NUCLEAR EXPORT OF BRCA1 IN MAMMARY EPITHELIAL CELLS

乳腺上皮细胞中 BRCA1 的核输出

基本信息

批准号：
6089881
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
项目类别：
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/6089881
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

项目摘要

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·汤普森接待了她博士 1994年获得南阿拉巴马大学药理学博士学位在 Jeffrey T. 博士的实验室接受博士后培训霍尔特在范德比尔特大学细胞生物学系。这环境为汤普森博士提供了充足的实验室、办公空间和设施。共享资源和设备包括液体闪烁和伽马能谱仪，几台制备型超速离心机。一个黑暗的房间和一个寒冷的房间。核心设施还可用于 DNA 测序、共聚焦激光显微镜和传统荧光显微镜。汤普森博士有并将继续利用这些资源，努力实现自己的长期目标成为一名成熟的癌症科学研究人员的长期职业目标研究。此外，汤普森博士将继续参加系研讨会、期刊俱乐部和每周实验室会议以及参加国家会议在她成长为一名独立科学家的过程中召开的会议。她的假设是： BRCA1 的生理效应是由两者之间的动态平衡决定的 BRCA1 的核导入和导出。具体目标 1：确定 BRCA1 是主动从细胞核输出并绘制介导此的序列出口。 BRCA1 DNA 瞬时转染和外源性显微注射 BRCA1 进入细胞系将用于表征 BRCA1 的输出机制乳腺癌1。将采用诱变来绘制核输出信号。具体的目标 2：阐明细胞核或细胞质 BRCA1 是否具有调节功能细胞增殖和DNA修复。稳定细胞系转染导出- 有缺陷的 BRCA1 将在 HCC1937 细胞中产生，并用于确定是否它们的细胞生长速度或修复辐射引起的 DNA 损伤的能力是与野生型 BRCA1 细胞相比有所改变。具体目标 3：验证 BRCA1 通过 MAP 激酶途径磷酸化，绘制 BRCA1 位点图磷酸化并确定 BRCA1 是否被 MAP 激酶磷酸化破坏BRCA1核灶。 MAP 激酶的药理学操作 MAP 激酶的活性、过度表达和体外磷酸化测定将用于评估 MAP 激酶对 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原理了解 BRCA1 发挥作用的过程。调节与作用这种蛋白质的结构很复杂，这些研究将提供重要的见解研究人乳腺上皮细胞中 BRCA1 的生理学。