ANTICANCER DRUG RESISTANCE BY BCL2 ONCOGENE EXPRESSION

BCL2 癌基因表达的抗癌药物耐药性

• 批准号: 6376190
• 负责人: RAYMOND E MEYN
• 金额: $ 24.86万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6376190
• 关键词: BCL2 gene /protein; antineoplastics; antioxidants; apoptosis; cell line; drug resistance; gel mobility shift assay; gene expression; glutathione; intracellular transport; mitochondria; northern blottings; nuclear factor kappa beta; oxidative stress; protein localization; protein structure function; thiols; western blottings

The goal of this research is to define the mechanism responsible for the resistance to cancer chemotherapy agents due to expression of the bcl-2 oncogene. Bcl-2, the prototypic member of a family of proteins that regulate apoptosis, is abnormally expressed in many different types of human cancer including lymphomas, and tumors of the breast, prostate, lung and colon. Bcl-2 blocks apoptosis and, thus, causes tumor cells to become resistant to anticancer treatments such as radiotherapy and chemotherapy. The mechanism by which bcl-2 blocks apoptosis is not understood, but it appears to be related to bcl-2's localization in intracellular membranes including mitochondrial membranes and the nuclear envelope. At these sites, bcl-2 appears to regulate the transport of substances or factors involved in the apoptotic cascade. However, additional activities of bcl-2 also require explanation. Other observations indicate that bcl-2 may have a role in the cell's antioxidant pathways, specifically those involving thiols such as glutathione (GSH). Progress related to this application has demonstrated that bcl-2, at the level of the nuclear envelope, redistributes GSH into the nucleus by active transport where it apparently inhibits the proteases that carry out the apoptotic process. If this model is further validated to indicate that GSH is the substance blocking apoptosis, strategies may be devised to overcome such a block and restore anticancer drug-sensitivity. In this application, the following questions that serve to test predictions of this model, will be addressed. 1. Are other members of the bcl-2 family of proteins also involved in GSH metabolism? 2. Does bcl-2-expression also alter GSH levels in mitochondria? 3. Will newly developed strategies that target bcl-2 function or GSH itself act to sensitize tumor cells to anticancer drugs? 4. Is bcl-2 expression in tumor cells under the control of the NF-kappaB transcription factor? The methods to be used include quantitative measurements of GSH in intracellular pools and determinations of relative expression of bcl-2 and other proteins by immunoblot analysis. Apoptosis and clonogenic survival will be used as relevant endpoints for cell response to chemotherapy agents.

本研究的目的是确定由于 bcl-2 癌基因表达而对癌症化疗药物产生耐药性的机制。 Bcl-2 是调节细胞凋亡的蛋白质家族的原型成员，在许多不同类型的人类癌症中异常表达，包括淋巴瘤以及乳腺癌、前列腺癌、肺癌和结肠癌。 Bcl-2 阻断细胞凋亡，从而导致肿瘤细胞对放疗和化疗等抗癌治疗产生耐药性。 bcl-2 阻断细胞凋亡的机制尚不清楚，但似乎与 bcl-2 在细胞内膜（包括线粒体膜和核膜）中的定位有关。 在这些位点，bcl-2 似乎调节参与细胞凋亡级联的物质或因子的运输。 然而，bcl-2 的其他活性也需要解释。 其他观察结果表明，bcl-2 可能在细胞的抗氧化途径中发挥作用，特别是涉及谷胱甘肽 (GSH) 等硫醇的途径。 与该应用相关的进展表明，bcl-2 在核膜水平上通过主动转运将 GSH 重新分配到细胞核中，并明显抑制执行细胞凋亡过程的蛋白酶。 如果该模型得到进一步验证，表明 GSH 是阻断细胞凋亡的物质，则可以设计策略来克服这种阻断并恢复抗癌药物的敏感性。在此应用中，将解决用于测试该模型预测的以下问题。 1. bcl-2 蛋白家族的其他成员是否也参与 GSH 代谢？ 2. bcl-2 表达是否也会改变线粒体中的 GSH 水平？ 3. 新开发的针对 bcl-2 功能或 GSH 本身的策略是否会使肿瘤细胞对抗癌药物敏感？ 4. 肿瘤细胞中bcl-2的表达是否受到NF-kappaB转录因子的控制？ 使用的方法包括细胞内谷胱甘肽的定量测量以及通过免疫印迹分析确定 bcl-2 和其他蛋白质的相对表达。 细胞凋亡和克隆存活将用作细胞对化疗药物反应的相关终点。