Pediatric and adult brain tumors are among the most therapeutically unresponsive and lethal of human cancers and their incidence continues to rise in the United States. A major reason for this therapeutic failure is the overexpression of O6-alkylguanine-DNA alkyltransferase (AGT), which prevents the formation of G-C cross-links in DNA by the chloroethylnitrosourea (CENU) class of drugs. Currently, a strategy involving the inactivation of AGT by O6-benzylguanine (BG) followed by CENU treatment has shown excellent promise, and clinical trials are underway for its exploitation. However, an extended suppression of AGT activity is necessary to achieve therapeutic efficacy and a rapid repletion of AGT occurring soon after BG treatment poses a potentially severe limitation to successful chemotherapy. Based on the applicant's recent studies of AGT proteolysis through the ubiquitin (ub)-proteasome pathway and preliminary studies showing the inhibition of AGT regeneration by proteasome blockers, the primary goal of this project is to further enhance the BG-based CENU therapy by preventing the repletion of AGT. The applicant's hypothesis is that ubiquitin-mediated break-down of inactive AGT triggers a regeneration of active AGT by enhancing its translation in BG-treated cells. The applicant proposes that the ub-proteasome pathway regulates both the proteolysis and subsequent regeneration of AGT after BG treatment and that specific inhibitors of this pathway will reduce the repletion of AGT to enable increased sensitization of glioma cells to BG-CENU regimen.
The specific aims are: 1) to quantitate the expression of ub-proteasome components in relation to AGT activity, the levels of AGT proteolysis after BG treatment, and the rate and extent of subsequent AGT regeneration in human glioma cell lines; 2) to examine the ub-requirement for AGT regeneration in a cell line, temperature-sensitive for ub-activation, and study increased translational efficiency of AGT in BG-treated glioma cells; 3) to evaluate ub-components and AGT in primary gliomas and lymphocytes, and to treat glioma cells and glioma xenografts in nude mice with specific inhibitors of the ub-proteasome pathway and examine AGT regeneration and BCNU cytotoxicity following BG treatment. Overall, this project promises to provide novel information on the biochemical modulation of AGT in BG-treated cells and rationalize alternative strategies to improve AGT-targeted chemotherapy of human brain tumors and other tumor types.
