UB-PROTEOLYSIS OF ALKYLTRANSFERASE IN GLIOMA THERAPY

描述：（申请人摘要）儿童和成人脑肿瘤是 是对治疗最无反应和最致命的人类癌症之一 在美国，其发病率持续上升。 一个主要原因是 这种治疗失败是由于 O6-烷基鸟嘌呤-DNA 的过度表达 烷基转移酶 (AGT)，可防止 G-C 交联的形成 DNA由氯乙基亚硝基脲（CENU）类药物组成。 目前，一个 随后采用 O6-苄基鸟嘌呤 (BG) 灭活 AGT 的策略 CENU 的治疗已显示出良好的前景，临床试验正在进行中 正在进行对其的利用。 然而，AGT 的长期抑制 活性对于实现治疗功效和快速补充是必要的 BG 治疗后不久发生的 AGT 可能会造成严重的后果 化疗成功的局限性。 根据申请人近期的情况 通过泛素 (ub)-蛋白酶体途径进行 AGT 蛋白水解的研究 初步研究显示蛋白酶体抑制 AGT 再生 拦截器，该项目的主要目标是进一步增强 基于 BG 的 CENU 疗法通过防止 AGT 的补充。 申请人的 假设是泛素介导的非活性 AGT 分解会触发 通过增强 BG 处理细胞中活性 AGT 的翻译来再生活性 AGT。 申请人提出，ub-蛋白酶体途径调节 BG 处理后 AGT 的蛋白水解和随后的再生 该途径的特异性抑制剂将减少 AGT 的补充 提高神经胶质瘤细胞对 BG-CENU 方案的敏感性。 的 具体目标是：1）定量ub-蛋白酶体的表达 与 AGT 活性相关的成分，AGT 蛋白水解后的水平 BG 治疗以及随后 AGT 再生的速率和程度 人神经胶质瘤细胞系； 2) 检查 AGT 的 ub 要求 细胞系再生，对 ub 激活温度敏感，并且 研究提高 AGT 在 BG 处理的神经胶质瘤细胞中的翻译效率； 3) 评估原发性神经胶质瘤和淋巴细胞中的ub成分和AGT，以及 用特定的方法治疗裸鼠中的神经胶质瘤细胞和神经胶质瘤异种移植物 ub-蛋白酶体途径的抑制剂并检查 AGT 再生和 BG 治疗后 BCNU 细胞毒性。 总体而言，该项目承诺 提供有关 BG 治疗中 AGT 生化调节的新信息 细胞并合理化替代策略以改善 AGT 靶向 人脑肿瘤和其他肿瘤类型的化疗。

DESCRIPTION: (Applicant's Abstract) 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.