Tumor resistance to the 2-chloroethylnitrosoureas still constitutes a formidable problem limiting the successful therapy of human malignant brain tumors with these agents. Our efforts over the past few years with human malignant glioma cell lines variably resistant to CENUs, have demonstrated that brain tumor CENU-resistance is associated with elevation of cellular glutathione (GSH) and over-expression of glutathione-S-transferases (GSTs). However, little is known about the exact nature of the GSTs present in CENU-resistant brain tumors, or about the level of transcription and/or amplification of GST genes in these cells. The goal of this proposal is to undertake the purification and characterization of GSTs from CENU-resistant human malignant astrocytoma cell lines and from primary biopsies of both adult and pediatric brain tumor patients. We will characterize these GSTs biochemically and functionally and determine their potential role in brain tumor CENU-resistance by establishing their ability to catalyze both the direct inactivation of CENUs and the quenching of CENU-induced DNA crosslink precursors by GSH, two processes that can result in cellular CENU-resistance. Using antibodies raised against the purified GSTs and against other known GSTs, as well as, cDNAs specific for the basic, near- neutral and acidic GSTs, we will determine the type and molecular expression of specific glutathione-S-transferases in brain tumors. We will also determine the level of transcription and/or amplification of specific GST subunit genes. The in vitro CENU-resistance of the cell lines and the response to therapy of patients treated with a CENU regimen will be correlated with the type and molecular expression of the specific GSTs in the rumors. The blood-brain-barrier poses a significant limitation to the delivery of water soluble agents to the brain and to brain tumors. Consequently, we propose to synthesize lipophilic analogues of a known GST inhibitor, ethacrynic acid, determine their ability to inhibit GSTs in CENU-resistant brain tumors, and determine the extent to which pretreatment of tumor cells with these lipophilic, brain-permeable GST inhibitors will reverse the CENU-resistance of the tumors. The results of this tightly- focused research will provide very important information on the specific types and role of GSTs mediating CENU-resistance of human malignant brain tumors and provide the basis for future attempts to circumvent this resistance clinically.
