Bleomycin (Blm) is one of 3-4 highly effective drugs used to treat human cancer that require metals for their action. The iron complex of B1m(F3B1m) causes single and double strand scission of DNA in vivo and in vitro. The chemical properties of the redox activation of FeB1m to HO(2)(-)-Fe(III)B1m have been described in solution, but less is know about the reactions of FeB1m initiates DNA damage leading to inhibition of tumor growth. The central hypothesis is that DNA significantly influences the chemistry of FeB1m because the DNA domain and metal domain of the drug interact with the DNA polymer.
The specific aims are (1) To carry out structural analyses of metallobleomycin adducts bound to DNA sites, focusing on interaction of the metal domain with DNA and the mechanism by which drugs locate specific binding sites. (3) To determine properties of reduction of FeB1m species bound to DNA. (4) To investigate the mechanism of DNA damage including double strand cleavage of DNA by FeB1m. Using NMR, ESR, ENDOR and resonance raman techniques, structural features of relevant metallobleomycin species in solution and bound to DNA will be determined. These include CoB1mDNA structures to model important forms of FeB1mDNA and the deglycometal domain (Co, Fe) to examine the interaction of the metal domain with DNA. The electro-chemistry of FeB1mDNA will be examined using square wave voltammetry to define properties of redox states of FeB1m nd to provide unique means to generate HO(2)(-) -Fe(III)B1mDNA for studies of its reaction with DNA. Assays of DNA damage will assess the reaction of FeB1m with DNA in terms of kinetics of single and double strand cleavage. Site specific and nonspecific DNA oligomers will be synthesized and used in these studies. These experiments should markedly increase the understanding of how FeB1m, the prototype metallonuclease, achieves high efficiency of reaction with DNA.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA022184-14
Application #
2712551
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Johnson, George S
Project Start
1977-09-30
Project End
2001-05-31
Budget Start
1998-06-01
Budget End
1999-05-31
Support Year
14
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Wisconsin Milwaukee
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Milwaukee
State
WI
Country
United States
Zip Code
53201
Petering, David H; Xia, Chuanwu; Antholine, William E (2004) Metal ion dependent antibiotics in chemotherapy. Met Ions Biol Syst 42:463-97
Rajani, Cynthia; Kincaid, James R; Petering, David H (2004) Resonance Raman studies of HOO-Co(III)bleomycin and Co(III)bleomycin: identification of two important vibrational modes, nu(Co-OOH) and nu(O-OH). J Am Chem Soc 126:3829-36
Xia, Chaunwu; Forsterling, F Holger; Petering, David H (2003) Identification of the internal axial ligand of HO2-cobalt(III)-bleomycin: 1H[15N] HSQC NMR investigation of bleomycin, deglycobleomycin, and their hydroperoxide-cobalt(III) complexes. Biochemistry 42:6559-64
Li, Wenbao; Antholine, William E; Petering, David H (2002) Kinetics of reaction of DNA-bound Fe(III)bleomycin with ascorbate: interplay of specific and non-specific binding. J Inorg Biochem 90:8-17
Zhao, Chunqing; Xia, Chuanwu; Mao, Qunkai et al. (2002) Structures of HO(2)-Co(III)bleomycin A(2) bound to d(GAGCTC)(2) and d(GGAAGCTTCC)(2): structure-reactivity relationships of Co and Fe bleomycins. J Inorg Biochem 91:259-68
Rajani, C; Kincaid, J R; Petering, D H (2001) Raman spectroscopy of an O(2)-Co(II)bleomycin-calf thymus DNA adduct: alternate polymer conformations. Biophys Chem 94:219-36
Li, W; Zhao, C; Xia, C et al. (2001) Comparative binding properties of metallobleomycins with DNA 10-mers. Biochemistry 40:7559-68
Li, W; Xia, C; Antholine, W E et al. (2001) Interactions of iron bleomycin, phosphate or cyanide, and DNA: sequence-dependent conformations and reactions. J Biol Inorg Chem 6:618-27
Rajani, C; Kincaid, J R; Petering, D H (1999) A systematic approach toward the analysis of drug-DNA interactions using Raman spectroscopy: the binding of metal-free bleomycins A(2) and B(2) to calf thymus DNA. Biopolymers 52:110-28
Rajani, C; Kincaid, J R; Petering, D H (1999) The presence of two modes of binding to calf thymus DNA by metal-free bleomycin: a low frequency Raman study. Biopolymers 52:129-46

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