Radiation enhances caspase 3 cleavage of Rad51 in BRCA2-defective cells.

After DNA damage, caspases cleave and activate proteins involved in cell death by apoptosis but also cleave and inactivate proteins implicated in DNA repair. Here we report a rapid onset of Rad51 cleavage by caspase 3 in BRCA2-defective mouse and human cells. This rapid cleavage was reduced markedly by transfer of full-length human BRCA2 into BRCA2-defective mouse or human cells, which also blocked the association of caspase 3 and Rad51 proteins. Overall caspase 3 activity was increased in BRCA2-defective cells, but the time course was much slower than that for Rad51 cleavage. We further showed that caspase 3 cleavage of Rad51 resulted in a functional decrease in Rad51 strand exchange activity and that inhibition of caspase 3 activity increased Rad51 protein levels and Rad51 foci. These findings indicate that BRCA2 inhibits Rad51 cleavage and subsequent apoptosis.

A transgenic model reveals important roles for the NF-kappa B alternative pathway (p100/p52) in mammary development and links to tumorigenesis.

A regulated pattern of nuclear factor kappaB (NF-kappaB) activation is essential for normal development of the mammary gland. An increase in NF-kappaB activity has been implicated in breast cancer. We have generated a novel transgenic mouse model to investigate the role of the alternative NF-kappaB pathway in ductal development and identify possible mediators of tumorigenesis downstream of p100/p52. By overexpressing the NF-kappaB p100/p52 subunit in mammary epithelium using the beta-lactoglobulin milk protein promoter, we found that transgene expression resulted in increased overall NF-kappaB activity during late pregnancy. During pregnancy, p100/p52 expression resulted in delayed ductal development with impaired secondary branching and increased levels of Cyclin D1, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and cyclo-oxygenase-2 (COX-2) in the mammary gland. After multiple pregnancies the p100 transgenics exhibited a ductal thickening accompanied by small hyperplastic foci. In tumors from mice expressing the polyoma middle T oncoprotein (PyVT) in the mammary gland, increased levels of p100/p52 were present at the time of tumor development. These results show that increased p100/p52 disrupts normal ductal development and provides insight into the mechanism by which this may contribute to human breast cancer.

An amino-terminal motif functions as a second nuclear export sequence in BRCA1.

Mutations in the breast cancer susceptibility gene 1 (BRCA1) account for a substantial percentage of familial breast and ovarian cancers. Although BRCA1 is thought to function within the nucleus, it has also been located in the cytoplasm. In addition, BRCA1 accumulates in the nucleus of cells treated with leptomycin B, an inhibitor of chromosome region maintenance 1-mediated nuclear export, indicative of its active nuclear export via this pathway. The nuclear export signal in BRCA1 has been described as consisting of amino acid residues 81-99. However, a number of other tumor suppressors have multiple nuclear export sequences, and we sought to determine whether BRCA1 did also. Here, we report that BRCA1 contains a second nuclear export sequence that comprises amino acid residues 22-30. By use of the human immunodeficiency virus-1 Rev complementation assay, this sequence was shown to confer export capability to an export-defective Rev fusion protein. The level of export activity was comparable with that of residues 81-99 comprising the previously reported nuclear export sequence in BRCA1. Mutation of leucine 28 to an alanine reduced nuclear export by approximately 75%. In MCF-7 cells stably transfected with a BRCA1 cDNA containing mutations in this novel sequence or the previously reported export sequence, BRCA1 accumulated in the nucleus. These data imply that BRCA1 contains at least two leucine-dependent nuclear export sequences.