Activation of the cyclin D1 promoter by arsenite

描述（由申请人提供）：在上一个资助期，我们表明用亚微摩尔浓度的亚砷酸盐处理培养的人类角质形成细胞会诱导细胞周期蛋白D1的表达，并且伴随着细胞周期进入G2区室。电泳迁移率变动分析（EMSA）证明了AP 1和CREB转录因子与细胞周期蛋白D1启动子中它们的同源结合基序的结合增强。在此，我们希望继续在先前SCORE资助期开始的工作，a）在荧光素酶报告基因分析中使用启动子缺失突变体来研究转录活性，B）通过使用染色质免疫沉淀（ChIP）作为亚砷酸盐对细胞周期蛋白D1表达的转录控制的体内测定。我们推测亚砷酸盐是MAP激酶应激激活通路的效应物，该通路最终激活JNK。为了验证这一点，我们将通过使用a）MAP激酶途径组分的化学抑制剂和B）显性失活突变体来更详细地检查ERK、JNK和p38途径的MAP激酶组分。由磷酸化引起的转录因子的活化将通过使用磷酸化特异性抗体的蛋白质印迹来证明。虽然我们的主要重点将是在信号通路的已知组件的可能性存在，亚砷酸盐也可能会带来磷酸化的新元素，可能会进一步上游。出于这个原因，我们也想研究更多的全球性变化的磷酸化模式，发生在响应亚砷酸盐使用抗体微阵列，以识别蛋白质的磷酸化模式的变化，这是相关的诱导细胞周期蛋白D1通过转录因子激活。最后，我们希望探索亚砷酸盐的诱导作用与活性氧有关的可能性。如果能够证明这一点，这一发现将成为未来研究的基础。 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砷在长期暴露于砷的个体中引起癌症的分子机制。这将最终提供目标，可能成为预防砷引起的癌症的新方法的基础。

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.