Chemotherapy of malignant brain tumors is currently severely limited by both inherent and acquired resistance of the brain tumor cells to the chloroethylnitrosoureas (CENU)s, the most effective agents available for the treatment of brain tumors. In this project, the role of glutathione (GSH) and its related enzymes, GSH-transferase, glutathione reductase, gamma-glutamylcysteine synthetase and glutathione synthetase in mechanisms of resistance of human brain tumor cells to chloroethylnitrosoureas will be investigated. Primary brain tumor specimens will be used to establish early passage cell lines with graded inherent resistance to bischloroethylnitrosourea (BCNU), a clinically active CENU. An acquired resistance model will be developed by treating a BCNU-sensitive cell line in vitro with increasing doses of BCNU over several passages. GSH levels and the activities of GSH-metabolizing enzymes in these cells will be determined and correlated with the degree of development resistance to BCNU. Agents that are capable of depleting intracellular GSH and/or inhibiting GSH-relatined enzymes will be used to cause GSH-depletion and enzyme inhibition in BCNU- resistant cells. Dose-effect and kinetic studies will be performed to establish the optimum inhibitor concentration and treatment times to achieve maximum GSH-depletion and enzyme inhibition in brain tumor cells of graded degrees of BCNU-resistance. Based on these results, strategies will be developed with which resistant brain tumor cells will be """"""""re-sensitized"""""""" to killing by BCNU. The degree of conversion of resistant cells to sensitive ones will be estimated in a clonogenic cell assay and the increase in DNA interstrand crosslinking will be measured by alkaline elution techniques. Although the glutathione system is one of the most important cellular pathways to detoxify carcinogens, free radicals and electrophiles, its role in the CENU-resistance of brain tumor cells has until now been little investigated. The findings in this project, therefore, promise to contribute substantially to our understanding of GSH-dependent mechanisms involved in both the sensitivity and resistance of brain tumor cells to the chloroethylnitrosoureas, and thus, provide potential guidelines with which clinical strategies for overcoming this resistance may be developed.
