Arsenic Induced Miotic Arrest Associated Apoptosis

说明(申请人提供)：砷是世界许多地区饮用水的天然污染物，是已知的人类致癌物质，在美国环保局的危险化学品清单上排名第一。最常与慢性砷中毒有关的癌症是皮肤鳞状细胞癌和基底细胞癌。砷是如何致癌的，目前尚不清楚。然而，国家研究委员会关于饮用水中砷的报告得出结论，最有可能的作用模式是诱导数字和结构染色体异常。亚砷酸盐是饮用水中发现的致癌形式的砷，它破坏有丝分裂，导致后期延迟，并诱导正常二倍体人成纤维细胞和外周血淋巴细胞的非整倍体，以及p53基因缺陷的人成纤维细胞的有丝分裂停滞相关凋亡(MAAA)。P53基因缺陷的人细胞对亚砷酸盐诱导的MAAA的敏感性表明，亚砷酸盐致癌的机制不同于阳光诱导的皮肤癌，其中P53突变是一种早期和常见的事件。需要研究的假设是，P53通过激活G2检查点反应来解除亚砷酸盐诱导的后期阻断，G2检查点反应使细胞周期蛋白B/cdc2失活，并降低有丝分裂退出网络的压力，使细胞能够逃避亚砷酸盐诱导的MAAA。正是对砷中毒细胞的凋亡的预防，使得有丝分裂中断后的遗传不稳定(非整倍体)。识别与P53或P53调控基因相互作用的细胞因子，以防止有丝分裂停滞相关的细胞凋亡，并允许细胞延迟进行有丝分裂，将为了解亚砷酸盐的作用模式提供有价值的信息。提出的具体目标是：1.在有丝分裂过程中，检测亚砷酸盐阻断的p53(+)和p53(-)细胞中G2检查点通路的激活；通过G2检查点蛋白的过度表达和靶向敲除来检验G2检查点激活在亚砷酸盐诱导的后期阻断逃逸中的作用；检测与砷相关的皮肤肿瘤是野生型还是突变型。这些研究的结果将有助于确定从亚砷酸盐诱导的有丝分裂抑制中释放的分子，并将为砷致癌机制提供有价值的信息，为亚砷酸盐作为化疗药物的有效性提供线索，并为有丝分裂干扰药物杀伤人类细胞的作用模式提供有价值的信息。

DESCRIPTION (provided by applicant): Arsenic is a natural contaminant of drinking water in many parts of the world, is a known human carcinogen and is #1 on the EPA list of hazardous chemicals. Cancers most often associated with chronic arsenism are squamous and basal cell carcinomas of the skin. How arsenic causes cancer is unknown. However, the National Research Council Report on Arsenic in Drinking Water concluded that the most likely mode of action is induction of numerical and structural chromosomal abnormalities. Arsenite, the carcinogenic form of arsenic found in drinking water, disrupts mitosis causing an anaphase delay and induces aneuploidy in normal diploid human fibroblasts and peripheral blood lymphocytes, and mitotic arrest associated apoptosis (MAAA) in p53 deficient human fibroblasts. The sensitivity of p53 deficient human cells to arsenite induced MAAA suggests that the mechanism of arsenite carcinogenesis is different than sunlight induced skin carcinogenesis in which p53 mutation is an early and common event. The hypothesis to be investigated is that p53 relieves the arsenite-induced anaphase block by activation of the G2 checkpoint response which inactivates cyclin B/cdc2 and derepresses the mitotic exit network and allow the cells to escape arsenite induced MAAA. It is the prevention of apoptosis in arsenic intoxicated cells that allows genetic instability (aneuploidy) after mitotic disruption. Identification of the cellular factors that interact with p53 or the p53 regulated genes to prevent mitotic arrest associated apoptosis and to allow cells to proceed through mitosis with a delay will provide valuable information regarding the mode of action of arsenite. The specific aims proposed are: 1.) Determine activation of the G2 checkpoint pathway in p53(+) and p53(-) cells arrested by arsenite in mitosis; 2.) Test by overexpression and targeted knockdown of G2 checkpoint proteins the role of G2 checkpoint activation in the escape from arsenite induced anaphase block; 3.) Test whether arsenic associated skin tumors are p53 wild type or mutant. The results of these studies will identify players mediating release from arsenite induced mitotic arrest, and will provide valuable information on the mechanism of arsenic induced carcinogenesis, clues to the usefulness of arsenite as a chemotherapeutic agent and valuable information on the mode of action of mitosis disrupting drugs in killing human cells.