to determine whether the known hypersensitivity of FA cells to MC is primarily due to ROS generation or to DNA adduct formation by the drug. These studies will better define the biochemical basis of the unique sensitivities of Faneoni's anemia cells, as well as providing insights into the activities of the mitomycins. The other specific aims serve to detect and characterize biochemical responses unique to the different DNA adducts of MC. In this context we shall investigate differential rates of repair of the adducts in living EMT6 tumor cells in cell culture. Differential modes of nucleotide excision repair of cross-link and monoadducts will be investigated in subcellular systems by constructing 91-mer oligonucleotide duplexes modified site-specifically with the various MC adducts, and using these as substrates for uvrABC excinuclease, or for the repair enzymes present in a mammalian whole cell extract. The same substrates will be used to search for """"""""adduct binding proteins"""""""", specific for the cross-link, in cell extracts. Differential inhibition of lesion bypass by DNA polymerases by the different mitomycin monoadducts will also be investigated, using synthetic adduct-template-primer complexes as substrates. These latter experiments are aimed at identifying the basis for the unusually low cytotoxicity of the monoadducts 5 and 6. This research will lead to a better understanding of the molecular and biochemical basis of the antitumor activity and toxicology of MC and, more generally, of other DNA cross-linking agents, a major class of drugs currently used in cancer chemotherapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA028681-24
Application #
7058819
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Lees, Robert G
Project Start
1980-07-01
Project End
2008-04-30
Budget Start
2006-05-01
Budget End
2008-04-30
Support Year
24
Fiscal Year
2006
Total Cost
$225,917
Indirect Cost
Name
Hunter College
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
620127915
City
New York
State
NY
Country
United States
Zip Code
10065
Paz, Manuel M; Ladwa, Sweta; Champeil, Elise et al. (2008) Mapping DNA adducts of mitomycin C and decarbamoyl mitomycin C in cell lines using liquid chromatography/ electrospray tandem mass spectrometry. Chem Res Toxicol 21:2370-8
Champeil, Elise; Paz, Manuel M; Ladwa, Sweta et al. (2008) Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene. J Am Chem Soc 130:9556-65
Boamah, Ernest K; White, David E; Talbott, Kathryn E et al. (2007) Mitomycin-DNA adducts induce p53-dependent and p53-independent cell death pathways. ACS Chem Biol 2:399-407
Paz, Manuel M; Kumar, Gopinatha Suresh; Glover, Mark et al. (2004) Mitomycin dimers: polyfunctional cross-linkers of DNA. J Med Chem 47:3308-19
Abbas, Tarek; Olivier, Magali; Lopez, Jaqueline et al. (2002) Differential activation of p53 by the various adducts of mitomycin C. J Biol Chem 277:40513-9
Palom, Yolanda; Suresh Kumar, Gopinatha; Tang, Li-Qian et al. (2002) Relative toxicities of DNA cross-links and monoadducts: new insights from studies of decarbamoyl mitomycin C and mitomycin C. Chem Res Toxicol 15:1398-406
Paz, M M; Das, A; Palom, Y et al. (2001) Selective activation of mitomycin A by thiols to form DNA cross-links and monoadducts: biochemical basis for the modulation of mitomycin cytotoxicity by the quinone redox potential. J Med Chem 44:2834-42
Paz, M M; Tomasz, M (2001) Reductive activation of mitomycin A by thiols. Org Lett 3:2789-92
Subramaniam, G; Paz, M M; Suresh Kumar, G et al. (2001) Solution structure of a guanine-N7-linked complex of the mitomycin C metabolite 2,7-diaminomitosene and DNA. Basis of sequence selectivity. Biochemistry 40:10473-84
Paz, M M; Das, T A; Tomasz, M (1999) Mitomycin C linked to DNA minor groove binding agents: synthesis, reductive activation, DNA binding and cross-linking properties and in vitro antitumor activity. Bioorg Med Chem 7:2713-26

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