Role of alphaB-Crystallin in Cancer Cell Death

αB-晶状体蛋白在癌细胞死亡中的作用

• 批准号: 6615904
• 负责人: VINCENT L. CRYNS
• 金额: $ 21.2万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-03 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6615904
• 关键词: apoptosis; cell line; clinical research; crystallins; cysteine endopeptidases; cytoprotection; drug resistance; enzyme activity; heat shock proteins; neoplastic cell; phosphorylation; protein structure function; restriction fragment length polymorphism; terminal nick end labeling

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.

描述（由申请人提供）：细胞凋亡紊乱在癌症中很常见，并赋予对细胞毒性治疗的抗性。 然而，人们对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 αB-晶状体蛋白是细胞凋亡的新型调节剂。 他们证明，αB-晶状体蛋白在癌细胞中的稳定表达至少部分地通过一种新机制赋予对化疗诱导的细胞凋亡的抵抗力：αB-晶状体蛋白在体外和体内特异性地结合 pro-caspase-3 并抑制其作用。顶端半胱天冬酶的蛋白水解激活。 然而，caspase-3 并不是 alphaB-crystallin 的唯一下游靶点：alphaB-crystallin 可以保护 MCF-7 乳腺癌细胞（缺乏 caspase-3）免受化疗诱导的细胞凋亡，尽管它在化疗中为 MCF-7 细胞提供了更大的保护。已稳定导入pro-caspase-3基因。 Cryns 博士实验室的初步研究还表明，αB-晶状体蛋白在化疗反应中被磷酸化，并且 αB-晶状体蛋白的磷酸化可能会损害其抗凋亡功能。 拟议实验的目的是检验以下假设：癌细胞中的 αB-晶状体蛋白表达通过抑制 caspase-3 和其他 caspase 的激活来抵抗化疗诱导的细胞凋亡。 具体目标是： 1) 确定αB-晶状体蛋白抑制化疗诱导的细胞凋亡的分子机制； 2) 描绘αB-晶状体蛋白介导其抗凋亡作用的功能域； 3) 评估αB-晶状体蛋白磷酸化的功能后果； 4)确定使用脱氧核酶选择性抑制αB-晶状体蛋白表达是否使癌细胞对化疗诱导的半胱天冬酶激活和细胞凋亡敏感。这些目标将使用多种技术来定量测量细胞凋亡（例如，TUNEL 染色和核碎片测定）和半胱天冬酶激活（例如，前半胱天冬酶蛋白水解加工和荧光底物的裂解）来实现。 这些研究将为癌症化疗耐药机制提供新的见解，并可能带来专门针对 αB-晶状体蛋白的癌症新疗法。