The BRCA2 gene is a cancer predisposition gene, which is matured in breast cancer, prostate cancer, ovarian cancer, esophageal cancer , and pancreatic cancer. In response to the critique, we have markedly modified this proposal to study mouse BRCA2 defective cells (BRCA2--) instead of human BRCA2 defective Vance cells, since the genetic defect in the mouse cells is simpler. Our preliminary data in mouse BRCA2-- cells shows that Rad51 is rapidly cleaved by caspase-3 in BRCA2 defective cells and that overexpression of BRCA2 or Rad51, or inhibition of caspase-3 reverse radioactivity and the DNA repair defect. A caspase-3 resistant Rad51 mutant (D- A187Rad51) reverses radio sensitivity and the DNA repair in brca2-- to a greater extent than wildtype Rad51 when both proteins are expressed at near physiologic levels. We have recently purified Rad51 and caspase-3 proteins to allow in vitro mechanistic studies. We present new data showing Rad51 is mostly cytoplasmic in brca2--, and that wildtype BRCA2 expression relocates Rad51 to the nucleus whereas mutant BRCA2 expression does not effect Rad51 localization. These fundings lead to the following hypotheses: 1. BRCA2 normally functions in double strand DNA break repair; 2. BRCA2 and Rda51 function in the same biochemical pathway mediating DNA repair; 3. BRCA2 stabilizes Rad51 by inhabiting degradation by caspase-3 and other proteases; 4. BRCA2 influences nuclear localization of Rad51. To test hypothesis we propose these aims.
Aim 1 : Determine the functional domains of BRCA2 protein which mediate double strand break repair and tumorigenesis.
Aim 2 : See if known additional mutations in BRCA2 defective cancer cells contribute to radiation sensitivity & DNA repair defects in brca2-- cells.
Aim 3 : Determine the mechanism and in vivo significance off BRCA2- Rad51 interactions by in vitro assays.
Aim 4 : Analyze mechanism of BRCA2- Rad51 localization.
Aim 5 : Develop and use in Vivo imaging methods to study Rad51 localization and DNA repair in living cells, in real time.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA085269-02
Application #
6514387
Study Section
Metabolic Pathology Study Section (MEP)
Program Officer
Pelroy, Richard
Project Start
2001-06-01
Project End
2006-05-31
Budget Start
2002-07-09
Budget End
2003-05-31
Support Year
2
Fiscal Year
2002
Total Cost
$373,468
Indirect Cost
Name
University of Colorado Denver
Department
Pathology
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
Nelson, Andrew C; Holt, Jeffrey T (2010) Impact of RING and BRCT domain mutations on BRCA1 protein stability, localization and recruitment to DNA damage. Radiat Res 174:1-13
Nelson, Andrew C; Lyons, Traci R; Young, Christian D et al. (2010) AKT regulates BRCA1 stability in response to hormone signaling. Mol Cell Endocrinol 319:129-42
Malone, J L; Nelson, A C; Lieberman, R et al. (2009) Oestrogen-mediated phosphorylation and stabilization of BRCA2 protein in breast. J Pathol 217:380-8
Brown, Erika T; Holt, Jeffrey T (2009) Rad51 overexpression rescues radiation resistance in BRCA2-defective cancer cells. Mol Carcinog 48:105-9
Brown, Erika T; Robinson-Benion, Cheryl; Holt, Jeffrey T (2008) Radiation enhances caspase 3 cleavage of Rad51 in BRCA2-defective cells. Radiat Res 169:595-601
Holt, Jeffrey T; Toole, William P; Patel, Vedang R et al. (2008) Restoration of CAPAN-1 cells with functional BRCA2 provides insight into the DNA repair activity of individuals who are heterozygous for BRCA2 mutations. Cancer Genet Cytogenet 186:85-94
Thompson, Marilyn E; Robinson-Benion, Cheryl L; Holt, Jeffrey T (2005) An amino-terminal motif functions as a second nuclear export sequence in BRCA1. J Biol Chem 280:21854-7