Mechanism of Action of Bifunctional Alkylating Agents

Ducore, Jonathan

Abstract

This study will examine in detail changes in DNA synthesis and cell cycle progression following bifunctional alkylating agent treatment and correlate the changes with cytotoxicity and DNA damage. Using a variety of cell types, both mono- and bifunctional alkylating agents, and drugs which modify the DNA damage and toxicity of the alkylating agents, we will be able to determine whih functional abnormalities of DNA synthesis and cell cycle result from the DNA damage of bifunctional alkylators and result in cell death. Alterations in rate and initiation and elongation of DNA synthesis will be measured by the pH step modification of the alkaline elution method. Alterations will be measured at a series of time points following drug treatment. Cell cycle changes will be measured utilizing flow microfluorometry. Alterations in cell cycling and percentage of cells in different cycle compartments will be measured at various times following drug treatment. Abnormalities of DNA synthesis and cell cycle progression will be correlated with known parameters of DNA damage (DNA interstrand crosslinking) and cytotoxicity. The toxic importance of bifunctional alkylations in the nitrosoureas will be measured by comparing mono- and bifunctional nitrosoureas. Platinum complex mono-alkylations can be compared to bifunctional alkylations by the addition of thiourea following cis-platinum treatments. The use of trans-platinum will further distinguish those functional aberrations which correlate with cytotoxicity. Modifications of mustard toxicity (L-PAM) will be made with thiourea. We will use methylxanthines (caffeine and theophylline) to modify cytotoxicity and study changes in DNA synthesis. Polyamine synthesis inhibitors modify drug effects with resultant changes in DNA synthesis and cytotoxicity as well as DNA damage parameters. We will develop and isolate cell lines with altered alkylating agent sensitivity and contrast these with their parent line as to DNA synthesis and cell cycle changes. Where cytotoxic or DNA damage data is lacking, this will be measured utilizing the alkaline elution technique. Finally, if major alterations in intracellular dNTP pools are seen, changes in pool sizes will be measured by an in vitro DNA synthesis rate technique. This information will allow identification of functional consequences of bifunctional alkylating agent treatment and enlarge our understanding of how DNA crosslinking affects DNA synthesis and cell cycling and hence cell death.