This project encompasses a wide variety of pharmacologic, cellular, biochemical and molecular procedures basic not only to an understanding of the mechanism of action of the new anticancer agents, but also to provide important input into the design of clinical protocols. Pharmacology studies will define serum pharmacokinetics and serum binding characteristics for mice as well as drug distribution and metabolism in normal and tumor tissues. We have a state-of-the-art instrumentation laboratory as a Core of the Cancer Center available for metabolite identification. Cellular pharmacokinetics will define the uptake, efflux and intracellular distribution of agents under study. This information will be correlated with cellular response and host toxicity. This project will carry out the corresponding clinical pharmacology studies and do the correlative clinical/preclinical analyses. Cellular studies will define dose-response relationships both in vitro and in vivo as well as age- response characteristics for cytotoxicity and progression delay. We have a sophisticated flow cytometry facility within our Cancer Center fully capable of these and other studies. A series of biochemical assays will examine drug effects on macromolecular synthesis and on specific enzymes and substrates. Molecular studies are directed at DNA effects examining binding as well as damage and repair kinetics; the latter assessed by alkaline elution. Depending upon the leads from the studies on macromolecular synthesis inhibition and DNA interactions, other assays will be employed to define precisely the locus or loci of actions of the new anticancer agent. An assay for drug cytotoxicity mediated through an apoptotic mechanism has been introduced into our array of assays for mechanism of action of new anticancer agents. Newer assays directed at drug effects on membrane structures will be assessed by a variety of fluorescent probes. Presently available resistant lines as well as specifically developed lines resistant to the new agent will be used to determine mechanism of drug action and patterns of cross resistance. Finally, specific laboratory studies will be built into the clinical protocols which address questions of mechanism of action as well as drug metabolism and tumor resistance.
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