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Drug Resistance due to loss of Beta2-microglobulin

由于β2-微球蛋白丢失而产生耐药性

基本信息

批准号：
6850103
负责人：
Ahmad R. Safa
金额：
$ 28.29万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-03-03 至 2007-02-28
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The loss or decreased expression of beta 2m-microglobulin (beta2m) causes cellular resistance to chemotherapy. Moreover, exogenous beta 2m protein causes caspase-dependent apoptosis, but independently of caspases-3, -8, and -9, in drug sensitive and resistant variants. In this application, we propose to explore the molecular mechanism(s) by which beta2m triggers apoptosis and how it increases the efficacy of anticancer agents. Our long-term objectives are to unravel the molecular mechanism(s) of beta2m-induced apoptosis, and to apply this knowledge to identify and/or develop new therapeutic regimens. The Specific Aims are to (1) restore beta2 expression in drug resistant cells and assess its influence on the cytotoxic effects of chemotherapeutic drugs, (2) identify specific caspases or non-caspase proteins involved in the execution of beta 2m-induced apoptosis, and (3) investigate the role of the tumor necrosis factor-alpha (TNF-alpha) receptor family and increased reactive oxygen species (ROS) in beta 2m-induced apoptosis. Specific Aim 1 will (a) determine whether the increase in endogenous J32m in MCF-7 and MCF-7/Adr-5 cells increases the efficacy of DOX, VCR, Taxol and VP-16, (b) determine whether exogenous beta 2m synergistically enhances apoptosis induced by these anticancer agents in these cells, (c) evaluate whether [beta 2m alone or in combination with these drugs exerts similar apoptotic effects on the estrogen receptor-negative breast cancer cell line MDA-MB-231 and its DOX-resistant derivative MDA-MB-231/Adr, and (d) examine whether [beta 2m modulates the effects of anticancer drugs on caspases during apoptosis and affects the cell cycle in these cells, and ascertain biochemically how apoptosis-triggering by [beta 2m and anticancer agents differs. Specific Aim 2 will (a) explore by Western blot analysis whether caspases-4, -7, -10, -11, -12 and -13 are activated during beta 2m-triggered apoptosis, (b) assess beta 2m-induced caspase activation by detecting cleavage of the caspase substrate Z-VAD-afc, (c) identify the proteins that bind specifically to the biotin-VAD-fmk and biotin-YVAD-cmk affinity probes and 125I-labeled YVAD-cmk in the cytosols from beta 2m-treated and untreated control cells, and (d) determine the identity of the proteins specifically labeled with these affinity probes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and N-terminal sequencing. Specific Aim 3 will (a) explore the mechanisms by which beta 2m induces apoptosis through death receptors, (b) determine whether [beta 2m induces increased ROS through the mitochondria, and (c) investigate whether cellular redox state plays a role in cytochrome c release by [beta 2m. These studies will aid in understanding the molecular mechanism(s) of beta 2m-induced apoptosis, and will be useful for the development of more effective chemotherapeutic or potential gene therapy strategies.

描述(由申请人提供)：β2M-微球蛋白(β2M)的缺失或表达降低会导致细胞对化疗产生耐药性。此外，在药物敏感和耐药变异体中，外源性β2M蛋白引起caspase依赖的细胞凋亡，但不依赖于caspase-3、-8和-9。在这一应用中，我们建议探索β2m触发细胞凋亡的分子机制(S)，以及它如何增加抗癌药物的疗效。我们的长期目标是揭开Beta2m诱导细胞凋亡的分子机制(S)，并应用这一知识来识别和/或开发新的治疗方案。其具体目的是：(1)恢复耐药细胞中β_2的表达并评价其对化疗药物细胞毒作用的影响；(2)鉴定参与β_2M诱导的细胞凋亡的特异性caspase或非caspase蛋白；(3)研究肿瘤坏死因子-α受体家族和增加的活性氧在β_2M诱导的细胞凋亡中的作用。具体目标1将(A)确定MCF-7和MCF-7/ADR-5细胞内源性J32M的增加是否增加了DOX、VCR、紫杉醇和VP-16的疗效，(B)确定外源性β2M是否协同增强这些抗癌剂诱导的这些细胞的凋亡，(C)评估[β2M]单独或与这些药物联合使用是否对雌激素受体阴性的乳腺癌细胞株MDA-MB-231及其DOX耐药衍生物MDA-MB-231/ADR具有类似的凋亡作用，以及(D)研究[β2M]是否调节抗癌药物在细胞凋亡过程中对caspase的影响并影响这些细胞的细胞周期，并从生物化学的角度确定[β2M]和抗癌剂触发细胞凋亡的不同之处。具体目标2将(A)通过Western印迹分析来探索caspase-4、-7、-10、-11、-12和-13在β2M诱导的细胞凋亡中是否被激活，(B)通过检测caspase底物Z-VAD-AFC的裂解来评估β2M诱导的caspase激活，(C)识别与生物素-VAD-fmk和生物素-YVAD-CMK亲和探针和125I标记的YVAD-CMK在β2M处理和未处理的对照细胞胞浆中特异结合的蛋白质，以及(D)使用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)和N-末端测序来确定用这些亲和探针特异性标记的蛋白质的身份。具体目标3将(A)探索β2M通过死亡受体诱导细胞凋亡的机制，(B)确定[β2M]是否通过线粒体诱导ROS增加，以及(C)研究细胞氧化还原状态是否在[β2M]释放细胞色素c中起作用。这些研究将有助于了解β2M诱导细胞凋亡的分子机制(S)，并将有助于开发更有效的化疗或潜在的基因治疗策略。