The applicant and others have described splice variants of BRCA1 that are widely expressed, in which the coding sequence corresponding to approximately 60% of the internal amino acids (encoded largely by exon 11) are spliced out, yielding a BRCA1-derived protein which is identical with full-length BRCA1 at its amino and carboxy termini, but which lacks important features including the nuclear localization signals and the Rad51-associating region. The overall objective of this proposal is to characterize the biological functions of the full-length and internally-deleted BRCA1 proteins. 1) The applicant proposes to transfect cDNA expression plasmids for BRCA1 splice variants into normal and malignant mammary epithelial cells. He proposes to use the tetracycline-responsive expression system, and to generate double BRCA1 knock-out cells from a human mammary epithelial cell line, in order to fine-tune and completely regulate the relative expression of the splice variants. The goal is to ascertain which elements of BRCA1 function depend on the specific isoform that is expressed, or on the relative abundance of BRCA1 isoforms, and which elements are common to all splice variants. Transfected cells will be analyzed with respect to their cell cycle distribution, rates of proliferation and apoptosis, anchorage-independent growth, radiation sensitivity, and capacity for DNA repair. 2) The second approach will be the identification of BRCA1-interacting proteins. The applicant has focused this search to proteins which display affinity for the carboxy terminus of BRCA1, a region which is shared among the splice variants and is critical for the anti-tumor effect of BRCA1. Using the yeast 2-hybrid system and a cDNA library from thymus, the applicant has recently identified IA-1 as a BRCA1 interaction protein. Experiments are now proposed to: i) confirm that these proteins interact in vivo in human cells; ii) map the regions required for the association of IA-1 with BRCA1; and iii) search for additional BRCA1-interacting proteins using a cDNA library from normal human mammary tissue.
| McEachern, Kristen A; Archey, William B; Douville, Karen et al. (2003) BRCA1 splice variants exhibit overlapping and distinct transcriptional transactivation activities. J Cell Biochem 89:120-32 |
| Archey, William B; McEachern, Kristen A; Robson, Mark et al. (2002) Increased CpG methylation of the estrogen receptor gene in BRCA1-linked estrogen receptor-negative breast cancers. Oncogene 21:7034-41 |
| Lu, M; Arrick, B A (2000) Transactivation of the p21 promoter by BRCA1 splice variants in mammary epithelial cells: evidence for both common and distinct activities of wildtype and mutant forms. Oncogene 19:6351-60 |
