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

描述（由申请人提供）： 砷是一种有据可查的人类致癌物质。我们的目标是要解决的核心假设，磷酸化组蛋白及其上游激酶在砷诱导的细胞转化和癌变中发挥重要的功能作用。具体目标1是研究组蛋白磷酸化在砷诱导的细胞转化中的作用，具体目标2是研究和鉴定磷酸化组蛋白H3和H2 B不同氨基酸残基的组蛋白激酶;具体目标3是研究组蛋白激酶RSK 2的晶体结构，进行计算机筛选并设计RSK 2抑制剂，用于抑制砷诱导的组蛋白磷酸化和细胞转化;具体目标4是研究RSK 2在砷/紫外线A（UVA）诱导的皮肤癌发生中的作用，并确定RSK 2作为癌症化学预防靶点的潜力。特定目标1的策略是在组蛋白H3和H2 B siRNA基因敲低的关键磷酸化位点使用点突变，并过表达稳定细胞系，以测试H3和H2 B在软琼脂细胞转化试验中的作用。对于特异性目标2，我们将使用体外激酶测定、特异性突变、LTQ Orbitrap混合质谱仪分析和RSK 2敲除细胞以及RSK 2抑制剂。在具体目标3中，我们将使用X射线晶体学来确定RSK 2的结构。然后，我们将使用超级计算机筛选250万种化学物质的数据库，以找到RSK 2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RSK 2抑制剂山奈酚和RSK 2基因敲除小鼠在UVA/砷诱导的小鼠皮肤癌发生中的作用。这些知识将有助于设计更有效和更具体的策略，减少副作用，用于砷诱导的癌症的化学预防。环境砷污染是世界许多地区的一个主要问题，是一种有充分记录的人类致癌物。我们将利用X射线晶体学、基于超级计算机的分子模拟和药物筛选、细胞和分子生物学以及基因敲除小鼠等最新技术，研究组蛋白磷酸化在砷诱导的细胞癌变过程中的新机制。这些研究将有助于开发更有效的药物，减少副作用，以化学预防环境致癌物，如砷诱导的癌症。

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. PUBLIC HEALTH RELEVANCE: Environmental arsenic contamination is a major problem in many parts of the world and is a well-documented human carcinogen. By using state-of-the-art technology such as x-ray crystallography, super computer based molecular modeling and drug screen, cellular and molecular biology and gene knockout mice, we will study the novel mechanism involved in histone phosphorylation on arsenic-induced cell transformation to cancer and the carcinogenesis process. These studies will facilitate the development of more effective agents with fewer side effects for chemoprevention against environmental carcinogens such as arsenic- induced cancer.