MODULATION OF DNA DAMAGE IN HEMATOPOIETIC STEM AND PROGENITOR CELLS

造血干细胞和祖细胞 DNA 损伤的调节

• 批准号: 6563888
• 负责人: DAVID A WILLIAMS
• 金额: $ 22.01万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6563888
• 关键词: DNA damage; DNA repair; Retroviridae; aldehyde dehydrogenases; alkylating agents; antineoplastics; cyclophosphamide; cytotoxicity; disease /disorder model; drug resistance; drug screening /evaluation; enzyme activity; gene expression; genetic manipulation; glutathione transferase; hematopoietic stem cells; human tissue; laboratory mouse; methyltransferase; neoplasm /cancer chemotherapy; neoplasm /cancer genetics; nitrosourea; pharmacokinetics; transfection; transfection /expression vector

DNA alkylating agents are an important part of most dose-intensified chemotherapy protocols. In spite of increased use of myeloid growth factor and stem cell support, myelosuppression continues to be a dose-limiting toxicity of many alkylating agents. This is particularly true in patients with relapsed disease previously treated with intensive chemotherapy during induction and consolidation phases of initial therapies. One approach to circumvent the dose-limiting myeloid toxicities of chemotherapy agents has been the use of gene transfer to introduce and express genes important in chemotherapy resistance in bone marrow-derived cells. Work in our laboratory has concentrated on retroviral vectors encoding DNA repair proteins as a mechanism to generate resistance to chloroethylnitrosourea (CENUs) and other alkylating agents. In our previous studies, we have developed a murine model of CENU-induced fatal bone marrow suppression. Reconstitution of murine bone marrow with hematopoietic stem cells expressing vector-derived MGMT protects mice from BCNU-induced bone marrow hypoplasia and peripheral blood pancytopenia. Bone marrow cells harvested from these mice are more resistant to CENU in vitro, and demonstrate a higher level of DNA repair activity compared to CENU-treated mock-infected control mice. In the studies outlined here, we propose to continue our work on CENU-resistance by developing a new series of retroviral vectors which encode DNA repair activity resistant to depletion by the free base 06-benzylguanine, which would allow differential depletion of tumor versus bone marrow repair activity. In addition, we propose to expand this approach to include proteins of the base excision repair (BER) pathway, which are important in the repair of DNA damage from CENUs and other alkylating agents. Finally, we wish to develop vectors which would potentially prevent DNA damage from alkylating agents by increasing the expression of detoxifying proteins in bone marrow cells. Overall, this project seeks to use gene transfer technology to increase resistance of normal bone marrow to the cytotoxic effects of alkylating agent damage. The specific aims include: 1) Develop retroviral vectors which increase the therapeutic window of chloroethylnitrosourea (CENU)-based tumor killing in vivo utilizing 6-benzylguanine resistant methylguanine DNA methyltransferases expressed in bone marrow cells; 2) Examine the effect of transgene expression of components of the base excision repair (BER) pathway on CENU and alkylating (bleomycin, cyclophosphamide and thiotepa) agent resistance; and, 3) Examine the role of increased expression of aldehyde dehydrogenase and glutathione-S- transferase in resistance to cyclophosphamide. These basic studies are likely to lead to innovative clinical studies utilizing gene transfer technology in the future.

DNA烷基化剂是大多数剂量强化的重要组成部分 化学疗法方案。尽管使用髓样生长因子增加了 和干细胞支撑，骨髓抑制继续是剂量限制 许多烷基化剂的毒性。患者尤其如此 先前用重症疗法治疗的复发性疾病 在初始疗法的诱导和巩固阶段。一 绕过限制剂量的髓样毒性的方法 化学疗法剂一直是使用基因转移来引入和 表达基因在骨髓衍生的化学疗法耐药性中很重要 细胞。我们的实验室工作集中于逆转录病毒载体 编码DNA修复蛋白作为产生抗性的机制 氯乙基硝酸（Cenus）和其他烷基化剂。在我们的 先前的研究，我们开发了CENU诱发的致命的鼠模型 骨髓抑制。鼠骨髓的重建 表达载体的MGMT的造血干细胞可保护小鼠免受 BCNU诱导的骨髓发育不全和周围血液全细胞减少症。 这些小鼠收获的骨髓细胞对CENU的抗性更大 与 CENU处理的模拟感染对照小鼠。在这里概述的研究中，我们 建议通过开发一个新系列来继续我们的CENU抗性工作 逆转录病毒载体编码抗DNA修复活性至 自由碱耗尽06-苯二唑氨酸，这将允许 肿瘤与骨髓修复活性的差异耗尽。在 此外，我们建议将这种方法扩展到包括 基础切除修复（BER）途径，这对于维修很重要 Cenus和其他烷基化剂的DNA损伤。最后，我们希望 开发向量，该向量有可能防止DNA损伤烷基化 通过增加骨髓中解毒蛋白的表达来增加剂 细胞。总体而言，该项目试图使用基因转移技术来 增加正常骨髓对细胞毒性作用的抗性 烷基化剂损伤。具体目的包括：1）发展逆转录病毒 增加氯乙基硝酸的治疗窗口的载体 （CENU）基于6-苯唑圭氨酸耐药的体内杀死肿瘤 在骨髓细胞中表达的甲基鸟氨酸DNA甲基转移酶； 2） 检查碱基分量的转基因表达的影响 CENU和烷基化的切除修复（BER）途径（博来霉素，， 环磷酰胺和硫代）耐药性； 3）检查角色 醛脱氢酶和谷胱甘肽-S-表达增加的表达 对环磷酰胺的抗性转移酶。这些基础研究是 可能导致利用基因转移的创新临床研究 将来的技术。