Role of alphaB-Crystallin in Cancer Cell Death

描述（由申请方提供）：细胞凋亡紊乱在癌症中很常见，并赋予细胞毒性治疗抗性。 然而，调节癌细胞死亡的机制知之甚少。 Cryns博士的实验室最近发现热休克蛋白alphaB-晶体蛋白是一种新的细胞凋亡调节因子。 他们已经证明，α B-晶状体蛋白在癌细胞中的稳定表达赋予对化疗诱导的细胞凋亡的抗性，至少部分是通过一种新的机制：α B-晶状体蛋白在体外和体内特异性结合胱天蛋白酶原-3，并抑制其通过顶端胱天蛋白酶的蛋白水解活化。 然而，半胱天冬酶-3不是alphaB-晶状体蛋白的唯一下游靶点：alphaB-晶状体蛋白保护MCF-7乳腺癌细胞（其缺乏半胱天冬酶-3）免于化疗诱导的凋亡，尽管它赋予稳定引入半胱天冬酶原-3基因的MCF-7细胞更大的保护。Cryns博士实验室的初步研究还表明，alphaB-晶状体蛋白在对化疗的反应中被磷酸化，并且alphaB-晶状体蛋白的磷酸化可能会损害其抗凋亡功能。 所提出的实验的目的是检验癌细胞中的α B-晶状体蛋白表达通过抑制半胱天冬酶-3和其它半胱天冬酶的活化而赋予对化疗诱导的细胞凋亡的抗性的假设。 具体目标是：1）确定α B-晶状体蛋白抑制化疗诱导的细胞凋亡的分子机制; 2）描绘α B-晶状体蛋白介导其抗细胞凋亡作用的功能结构域; 3）评估α B-晶状体蛋白磷酸化的功能后果;和4）为了确定使用脱氧核酶选择性抑制α B-晶状体蛋白表达是否使癌细胞对化疗敏感，诱导caspase活化和凋亡。这些目的将使用几种定量测量细胞凋亡的技术来实现（例如，TUNEL染色和核碎裂测定）和半胱天冬酶活化（例如，半胱天冬酶原蛋白水解加工和荧光底物的裂解）。 因此，这些研究将为癌症的耐药性机制提供新的见解，并可能导致专门针对α B-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.