MODULATION OF DNA REPAIR TO ENHANCE CHEMOTHERAPY

压倒性的证据表明，肿瘤的主要机制 细胞对氯乙基亚硝脲(CENU)的抗性是由DNA引起的 6-甲基鸟嘌呤DNA甲基转移酶(MGMT)的修复活性。这个DNA 修复蛋白被认为可以保护细胞免受细胞毒DNA相互作用的影响 由CENU通过去除氯乙基产生的链交联剂(ISC) 鸟嘌呤0-6位的加合物在这些加合物可以 重新排列以形成致命的交联物。在过去一年中进行的研究 八年的时间证明，这种DNA修复系统可以 暂时被各种生化策略抑制，包括 用DNA甲基化试剂预先孵育肿瘤细胞 产生MGMT，06-天然底物的链脲佐菌素(STZ) 甲基鸟嘌呤。这种损伤的修复耗尽了MGMT的肿瘤细胞 MGMT的自杀式修复活动。此外，自由碱基06- 甲基鸟嘌呤(MG)和6-苯基鸟嘌呤(6-BG)也可耗尽细胞 MGMT活性，并随后使肿瘤细胞对 BCNU.最近，我们已经证明了STZ联合6-BG和BCNU 可以在体外对耐药肿瘤细胞产生长期的敏感化 以及体内异种移植瘤中。目前，6-BG加BCNU正在测试中 在其他机构和6-BG+STZ Plus进行I期临床试验 北大将在该机构进行测试。然而，生化调节 与正常细胞相比，针对肿瘤细胞的治疗策略并不具有选择性。在这 我们建议开发基因治疗策略，使 在调节肿瘤耐药性的同时对关键正常组织的保护 送到中央师范大学。其具体目标是：1.在体外和体内开发一种 6-BG连续曝光计划，使用6-BG推注，然后是 低剂量连续暴露以最大限度地延长MGMT耗竭的持续时间。 2.检测转导的DNA修复基因的保护能力 BCNU联合应用对小鼠和人细胞的杀伤作用 用含6-BG或6-BG+STZ的预处理方案。3.使用 NOD/SCID小鼠模型我们将确定6-BG(使用和不使用STZ) BCNU化疗可选择性地用于异种移植瘤 在移植了人骨髓的小鼠身上。4)分析MGMT活动并 复发B细胞患者肿瘤细胞中DNA的交联 来自检查持续输注6-BG的1期试验的恶性肿瘤，或 6-BG联合STZ、BCNU。

Overwhelming evidence has demonstrated that the major mechanism of tumor cell resistance to the chloroethylnitrosoureas (CENU) results from the DNA repair activity of 06-methylguanine DNA methyltransferase (MGMT). This DNA repair protein is thought to protect cells from the cytotoxic DNA inter- strand crosslink (ISC) produced by the CENU by removing chloroethyl adducts from the 0-6 position of guanine before these adducts can rearrange to form a lethal crosslink. Studies conducts over the previous eight years have demonstrated that this DNA repair system can be temporarily inhibited by a variety of biochemical strategies including pre-incubation of tumor cells with DNA methylating agents such as streptozotocin (STZ) which product the natural substrate of MGMT, 06- methylguanine. Repair of this lesion depletes the tumor cell of MGMT due to MGMT's suicide repair activity. In addition, the free bases 06- methylguanine (MG) and 06-benzylguanine (6-BG) can also deplete cells of MGMT activity and subsequently sensitize tumor cells to treatment with BCNU. Recently, we have demonstrated that STZ combined with 6-BG and BCNU can produce a prolonged sensitization of resistant tumor cells in vitro and in xenograft tumors in vivo. Currently 6-BG plus BCNU is being tested in Phase I clinical trials at other institutions and 6-BG plus STZ plus BCNU will be tested at this institution. However, biochemical modulation strategies are not selective for tumor cells over normal cells. In this Project we propose to develop gene therapy strategies that will allow the protection for critical normal tissues while modulating tumor resistance to the CENU. The specific aims are: 1. To develop a in vitro and in vivo a 6-BG continuous exposure schedule using a bolus of 6-BG followed by a low dose continuous exposure to maximize the duration of MGMT depletion. 2. To determine the ability of transduced DNA repair genes to protect mouse and human cells from the cytotoxic killing by BCNU when combined with pretreatment regimens containing 6-BG or 6-BG plus STZ. 3. Using the NOD/SCID mouse model we will determine whether 6-BG (with and without STZ) and BCNU chemotherapy can be selectively administered to xenograft tumors in mice transplanted with human marrow. 4) To analyze MGMT activity and DNA cross linking in tumor cells from patients with relapsed B-cell malignancies from Phase 1 trials examining continuous infusion of 6-BG, or 6-BG in combination with STZ and BCNU.