The use of bone marrow transplantation and various marrow stimulating factors has ensured that alkylating agents will remain a mainstay in cancer chemotherapy for the next few years. Resistance to these drugs can be manifest by a plurality of factors, amongst which, enhanced detoxification through elevated glutathione S-transferases (GST) is a particular phenotype. GST have evolved into a multigene family, with complex substrate specificities and regulation controls. This year 12-16 continuation application seeks to extend our previous efforts to define the importance of GST to the acquisition of resistance. Using a number of resistant cell lines, isozyme specific cDNA probes and antibodies and some human biopsies, the following issues will be addressed: (i) Increased GST activity may be a function of gene amplification, increased half-life of mRNA or isozyme, enhanced transcriptional rate or a combination thereof. Southern analysis, isotope dilution, metabolic inhibitors and nuclear run-ons will distinguish these possibilities. (ii) The importance of the aziridinium species to GST substrate specificity will be gauged by measuring GST catalysis of glutathione (GSH) with busulfan and cis-Pt, two drugs which do not form this intermediate. In vivo conjugation of chlorambucil to GST will be studied in cells expressing high and low GST alpha levels. Additional pre-clinical studies will extend previous work with ethacrynic acid and characterize potential OST inhibitors as modulators of drug resistance. (iii) Because GST alpha catalysis is implicated in alkylating agent conjugation to GSH, human polymorphisms of this gene will be considered by Southern analyses. (iv) Vascular endothelial cells (EC) form a primary barrier to drug delivery to tumors in vivo. How these cells regulate GST may have an impact on how much drug reaches the tumor. Stress response and GST expression in human EC both in situ and in culture will determine its importance in drug metabolism and its potential role in contributing to resistance.
Showing the most recent 10 out of 12 publications
