MODULATION OF DNA REPAIR TO ENHANCE CHEMOTHERAPY

大量证据表明，肿瘤的主要机制 细胞对氯乙基亚硝基脲（CENU）的抗性来自DNA 06-甲基鸟嘌呤DNA甲基转移酶（MGMT）的修复活性。该DNA 修复蛋白被认为可以保护细胞免受细胞毒性DNA的侵害。 链交联（ISC）由CENU通过除去氯乙基 在这些加合物可以从鸟嘌呤的0-6位加合之前， 重新排列形成致命的交叉连接在过去的研究中， 八年来的研究表明，这种DNA修复系统可以 被各种生化策略暂时抑制，包括 肿瘤细胞与DNA甲基化剂预孵育， 链脲佐菌素（STZ）是MGMT的天然底物，06- 甲基鸟嘌呤这种损伤的修复耗尽了肿瘤细胞的MGMT， MGMT的自杀修复活动此外，游离碱06- 甲基鸟嘌呤（MG）和06-苄基鸟嘌呤（6-BG）也可以消耗细胞中的 MGMT活性和随后使肿瘤细胞对用 BCNU.最近，我们证明了STZ联合6-BG和BCNU 可在体外对耐药肿瘤细胞产生延长的致敏作用 以及在体内异种移植肿瘤中。目前正在测试6-BG加BCNU 在其他机构的I期临床试验和6-BG + STZ + BCNU将在该机构进行测试。然而，生化调节 相对于正常细胞，这些策略对肿瘤细胞没有选择性。在这 项目我们建议开发基因治疗策略， 在调节肿瘤抗性的同时保护重要的正常组织 到CENU。具体目标是：1.为了开发一种体外和体内 6-BG连续暴露时间表，使用6-BG推注，然后 低剂量持续暴露以最大化MGMT耗竭的持续时间。 2.为了确定转导的DNA修复基因的保护能力， 小鼠和人细胞联合使用时， 预处理方案包括6-BG或6-BG加STZ。3.使用 NOD/SCID小鼠模型，我们将确定6-BG（有和无STZ） 和BCNU化疗可以选择性地施用于异种移植肿瘤， 在移植了人类骨髓的老鼠身上。4)分析MGMT活动， 复发性B细胞白血病患者肿瘤细胞中的DNA交联 来自检查6-BG连续输注的I期试验的恶性肿瘤，或 6-BG联合STZ和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.