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: 7032982
• 负责人: VINCENT L. CRYNS
• 金额: $ 20.66万
• 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/7032982
• 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; 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）：凋亡中的扰动在癌症中很常见，并赋予了对细胞毒性疗法的抗性。 但是，调节癌细胞死亡的机制知之甚少。 Cryns博士的实验室最近将热休克蛋白字母 - 晶状体蛋白确定为凋亡的新调节剂。 他们已经证明，癌细胞中字母晶状体蛋白的稳定表达赋予了对化学疗法诱导的凋亡的抗性，至少部分通过一种新型机制：alphab-crystallin在体外和体内特异性与pro-caspase-3特异性结合，并抑制其蛋白质质量的蛋白质质量激活。 然而，caspase-3并不是α-晶状体蛋白的唯一下游靶标：α-晶格蛋白保护MCF-7乳腺癌细胞（缺乏caspase-3）免受化学疗法诱导的凋亡，尽管它赋予了对MCF-7细胞的更大保护，其中Pro-Caspase-3基因已稳定地引入了Pro-Caspase-3基因。 Cryns博士实验室的初步研究还表明，α-晶状体蛋白会响应化学疗法而磷酸化，而Alphab-Crystallin的磷酸化可能会损害其抗凋亡功能。 该提出的实验的目的是检查癌细胞中字母 - 晶状体蛋白酶表达的假设，通过抑制caspase-3和其他caspase（S）的激活来赋予对化学疗法诱导的凋亡的抵抗力。 具体目的是：1）确定字母 - 晶状体抑制化学疗法诱导的细胞凋亡的分子机制； 2）描绘介导其抗凋亡作用的字母晶状体的功能域； 3）评估α-晶状体蛋白磷酸化的功能后果； 4）确定是否使用脱氧核酶对化学疗法诱导的caspase激活和凋亡进行选择性抑制α-晶状体表达。这些目标将使用多种技术来定量测量细胞凋亡（例如，TUNEL染色和核碎片化测定）和caspase激活（例如，促蛋白酶蛋白水解处理和荧光底物的裂解）。 因此，这些研究将提供对癌症化学抗性机制的新见解，并可能导致针对癌症的新疗法，该癌症专门针对α-晶状体蛋白。