Germline mutations in the BRCA1 gene are associated with an increased susceptibility to the development of breast and ovarian cancers. Evidence suggests that BRCA1 protein plays a key role in mediating DNA damage-induced checkpoint responses. Several studies have shown that ectopic expression of BRCA1 in human cells can trigger cellular responses similar to those induced by DNA damage, including G2/M cell cycle arrest and apoptosis. While the effects of ectopic BRCA1 expression on the G2/M transition and apoptosis have been extensively studied, the factors that dictate the balance between these two responses remain poorly understood. We have recently shown that ectopic expression of BRCA1 in MCF-7 human breast cancer cells resulted in activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and G2/M cell cycle arrest. Furthermore, inhibition of BRCA1-induced ERK1/2 activation using mitogen-activated protein kinase kinase 1 and 2 (MEK1/2)-specific inhibitors resulted in increased apoptosis, suggesting a potential role of ERK1/2 kinases in BRCA1-mediated G2/M checkpoint response. In this study, we assessed the role of ERK1/2 kinases in the regulation of BRCA1-mediated G2/M cell cycle arrest. Results indicate that BRCA1-induced G2/M cell cycle arrest and ERK1/2 activation correlate with changes in the level and/or activity of several key regulators of the G2/M checkpoint, including activation of Chk1 and Wee1 kinases, induction of 14-3-3, and down-regulation of Cdc25C. Furthermore, inhibition of ERK1/2 kinases using MEK1/2-specific inhibitors results in a marked attenuation of the BRCA1-induced G2/M arrest. Biochemical studies established that ERK1/2 inhibition abolished the effects of BRCA1 on components of the G2/M checkpoint, including regulation of Cdc25C expression and activation of Wee1 and Chk1 kinases. These results implicate a critical role of ERK1/2 signaling in the regulation of BRCA1 function on controlling the G2/M checkpoint responses.

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=1211 KEY WORDSBcra1-deficiency, DNA-damage and repair, breast cancer, CDDP, MMS ABBREVIATIONSMMECs murine mammary epithelial cells DAPI 4-6-diamidine-2-phenylidone dihydrochloride CDDP cis-platinum (II) diamine dichloride MTT 3-(4-5 dimethylthiozol-2-yl) 2-5diphenyl-tetrazolium bromide PBS phosphate-buffered saline MMS methylmethane sulfonate ACKNOWLEDGEMENTSWe like to thank Kevin Olson for technical assistance and Joerg Rahnenfuehrer (UC Berkeley) for statistical analysis. We also like to thank Dr. Charles A. Kuszynski and Linda M. Wilkie for the flow cytometry analysis. Report Brca1-Deficient Murine Mammary Epithelial Cells Have Increased Sensitivity to CDDP and MMS ABSTRACTIn this report we describe the isolation of an isogenic pair of Brca1 ++ and Brca1 -/-murine mammary epithelial cells (MMECs). These cells were isolated from Brca1 conditional knock-out mice which contained loxP sites flanking exon 11 of the Brca1 gene (Brca1 fl/f1 ) and then immortalized by infection with HPV-16E6 retrovirus to degrade p53 protein.Brca1 -/-MMECs were generated by deletion of exon 11 following transduction of Brca1 fl/f1 MMECs with a retroviral vector expressing Cre recombinase. Brca1-deficiency rendered MMECs sensitive to cis-platinum (II) diamine dichloride (CDDP) and methylmethane sulfonate (MMS). The Brca1 +/+ and Brca1 -/-MMECs is the only known pair of isogenic mammary epithelial cell lines. The understanding of the mechanisms of the CDDP sensitivity of the BRCA1-deficient mammary epithelial cells would be very important in understanding how BRCA1-deficiency plays a role in tissue specific breast cancer chemotherapy. These studies support the role of BRCA1 in the CDDP-induced and MMS-induced DNA damage and repair by p53-independent pathways.

No abstract