The major objective of this research is to elucidate the mechanism of action of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP. Detailed information about how this leading anticancer drug functions will facilitate both clinical protocols for its use in cancer chemotherapy as well as the design of more effective drugs. Several new reagents to modulate and map the platinum binding sites on DNA, its most likely biological target, will be prepared and characterized. These include chelating diamine platinum(II) complexes linked by a polymethylene or polyamine chain to intercalators or to a pendant EDTA-containing side chain. Addition of Fe(II), O2, and dithiothreitol to the latter will cleave the DNA backbone near the platinum binding sites, providing an internal """"""""molecular scissors"""""""" by which the platinum loci on DNA can be mapped through subsequent electrophoresis on DNA sequencing gels. Platinum-DNA binding locations will also be mapped by using existing and proposed new mono- and polyclonal antibodies prepared agaist cis-DDP modified DNAs. To examine the hypothesis that cis-DDP is selectively cytotoxic to cancer cells because of their lesser ability, compared with normal cells, to repair a specific DNA-Pt lesion, such as the intrastrand d(GpG) crosslink, the genome of M13 virus will be modified with cis-DDP at a unique, chemically programmable site and introduced into normal and repair-deficient cells. Both repair of, and mutagenesis directed by, the platinum lesion will be studied. Replication of the cis-DDP modified M13 viral genome will also be investigated in vitro, and in vivo using E. coli cells mutant in their ability to repair and/or replicate DNA. These experiments require the synthesis of oligonucleotides containing bound cis-diammineplatinum(II). Platinated oligonucleotides from 2 to 21 monomer units in length will be synthesized, purified by HPLC chromatography, and structurally characterized in solution by NMR spectroscopic methods. Where possible, X-ray crystal structures of crystalline complexes will be determined. Mammalian cells will be infected with SV40 virus, treated with cis-DDp, and the platinum binding sites on the SV40 minichromosome mapped. Platinum binding at critical control regions, e.g. enhancer sequences, will be correlated with inhibition of viral directed processes. The speciation of cis-DDp in vivo and in buffers constructed to simulate intracellular conditions will be studied chemically by 195Pt NMR spectroscopy. The chemical characterization of cis-diammine- and ethylenediamineplatinum Alpha-pyridone blues and their Pt(III) derivatives will be completed.
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