Drug Resistance due to loss of Beta2-microglobulin

描述（由申请方提供）：β 2 m-微球蛋白（β 2 m）表达缺失或降低导致细胞对化疗产生耐药性。此外，外源性β 2 m蛋白在药物敏感和耐药变体中引起半胱天冬酶依赖性细胞凋亡，但独立于半胱天冬酶-3、-8和-9。在本申请中，我们提出探索β 2 m触发细胞凋亡的分子机制以及它如何增加抗癌剂的功效。我们的长期目标是阐明β 2 m诱导细胞凋亡的分子机制，并应用这些知识来识别和/或开发新的治疗方案。具体目的是（1）恢复β 2在耐药细胞中的表达，并评估其对化疗药物细胞毒性作用的影响，（2）鉴定参与β 2 m诱导的细胞凋亡的执行的特异性半胱天冬酶或非半胱天冬酶蛋白，（3）探讨肿瘤坏死因子-α（TNF-α）在肿瘤细胞凋亡中的作用（TNF-α）受体家族和增加活性氧（ROS）在β 2 m诱导的细胞凋亡。具体目标1将（a）确定MCF-7和MCF-7/Adr-5细胞中内源性J32 m的增加是否增加DOX、VCR、Taxol和VP-16的功效，（B）确定外源性β 2 m是否协同增强这些抗癌剂在这些细胞中诱导的细胞凋亡，（c）评估β 2 m单独或与这些药物组合是否对雌激素受体阴性乳腺癌细胞系MDA-MB-231及其DOX-231发挥类似的凋亡作用。耐药衍生物MDA-MB-231/Adr，和（d）检查β 2 m是否调节抗癌药物在细胞凋亡期间对半胱天冬酶的作用并影响这些细胞中的细胞周期，并从生物化学上确定β 2 m和抗癌剂如何不同地触发细胞凋亡。具体目标2将（a）通过蛋白质印迹分析探索半胱天冬酶-4、-7、-10、-11、-12和-13在β 2 m触发的细胞凋亡期间是否被激活，（B）通过检测半胱天冬酶底物Z-VAD-afc的切割来评估β 2 m诱导的半胱天冬酶激活，（c）鉴定来自β 2 m处理的和未处理的对照细胞的胞质溶胶中特异性结合生物素-VAD-亲和探针和生物素-YVAD-cmk亲和探针和125 I标记的YVAD-cmk的蛋白质，和（d）使用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）和N-末端测序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β 2 m通过死亡受体诱导细胞凋亡的机制，（B）确定β 2 m是否通过线粒体诱导ROS增加，（c）研究细胞氧化还原状态是否在β 2 m释放细胞色素c中发挥作用。这些研究将有助于理解β 2 m诱导细胞凋亡的分子机制，并将有助于开发更有效的化疗或潜在的基因治疗策略。

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.