The long range objective of the proposed work is to relate the potent biological effects of the antitumor antibiotic CC-1065 to the structural modification of DNA which occurs upon covalent binding. CC-1065 is a potent antitumor agent produced by Streptomyces zelensis which was isolated by scientists at the Upjohn Company. Although the lead compound CC-1065 is too toxic for clinical use as an antitumor agent, a synthetic analog has been prepared which has improved antitumor efficacy in mice, and lacks the delayed death effects produced by CC-1065. During the next grant period we will continue studies in two areas; correlation of DNA binding and sequence specificity with biochemical and biological consequences, and structural investigations on the CC-1065-DNA adducts. In addition, we will initiate two new research areas: effect of CC-1065 binding to DNA on DNA protein recognition and function, and isolation and identification of proteins which bind specifically to DNA sequences modified with CC-1065. Correlation of DNA binding with biological effects will rely upon DNA sequencing techniques, and determination of cytotoxicity, antitumor efficacy and delayed death. Structural studies will utilize high-field NMR and gel electrophoresis. The effect on transcription factor binding and function will be assayed using immunoprecipitation, DNA footprinting techniques, and microinjections into frog oocyte cells. Proteins that bind to CC-1065-DNA adducts will be isolated by affinity techniques and identified by microsequencing and gene cloning.
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