The long-term goal of this research is to elucidate the mechanism of action of cis-diammineddichloroplatinum (II), cis-DDP or cisplatin, a leading anti-cancer drug used to treat testicular tumors and a paradigm for successful chemotherapy. The major underlying hypothesis is that the biological activity derives from the formation and persistence of cisplatin- DNA adducts, mainly 1,2-intrastrand d(GpG) and d(ApG) cross-links. Geometric information about these adducts in several of 16 possible N1GGN2 and N11AGN2 sequence contexts and their binding to cellular proteins will be provided. Novel synthetic routes to site-specifically platinated duplex DNAs are presented as well as thermodynamic, kinetic, and NMR and X-ray structural methods for characterizing their complexes with proteins. Among proteins that bind specifically major cisplatin-DNA adducts and affect cellular processing are those containing high-mobility group (HMG) domains. HMG domain proteins shield platinum adducts from nucleotide excision repair (NER) and help elongate transcripts from chromatin. The ability of cisplatin-DNA intrastrand cross-links to be excised by NER and to block transcription will be investigated with reconstituted in vitro assays. Probes containing specific adducts and strategically placed photocross-linking agents will be introduced to identify factors contacting platinated DNA. Extracts from tumor and normal testis tissue from a new breed of mice that develops testicular cancer will be prepared for NER studies to test the hypothesis that poor repair of cisplatin adducts underlies its selective toxicity. Parallel work will be performed with cultured human testis cell. The (TTAGGG)n repeating sequence in human telomeres is a likely target for cisplatin. We shall investigate telomere length maintenance as a contributing factor to the cisplatin molecular mechanism by studying the platination of this sequence in duplex DNAs. The effect of cisplatin on telomere binding proteins (TBPs) such as TRF1 and TRF2 will be studied in cells or in situ immunofluorescence techniques. In vitro binding of TBPs to probes with embedded site-specifically platinated telomere sequences will also be examined. An additional aim, to improve the selective binding of HMG-domain proteins to cisplatin 1,2-cross-links, will be met by site-directed mutagenesis based on a recent structure of a complex between HMG1 domain A and a platinated 16-mer duplex. Phage display will provide additional peptides selected for their strong binding to cis0platin-cross-links. These protein constructs will be over- expressed in mammalian cells to evaluate whether they affect cisplatin sensitivity, ultimately for combined chemotherapy/gene therapy applications in human cancer. With a newly devised transcription inhibition assay have a fast fluorescent readout, combinatorial libraries of platinum compounds will be screened for anti-cancer drug candidates.
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