Role of alphaB-Crystallin in Cancer Cell Death

描述(由申请人提供)：细胞凋亡紊乱在癌症中很常见，并使人对细胞毒治疗产生抵抗。然而，调控癌细胞死亡的机制却知之甚少。克里恩斯博士的实验室最近发现，热休克蛋白α-晶状体蛋白是一种新的细胞凋亡调节因子。他们已经证明，α-晶体蛋白在癌细胞中的稳定表达至少在一定程度上通过一种新的机制使人对化疗诱导的细胞凋亡产生抵抗：在体外和体内，α-晶体蛋白特异性地与caspase-3原结合，并通过顶端caspase抑制其蛋白分解激活。然而，caspase-3并不是α-晶体蛋白唯一的下游靶点：α-晶体蛋白保护MCF-7乳腺癌细胞(缺乏caspase-3)免受化疗诱导的凋亡，尽管它对稳定导入前caspase-3基因的MCF-7细胞有更大的保护作用。克赖恩斯博士实验室的初步研究还表明，α-晶体蛋白在化疗后会被磷酸化，而α-晶体蛋白的磷酸化可能会削弱其抗细胞凋亡的功能。拟议实验的目的是检验一种假说，即癌细胞中α-晶体蛋白的表达通过抑制caspase-3和其他caspase的激活而对化疗诱导的细胞凋亡产生抵抗(S)。其具体目的是：1)确定α-晶体蛋白抑制化疗诱导的细胞凋亡的分子机制；2)描绘介导其抗凋亡作用的α-晶体蛋白的功能结构域；3)评估α-晶体蛋白磷酸化的功能后果；以及4)确定使用脱氧核酶选择性抑制α-晶体蛋白的表达是否使癌细胞对化疗诱导的caspase激活和凋亡敏感。这些目标将通过几种技术来实现，以定量测量细胞凋亡(例如，TUNEL染色和核碎裂分析)和caspase激活(例如，caspase酶原蛋白分解过程和荧光底物的切割)。因此，这些研究将为癌症的化疗耐药机制提供新的见解，并可能导致针对α-晶体蛋白的癌症新疗法。

DESCRIPTION (provided by applicant): Derangements in apoptosis are common in cancer and confer resistance to cytotoxic therapy. However, the mechanisms that regulate cancer cell death are poorly understood. Dr. Cryns' laboratory has recently identified the heat shock protein alphaB-crystallin as a novel regulator of apoptosis. They have demonstrated that stable expression of alphaB-crystallin in cancer cells confers resistance to chemotherapy-induced apoptosis, at least in part, by a novel mechanism: alphaB-crystallin specifically binds to pro-caspase-3 in vitro and in vivo and inhibits its proteolytic activation by apical caspases. However, caspase-3 is not the only downstream target of alphaB-crystallin: alphaB-crystallin protects MCF-7 breast cancer cells (which lack caspase-3) from chemotherapy-induced apoptosis, although it confers greater protection to MCF-7 cells in which the pro-caspase-3 gene has been stably introduced. Preliminary studies from Dr. Cryns' laboratory also suggest that alphaB-crystallin is phosphorylated in response to chemotherapy and that phosphorylation of alphaB-crystallin may impair its anti-apoptotic function. The goal of the proposed experiments is to examine the hypothesis that alphaB-crystallin expression in cancer cells confers resistance to chemotherapy-induced apoptosis by inhibiting the activation of caspase-3 and other caspase(s). The specific aims are: 1) To determine the molecular mechanisms by which alphaB-crystallin inhibits chemotherapy-induced apoptosis; 2) To delineate the functional domains of alphaB-crystallin that mediate its anti-apoptotic actions; 3) To assess the functional consequences of phosphorylation of alphaB-crystallin; and 4) To determine whether selective inhibition of alphaB-crystallin expression using a deoxyribozyme sensitized cancer cells to chemotherapy-induced caspase activation and apoptosis. These aims will be accomplished using several techniques to quantitatively measure apoptosis (e.g., TUNEL staining and nuclear fragmentation assays) and caspase activation (e.g., pro-caspase proteolytic processing and cleavage of fluorogenic substrates). These studies, then, will provide novel insights into the mechanisms of chemoresistance in cancer and may lead to new therapies for cancer that specifically target alphaB-crystallin.