Activation of the cyclin D1 promoter by arsenite

亚砷酸盐激活细胞周期蛋白 D1 启动子

基本信息

批准号：
7896859
负责人：
MARK L STEINBERG
金额：
$ 11.55万
依托单位：
CITY COLLEGE OF NEW YORK
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7896859
关键词：
Animal Model Antibodies Arsenic Arsenites Binding Biological Assay Biological Models Burn injury CREB1 gene Cancer Etiology Carcinogens Cell Cycle Cell Cycle Regulation Chemicals Chemosensitization Cyclin D1 Cyclins Development Dominant-Negative Mutation Electrophoretic Mobility Shift Assay Elements Environmental Pollutants Event Exposure to Funding Future Genetic Transcription Global Change Goals Health Human Individual Light Luciferases MAP Kinase Modules MAPK14 gene MAPK8 gene Malignant Neoplasms Measures Mitogen-Activated Protein Kinases Molecular Oncogenic Pathway interactions Pattern Phosphorylation Populations at Risk Prevention Process Proteins Public Health RNA Reactive Oxygen Species Reporter Research Role Signal Pathway Skin Solar Energy Staging Stress System Testing Time Transcription Factor AP-1 Transcriptional Regulation Up-Regulation Western Blotting Work Writing base carcinogenesis chromatin immunoprecipitation in vitro Model in vivo inhibitor/antagonist insight keratinocyte mutant novel novel strategies overexpression promoter public health relevance research study response transcription factor tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): In the previous funding period we showed that treatment of cultured human keratinocytes with arsenite at submicromolar concentrations induced expression of cyclin D1 and this was accompanied by a shift into the G2 compartment of the cell cycle. Electrophoretic mobility shift assays (EMSA) demonstrated enhanced binding of AP1 and CREB transcription factors to their cognate binding motifs in the cyclin D1 promoter Here we wish to continue the work begun in the previous SCORE funding period by, a) using promoter deletion mutants in luciferase reporter assays to study transcriptional activity and, b) by using chromatin immunoprecipitation (ChIP) as an in vivo assay of transcriptional control of cyclin D1 expression by arsenite. We hypothesize that arsenite is an effector of the MAP kinase stress activated pathway which ultimately activates JNK. To test this we will examine the MAP kinase components of the ERK, JNK and p38 pathways in more detail by using a) chemical inhibitors of components of the MAP kinase pathways and b) dominant negative mutants. Activation of transcription factors resulting from phosphorylation will be demonstrated by western blotting using phosphorylation-specific antibodies. Although our main focus will be on the known components of signaling pathways the possibility exists that arsenite may also bring about phosphorylation of novel elements that may act further upstream. For this reason we also want to examine more global changes in patterns of phosphorylation that occur in response to arsenite using antibody microarrays with a view towards discerning changes in patterns of phosphorylation of proteins that is correlated with the induction of cyclin D1 via transcription factor activation. Finally, we wish to explore the possibility that the inductive effects of arsenite are related to reactive oxygen species as preliminary evidence indicates. If this can be demonstrated this finding will form the basis for future research. PUBLIC HEALTH RELEVANCE: Arsenic is a toxic environmental pollutant and carcinogen. The experiments described are intended to shed light on the molecular mechanism by which arsenic causes cancer in individuals exposed to arsenic over long periods of time. This will ultimately provide targets that may form the basis for new approaches for the prevention of arsenic-caused cancers.

描述（由申请人提供）：在上一个资金期间，我们表明在亚摩尔摩尔浓度下用砷的培养的人角质形成细胞诱导了Cyclin d1的表达，这伴随着转移到细胞周期的G2室的转移。电泳迁移率转移测定法（EMSA）表明，AP1和CREB转录因子的结合增强了其与它们在Cyclin d1启动子中的相关结合基序的结合，我们希望继续在上一个得分资金中开始的工作开始，a）通过使用Luciferase reptrip Anditor contriptions Andive and chromatition Andemation（a）通过研究转录术的chromation contriptiation（b），并通过研究（b）进行转录式（b），并通过chrip chromatition（b）进行转录式（B），并通过chromate contriptions（b）进行转录式（b）。通过砷对细胞周期蛋白D1表达的转录控制的测定。我们假设砷是MAP激酶应力激活途径的效应子，该途径最终激活JNK。为了测试这一点，我们将通过使用a）MAP激酶途径和b）显性阴性突变体的MAP激酶途径成分的化学抑制剂来更详细地研究ERK，JNK和P38途径的MAP激酶成分。磷酸化引起的转录因子的激活将通过使用磷酸化特异性抗体的蛋白质印迹来证明。尽管我们的主要重点将放在信号通路的已知组成部分上，但存在砷也可能引起可能在上游进一步起作用的新元素的磷酸化的可能性。因此，我们还希望检查使用抗体微阵列响应磷酸化模式的更多全球变化，以鉴定蛋白质磷酸化模式的变化，而蛋白质的磷酸化模式与通过转录因子激活诱导的细胞周期蛋白D1相关。最后，我们希望探讨砷的归纳作用与初步证据所表明的那样，砷的感应作用与活性氧有关。如果可以证明这一点，这一发现将构成未来研究的基础。公共卫生相关性：砷是一种有毒的环境污染物和致癌物。所描述的实验旨在阐明分子机制，该机制在长时间内暴露于砷的个体中引起砷的癌症。这最终将提供目标，这可能是预防砷引起的癌症的新方法的基础。