The role of histone phosphorylation in arsenic-induced cell transformation and sk

描述(由申请人提供)： 砷是一种公认的人类致癌物质。我们的目标是解决这一中心假设，即组蛋白及其上游蛋白的磷酸化在砷诱导的细胞转化和致癌过程中发挥重要的功能作用。特异性目的1是研究组蛋白磷酸化在砷诱导的细胞转化中的作用；特异性目的2是研究和鉴定在不同氨基酸残基磷酸化组蛋白H3和组蛋白H 2B的组蛋白激酶；特异性目的3是研究组蛋白激酶RSK2的晶体结构，进行电子筛选和设计RSK2抑制剂，抑制砷诱导的组蛋白磷酸化和细胞转化；特异性目的4是研究RSK2在砷/紫外线A诱导的皮肤癌变中的作用，并确定RSK2作为化学预防癌症的靶点的潜力。针对特定目的1的策略是利用组蛋白H3和H 2B siRNA基因敲除和高表达稳定细胞系的关键磷酸化位点的点突变来测试H3和H 2B在软琼脂细胞转化试验中的作用。对于特定的目的2，我们将使用体外激酶分析、特定突变、LTQ Orbitrap杂交质谱仪分析和RSK2基因敲除细胞以及RSK2的抑制剂。在具体目标3中，我们将使用X射线结晶学来确定RSK2的结构。然后，我们将使用一台超级计算机来筛选一个包含250万种化学物质的数据库，以寻找RSK2的抑制剂，并在体外激酶试验中进行测试。在具体目标4中，我们将测试RSK2抑制剂山奈酚和RSK2基因敲除小鼠在UVA/砷诱导的小鼠皮肤癌变中的作用。这些知识将有助于设计更有效、更具体、副作用更少的砷诱导癌症的化学预防策略。

DESCRIPTION (provided by applicant): Arsenic is a well-documented human carcinogen. Our goal is to address the central hypothesis that phosphorylation of histones and their upstream kinases play an important functional role in arsenic-induced cell transformation and carcinogenesis. Specific Aim 1 is to study the role of histone phosphorylation in arsenic-induced cell transformation; Specific Aim 2 is to investigate and identify the histone kinases that phosphorylate histone H3 and H2B at different amino acid residues; Specific Aim 3 is to study the crystal structure of histone kinase RSK2, perform in- silico screening and design RSK2 inhibitors for suppressing arsenic-induced histone phosphorylation and cell transformation; and Specific Aim 4 is to study the role of RSK2 in arsenic/ultraviolet A (UVA)-induced skin carcinogenesis and determine RSK2's potential as a target for chemoprevention of cancer. The strategy for Specific Aim 1 is to use point mutations at key phosphorylation sites of histone H3 and H2B siRNA gene knockdown and overexpressing stable cell lines to test the role of H3 and H2B in soft agar cell transformation assays. For Specific Aim 2, we will use in vitro kinase assays, specific mutations, LTQ Orbitrap hybrid mass spectrometer analysis and RSK2 knockout cells as well as inhibitors of RSK2. In Specific Aim 3, we will use x-ray crystallography to determine the structure of RSK2. Then we will use a super computer to screen a database of 2.5 million chemicals to find inhibitors for RSK2 to be tested in an in vitro kinase assay. In Specific Aim 4, we will test the effect of the RSK2 inhibitor kaempferol and RSK2 knockout mice in UVA/arsenic-induced mouse skin carcinogenesis. Such knowledge will facilitate the design of more effective and specific strategies with fewer side effects for chemoprevention of arsenic- induced cancer.