MOLECULAR MODULATION OF DRUG RESISTANCE IN TUMOR CELLS

描述：(申请人摘要)压倒性的证据表明 肿瘤细胞耐药的主要机制是 氯乙基亚硝脲(CENU)来源于O-6的DNA修复活性 甲基鸟嘌呤DNA甲基转移酶(MGMT)。这种DNA修复蛋白是 认为可以保护细胞免受细胞毒性DNA链间交联(ISC)的影响 由CENU通过从O-6位除去氯乙基加合物而产生 在这些加合物可以重排形成致命的交联物之前。 在过去八年的支持下进行的研究表明 这种DNA修复系统可以被多种基因暂时抑制 包括肿瘤细胞与DNA预孵化在内的生化策略 甲基化试剂，如链脲佐菌素(STZ)，可产生天然的 底物为MGMT，O-6甲基鸟嘌呤。修复这一损伤会耗尽 MGMT的自杀性修复活性所致的肿瘤细胞。此外， 游离碱O-6甲基鸟嘌呤(MG)和O-6苯基鸟嘌呤(BG)也可以 耗尽细胞的MGMT活性，继而使肿瘤细胞对 用BCNU治疗。在最近的资助期内，申请人 已经证明STZ与BG和BCNU联合使用可以产生延长的 耐药肿瘤细胞体外和移植瘤的增敏作用 活着。不久，BG联合BCNU将在其他公司进行I期临床试验 机构和BG+STZ+BCNU将在该机构进行测试。 然而，生化调节策略对肿瘤细胞并不具有选择性。 而不是正常细胞。在本申请中，申请人提议开发 调控对CENU抗性的分子策略可能是 应用于未来的肿瘤特异性治疗。目标1将决定 转染人肿瘤细胞的最佳核酶靶向序列(S) 聚合型MGMT核酶的高表达 推动者。这种方法将确定最佳的核酶靶标。 测序(S)，鉴定抗性缺失表型 对BCNU敏感的克隆。目标2将优化反义基因的表达 利用诱导子降解长寿命MGMT基因的核酶 推动者。这些研究是从上一次支助时期继续进行的， 将利用从可诱导启动子转录的核酶，如 热休克促进剂(HSP)或糖皮质激素促进剂(GCP)。目标3将 鉴定能直接抑制MGMT的随机寡核苷酸 使用由科德和同事开发的SELEX系统。SELEX (指数富集法配基的系统进化)程序 从异构池中识别高度特定、高亲和力的配体 随机的核糖核酸分子。目标4将识别基因抑制元件 (GSE)由随机的MGMT Edna序列产生，导致抑制或 MGMT功能或表达下调。GSE技术利用 MGMT部分cDNA序列表达部分多肽的研究 定向，反义RNA在相反的方向。申请人 坚信任何分子试剂(核酶、反义RNA、GSE、 或SELEX OLOGO)在上述特定目标中确定的将是强大的 作为肿瘤特异性递送载体的未来基因治疗候选药物 都是由别人开发的。他的分子试剂将准备进行测试 在这些系统中。与实验室建立了合作关系 在脂质体和逆转录病毒载体开发方面拥有卓越的专业知识 未来对他的抗MGMT试剂的研究。

DESCRIPTION: (Applicant's Abstract) Overwhelming evidence has demonstrated that the major mechanism of tumor cell resistance to the chloroethyl-nitrosoureas (CENU) results from the DNA repair activity of O-6 methylguanine DNA methyltransferase (MGMT). This DNA repair protein is thought to protect cells from the cytotoxic DNA interstrand crosslink (ISC) produced by the CENU by removing chloroethyl adducts from the O-6 position of guanine before these adducts can rearrange to form a lethal crosslink. Studies conducted over the previous eight years of support 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 produce the natural substrate for MGMT, O-6 methylguanine. Repair of this lesion depletes the tumor cell of MGMT due to MGMT's suicide repair activity. In addition, the free bases O-6 methylguanine (MG) and O-6 benzylguanine (BG) can also deplete cells of MGMT activity and subsequently sensitize tumor cells to treatment with BCNU. During the most recent funding period the applicant has demonstrated that STZ combined with BG and BCNU can produce a prolonged sensitization of resistant tumor cells in vitro and in xenograft tumors in vivo. Soon BG plus BCNU will be tested in Phase I clinical trials at other institutions and 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 application the applicant proposes to develop molecular strategies for modulating resistance to the CENU which might be applied to tumor-specific therapy in the future. Aim 1 will determine the optimum ribozyme target sequence(s) by transfecting human tumor cells with pooled MGMT ribozymes constitutively expressed by a high expression promoter. This approach will identify the optimum ribozyme target sequence(s), and examine the loss of resistance phenotype by identification of clones sensitive to BCNU. Aim 2 will optimize expression of antisense ribozymes designed to degrade long-lived MGMT mRNA using inducible promoters. These studies, continued from the previous period of support, will utilize ribozymes transcribed from inducible promoters such as the heat-shock promoter (HSP) or the glucocorticoid promoter (GCP). Aim 3 will identify random oligoribonucleotides capable of direct inhibition of MGMT using the SELEX system developed by Cold and co-workers. The SELEX (Systematic Evolution of Ligands by Exponential enrichment) procedure identifies highly specific, high affinity ligands from a heterogeneous pool of random RNA molecules. Aim 4 will identify Genetic Suppressor Elements (GSEs) generated from random MGMT EDNA sequences which lead to inhibition or down-regulation of MGMT function or expression. GSE technology utilizes partial cDNA sequences of MGMT to express partial peptides in the sense orientation, and anti-sense RNAs in the opositer orientation. The applicant strongly believes that any molecular reagents (ribozyme, antisense RNA, GSE, or SELEX oligo) identified in the above Specific Aims will be powerful candidates for gene therapy in the future as tumor-specific delivery vectors are developed by others. His molecular reagents will be poised for testing in those systems. Collaborations have been established with laboratories with outstanding expertise in liposome and retroviral vector development for future studies of his anti-MGMT reagents.