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，并赋予细胞毒性治疗耐药性。然而，调控癌细胞死亡的机制尚不清楚。crns博士的实验室最近发现热休克蛋白alphaB-crystallin是一种新的细胞凋亡调节因子。他们已经证明，alphaB-crystallin在癌细胞中的稳定表达，至少在一定程度上，通过一种新机制赋予了化疗诱导的细胞凋亡的抗性：在体外和体内，alphaB-crystallin特异性地与caspase-3结合，并抑制其被顶端caspase激活的蛋白水解。然而，caspase-3并不是alphaB-crystallin的唯一下游靶点：alphaB-crystallin保护MCF-7乳腺癌细胞（缺乏caspase-3）免受化疗诱导的凋亡，尽管它对前caspase-3基因稳定引入的MCF-7细胞提供了更大的保护。Cryns博士实验室的初步研究也表明，alphaB-crystallin在化疗反应中被磷酸化，而alphaB-crystallin的磷酸化可能会损害其抗凋亡功能。本实验的目的是检验癌细胞中α -晶体蛋白表达通过抑制caspase-3和其他caspase(s)的激活来抵抗化疗诱导的凋亡的假设。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)明确α -晶体蛋白的抗凋亡功能域；3)评估α -晶体蛋白磷酸化的功能后果；4)确定脱氧核酶选择性抑制alphaB-crystallin表达是否会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定量测量细胞凋亡（例如，TUNEL染色和核碎片分析）和caspase激活（例如，前caspase蛋白水解加工和荧光底物的裂解）来实现。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 -晶体蛋白的癌症的新疗法。

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.