NUCLEAR EXPORT OF BRCA1 IN MAMMARY EPITHELIAL CELLS

乳腺上皮细胞中 BRCA1 的核输出

• 批准号: 6378198
• 负责人: MARILYN E. THOMPSON
• 金额: $ 9.18万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6378198
• 关键词: DNA repair; MCF7 cell; brca gene; cell growth regulation; cell nucleus; cell proliferation; complementary DNA; enzyme activity; enzyme inhibitors; intracellular transport; mammary epithelium; mitogen activated protein kinase; phosphoproteins; protein signal sequence; protein transport; site directed mutagenesis; transfection; western blottings

DESCRIPTION (Applicant's Description): Marilyn Eileen Thompson received her Ph.D. in Pharmacology from the University of South Alabama in 1994 and matriculated her postdoctoral training in the laboratory of Dr. Jeffrey T. Holt at Vanderbilt University in the Department of Cell Biology. This environment provides Dr. Thompson with adequate laboratory, office space and facilities. Shared resources and equipment include liquid scintillation and gamma spectrometers, several preparative ultracentrifuges. a dark room and a cold room. Core facilities are also available for DNA sequencing, confocal laser microscopy and conventional fluorescence microscopy. Dr. Thompson has and will continue to utilize these resources as she works towards her long term career goal of becoming an established scientific investigator in cancer research. In addition, Dr. Thompson will continue to attend departmental seminars, journal clubs and weekly lab meetings as well as attending national meetings as she develops as an independent scientist. Her hypothesis is: The physiological effects of BRCA1 are determined by a dynamic equilibrium between nuclear import and export of BRCA1. Specific Aim 1: To establish that BRCA1 is actively exported from the nucleus and map the sequence that mediates this export. Transient transfection of BRCA1 DNA and microinjection of exogenous BRCA1 into cell lines will be used to characterize the export mechanism of BRCA1. Mutagenesis will be employed to map the nuclear export signal. Specific Aim 2: To elucidate whether nuclear or cytoplasmic BRCA1 functions to regulate cell proliferation and DNA repair. Stable cell lines transfected with export- defective BRCA1 will be generated in HCC1937 cells and used to determine if their rate of cell growth or ability to repair radiation-induced DNA damage is altered from that of cells with wildtype BRCA1. Specific Aim 3: To verify that BRCA1 is phosphorylated via the MAP kinase pathway, map the sites of BRCA1 phosphorylation and determine if phosphorylation of BRCA1 by MAP kinase disrupts BRCAl nuclear foci. Pharmacological manipulation of MAP kinase activity, overexpression of MAP kinase and in vitro phosphorylation assays will be used to assess the phosphorylation of BRCA1 by MAP kinase. These studies will use molecular and immunological approaches to gain fundamental knowledge of the process by which BRCA1 functions. The regulation and function of this protein are complex and these studies will provide important insight into the physiology of BRCA1 in human mammary epithelial cells.

描述（申请人的描述）：玛丽莲·艾琳·汤普森（Marilyn Eileen Thompson）收到了她 博士 1994年南阿拉巴马大学的药理学专业 在Jeffrey T. 细胞生物学系范德比尔特大学的霍尔特。这 环境为汤普森博士提供了足够的实验室，办公空间和 设施。 共享资源和设备包括液体闪烁和 伽马光谱仪，几种制备超速离心。一个黑暗的房间和一个 寒冷的房间。 核心设施也可用于DNA测序，共焦 激光显微镜和常规荧光显微镜。汤普森博士有 并将继续利用这些资源，因为她努力长期 成为癌症的既定科学研究者的职业职业目标 研究。 此外，汤普森博士将继续参加部门 研讨会，期刊俱乐部和每周实验室会议以及参加全国 当她成长为独立科学家时会议。她的假设是： BRCA1的生理效应取决于动态平衡 BRCA1的核进口和出口。特定目的1：确定BRCA1是 积极从细胞核中导出并绘制介导的序列 出口。 BRCA1 DNA的瞬时转染和外源的显微注射 BRCA1进入细胞系将用于表征 BRCA1。诱变将用于绘制核出口信号。具体的 目标2：阐明核还是细胞质BRCA1功能来调节 细胞增殖和DNA修复。稳定的细胞系用出口转染 BRCA1有缺陷将在HCC1937细胞中产生，并用于确定是否 它们的细胞生长速率或修复辐射引起的DNA损伤的能力为 与WildType BRCA1的细胞改变了。特定目标3：验证 BRCA1通过MAP激酶途径磷酸化，映射BRCA1的位点 磷酸化并确定MAP激酶的BRCA1磷酸化是否磷酸化 破坏Brcal核局灶性。 地图激酶的药理操作 活性，MAP激酶的过表达和体外磷酸化测定 将使用MAP激酶评估BRCA1的磷酸化。这些 研究将使用分子和免疫学方法来获得基本 了解BRCA1功能的过程。调节和功能 该蛋白质很复杂，这些研究将提供重要的见解 进入人类乳腺上皮细胞中BRCA1的生理学。