Activation of the cyclin D1 promoter by arsenite

描述（由申请人提供）：在之前的资助期内，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可诱导细胞周期蛋白D1的表达，并伴有细胞周期的G2区转移。电泳淌度转变化验(EMSA)展示了增强约束力的AP1和转录因子分子同源绑定图案的细胞周期蛋白D1启动子在这里我们希望继续工作开始之前的分数融资期间,a)使用荧光素酶启动子缺失突变体记者化验研究转录活动和b)通过染色质免疫沉淀反应(芯片)作为体内转录控制细胞周期蛋白D1表达分析的亚砷酸盐。我们假设亚砷酸盐是MAP激酶应激激活途径的一种效应物，最终激活JNK。为了验证这一点，我们将通过使用a) MAP激酶途径成分的化学抑制剂和b)显性阴性突变体来更详细地研究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磷酸化特异性抗体的western blotting来证明。虽然我们的主要重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上游作用的新元素的磷酸化。出于这个原因，我们还希望使用抗体微阵列来研究更多针对亚砷酸盐的磷酸化模式的全局变化，以期识别与通过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，我们希望探讨亚砷酸盐的诱导效应与活性氧有关的可能性。如果能证明这一点，这一发现将为未来的研究奠定基础。

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.