The long-term goal of this research is to elucidate the molecular mechanism of action of cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin). Cisplatin is currently a leading anticancer drug, and detailed information about how it functions will facilitate both clinical protocols for its use in chemotherapy as well as the design of more effective drugs. A major hypothesis addressed is that the selective toxicity of cisplatin for tumor versus normal cells, and its greater efficacy for certain tissues such as solid testicular tumors, are a consequence of the formation and survival of specific adducts with DNA, its acknowledged biological target. Accordingly, duplex DNA fragments containing each of the major platinum adducts, especially intrastrand d(GpG) and d(ApG) crosslinks that account for >90% of all drug binding, will be synthesized and structurally characterized by X-ray crystallography, NMR spectroscopy, electron microscopy, and in electrophoresis gel mobility shift assays. These adducts will then be incorporated site-specifically into biologically viable genomes to study their survival and repair in a variety of bacterial and mammalian cells and cell extracts. Parallel studies with globally platinated DNA will be carried out. A further hypothesis to be tested is that a recently discovered factor, present in human cells, that binds specifically to DNA modified with cis-DDP, but not the inactive trans-DDP or [Pt(dien)Cl]+ complexes, might account for the selective toxicity of cisplatin in cells of different origin (normal vs tumor; testicular vs breast; parental vs resistant). The factor may be part of the DNA repair apparatus, a putative function addressed in this application. The protein and RNA components of the factor will be purified and characterized. Clones for the same or a related protein, already in hand, will be used to screen cells for DNA and RNA levels that encode for the factor. Antibodies raised against the protein will be employed to monitor its concentration in cells. If the sensitivity of cells to cisplatin correlates with levels of the protein, and especially if the functional link to repair of specific Pt-DNA adducts can be established, important details of the molecular mechanism of the drug will be known. Moreover, a powerful tool for clinical medicine should evolve, since tumor and patient response would be predictable based on levels of antibody-detectable DNA-damage-specific protein. Comparative studies will be made with active analogs of cis-DDP, especially monofunctional cis-[Pt(NH3)2ClL]+ cations (L = cytosine or substituted pyridines), and with the inactive trans-DDP and [Pt(dien)Cl]+ complexes. Complexes having an intercalator directly coordinated or attached by a variable length linker chain to the platinum atom will also be investigated to afford new tools for exploring how DNA stereochemistry affects platinum binding and to provide better insight into how platinum-induced DNA structural changes relate to the biological processing of adducts. The anticancer potential of this new class of compounds will be evaluated. The chemical, structural, and spectroscopic characterization of thermochromic {Pt(NH3)2}2+ complexes of ethidium and the DNA-binding chemistry of these novel complexes will also be studied.
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