Activation of the cyclin D1 promoter by arsenite

描述(申请人提供)：在之前的资助期间，我们发现用亚微摩尔浓度的亚砷酸盐处理培养的人角质形成细胞诱导细胞周期蛋白D1的表达，并伴随着细胞周期的G2转变。凝胶迁移率改变分析(EMSA)显示AP1和CREB转录因子与细胞周期蛋白D1启动子的同源结合基序结合增强。在此，我们希望通过在荧光素酶报告实验中使用启动子缺失突变体来研究转录活性，以及利用染色质免疫沉淀(ChIP)作为亚砷酸盐体内转录调控细胞周期蛋白D1表达的实验来继续这项工作。我们假设亚砷酸盐是最终激活JNK的MAP激酶应激激活途径的效应者。为了测试这一点，我们将更详细地检测ERK、JNK和p38途径的MAP激酶成分，方法是使用a)MAP激酶途径成分的化学抑制剂和b)显性负性突变体。由磷酸化导致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实。虽然我们的主要关注点将放在信号通路的已知成分上，但亚砷酸盐也可能导致可能进一步作用于上游的新元素的磷酸化。出于这个原因，我们还希望使用抗体微阵列来检测亚砷酸盐引起的磷酸化模式的更多全球变化，以期识别与转录因子激活诱导细胞周期蛋白D1相关的蛋白质磷酸化模式的变化。最后，如初步证据所示，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。 与公众健康相关：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长期接触砷的个人中砷导致癌症的分子机制。这最终将提供目标，可能形成预防砷引起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.