UB-PROTEOLYSIS OF ALKYLTRANSFERASE IN GLIOMA THERAPY

描述：（申请人的摘要）儿童和成人脑肿瘤是 在治疗上最无反应和最致命的人类癌症中， 其发病率在美国持续上升。 的一大原因 这种治疗失败是O 6-烷基鸟嘌呤-DNA的过度表达 烷基转移酶（AGT），其防止在细胞中形成G-C交联。 DNA由氯乙基亚硝基脲（CENU）类药物。 现时 策略包括通过O 6-苄基鸟嘌呤（BG）灭活AGT， 通过CENU治疗已经显示出良好的前景，临床试验是 正在开发中。 然而，AGT的长期抑制 活性是达到治疗效果和快速补充所必需的 BG治疗后不久发生的AGT可能造成严重的 限制化疗成功。 根据申请人最近的 AGT通过泛素（ub）-蛋白酶体途径蛋白水解的研究， 初步研究显示蛋白酶体抑制AGT再生 阻滞剂，该项目的主要目标是进一步提高 通过预防AGT的补充，以BG为基础的CENU治疗。 申请人的 有一种假说认为，泛素介导的非活性AGT的分解触发了一种 通过增强BG处理的细胞中的活性AGT的翻译来再生。 申请人提出，亚蛋白酶体途径调节两种蛋白酶， 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， 能够增加胶质瘤细胞对BG-CENU方案的敏感性。 的 具体目的是：1）定量表达亚蛋白酶体 与AGT活性相关的组分，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， 人胶质瘤细胞系; 2）检测AGT的亚需要量 在细胞系中再生，对亚活化温度敏感，和 研究BG处理的胶质瘤细胞中AGT的翻译效率增加; 3)评价原发性胶质瘤和淋巴细胞中的亚组分和AGT，以及 在裸鼠中用特异性抗肿瘤药物治疗胶质瘤细胞和胶质瘤异种移植物， 亚蛋白酶体途径的抑制剂，并检查AGT再生， BG处理后的BCNU细胞毒性。 总的来说，该项目承诺 提供了新的信息AGT的生化调节BG处理 细胞和合理化替代战略，以提高AGT靶向 人类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.