This research is aimed at providing an understanding of how the anthracycline antitumor drugs, particularly daunomycin, interact with DNA in the state it is found in all nuclei, namely chromatin, its subunits, and possible aggregates. In addition, experiments designed to discover the targets actually responsible for cell toxicity of the drug are planned. Finally, efforts will be made to prepare drug derivatives capable of rebinding to specific DNA sequences, with the long range objective of targeting antitumor drugs to DNA onc gene and related sequences.
Specific aims i nclude study of binding affinities by equilibrium dialysis and optical methods, and examination of drug-induced conformational changes using transient electric dichroism, sedimentation velocity, and light scattering. Drug binding kinetics will be studied using the temperature jump method, with fluorescence detection. Ethidium will serve as a comparison compound for the physical studies. Possible protein targets for drug action in cells and cell nuclei will be examined using a new photoaffinity analog of daunomycin (DNG), which reacts specifically with typical protein nucleophiles. Excess production of drug-protein crosslinked species will be sought in cell lines which are resistant to the drug. The effect of daunomycin on transcription initiation, propagation, termination, and promoter selection will be examined using gel electrophoresis methods, and posssible DNG-RNA polymerase photoadducts will be investigated. DNG-lac repressor and DNG-CAP protein photoadducts will serve as the starting point for isolation of daunomycin-peptide conjugates capable of specific rebinding to target DNA sequences. New methods of derivatizing daunomycin will be sought, and monitored by cell uptake and toxicity studies.
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