The breast cancer tumor-suppressor gene, BRCA1, encodes a protein with a BRCT domain-a motif that is found in many proteins that are implicated in DNA damage response and in genome stability. Phosphorylation of BRCA1 by the DNA damage-response proteins ATM, ATR and hCds1/Chk2 changes in response to DNA damage and at replication-block checkpoints. Although cells that lack BRCA1 have an abnormal response to DNA damage, the exact role of BRCA1 in this process has remained unclear. Here we show that BRCA1 is essential for activating the Chk1 kinase that regulates DNA damage-induced G2/M arrest. Thus, BRCA1 controls the expression, phosphorylation and cellular localization of Cdc25C and Cdc2/cyclin B kinase-proteins that are crucial for the G2/M transition. We show that BRCA1 regulates the expression of both Wee1 kinase, an inhibitor of Cdc2/cyclin B kinase, and the 14-3-3 family of proteins that sequesters phosphorylated Cdc25C and Cdc2/cyclin B kinase in the cytoplasm. We conclude that BRCA1 regulates key effectors that control the G2/M checkpoint and is therefore involved in regulating the onset of mitosis.

The breast and ovarian cancer susceptibility gene product BRCA1 has been reported to be expressed in a cell cycle-dependent manner; possess transcriptional activity; associate with several proteins, including the p53 tumor suppressor; and play an integral role in certain types of DNA repair. We show here that ectopic expression of BRCA1 using an adenovirus vector (Ad-BRCA1) leads to dephosphorylation of the retinoblastoma protein accompanied by a decrease in cyclin-dependent kinase activity. Flow cytometric analysis on Ad-BRCA1-infected cells revealed a G 1 or G 2 phase accumulation. High density cDNA array screening of colon, lung, and breast cancer cells identified several genes affected by BRCA1 expression in a p53-independent manner, including DNA damage response genes and genes involved in cell cycle control. Notable changes included induction of the GADD45 and GADD153 genes and a reduction in cyclin B1 expression. Therefore, BRCA1 has the potential to modulate the expression of genes and function of proteins involved in cell cycle control and DNA damage response pathways.

The hereditary breast and ovarian tumor suppressor BRCA1 can activate p53-dependent gene expression. We show here that BRCA1 increases p53 protein levels through a post-transcriptional mechanism. BRCA1-stabilized p53 has increased sequence-speci®c DNAbinding and transcriptional activity. BRCA1 does not stabilize p53 in p14 ARF -de®cient cells. A deletion mutant of BRCA1 which inhibits p53-dependent transcription confers resistance to topoisomerase II-targeted chemotherapy. Our results suggest that BRCA1 may trigger the p53 pathway through two potentially separate mechanisms: accumulation of p53 through a direct or indirect induction of p14 ARF as well as direct transcriptional coactivation of p53. BRCA1 may also enhance chemosensitivity and repair of DNA damage through binding to and coactivation of p53.

Cell proliferation, differentiation, and survival are highly coordinated processes during the development and maturation of the mammary gland, and control of these mechanisms is critical for the prevention of breast cancers (reviewed in reference 39). Aberrant regulation of the HER/ErbB family of receptor tyrosine kinases (RTKs) and their ligands is a common occurrence in many human cancers, including breast cancer (14,15,45). This family consists of four related members, HER1/ErbB1/EGFR (epidermal growth factor receptor), HER2/ErbB2/Neu, HER3/ErbB3, and HER4/ErbB4. Each protein is comprised of a large amino-terminal extracellular domain, a transmembrane domain, and a large intracellular domain with a tyrosine-rich carboxy-terminal region and a tyrosine kinase-like sequence (27,33,56). The tyrosine kinase activity of the ErbBs is induced upon ligand interaction, leading to receptor dimerization (homo-and heterodimerization) and subsequent receptor transphosphorylation. Although HER2 is the preferential heterodimeric partner, HER2 does not bind any conventional ligand within the two major families of ErbB ligands (EGF-like ligands and heregulin [HRG]/neuregulin-like ligands) and therefore relies on HER1, HER3, or HER4 for activation of its tyrosine kinase activity.The well-documented growth stimulatory effects of HER1 and HER2 have driven the investigation of ErbB signaling in breast cancer. HER1 is expressed in nearly all human carcinomas, including breast cancers, while nearly 20 to 25% of breast cancers overexpress HER2 and/or exhibit gene amplification at the her2 locus (26,37,40,48). Expression of either HER1 or HER2 in tumor specimens correlates with a shorter survival period, a higher grade of malignancy, and an overall poor prognosis (10, 12-14, 19, 24). Genetically engineered animal models of breast cancer confirm the role of HER1 and HER2 in driving proliferation of the mammary epithelium (reviewed in reference 39). Recent evidence suggests that increased expression of HER3 in breast cancers also correlates with a poor prognosis. HER3 is overexpressed in about 20% of all breast cancers and is frequently coexpressed with HER2 (2,5,10,23,31,52,53). This has generated the hypothesis that HER2/ HER3 heterodimers may function to simultaneously drive cellular proliferation and survival in breast cancer cells.In contrast, there is evidence that HER4 expression correlates with a more differentiated tumor grade, longer survival, and positive prognostic indicators, such as estrogen receptor expression (1,17,29,38,41,44). Women whose breast tumors express HER4 exhibit the lowest risk of death due to cancer compared to women whose tumors express HER1, HER2, or HER3. During breast development, HER4 expression and activity (measured by tyrosine phosphorylation) are lowest during phases of epithelial cell proliferation and highest during phases of differentiation (35). Mammary glands from mice that lack HER4 activity, either by Cre-Lox technology, cardiac-specific transgene rescue of a HER4 knockout (with the mammary ...

Tumor-infiltrating lymphocytes (TIL) were derived from primary breast tumors, metastatic lymph nodes and malignant pleural effusions from 34 patients with breast cancer. TIL were cultured for approximately 30 days and studied for phenotype, cytotoxicity, and the ability to secrete cytokines in response to autologous tumor stimulation. Tumor specimens were obtained from two different sites in 7 patients, resulting in 41 samples from which 38 TIL cultures were established. In addition to screening 38 bulk TIL cultures, TIL from 21 patients were separated into CD4+ and CD8+ subsets and extensively studied. Three CD4+ TIL were found specifically to secrete granulocyte macrophage-colony-stimulating factor and tumor necrosis factor alpha when stimulated by autologous tumor and not by a large panel of stimulators (24-34) consisting of autologous normal cells, allogeneic breast or melanoma tumors and EBV-B cells. This cytokine release was found to be MHC-class-II-restricted, as it was inhibited by the anti-HLA-DR antibody L243. These 3 patients' EBV-B cells, when pulsed with tumor lysates, were unable to act as antigen-presenting cells and induce cytokine secretion by their respective CD4+ TIL. These findings demonstrate that MHC-class-II-restricted CD4+ T cells recognising tumor-associated antigens can be detected in some breast cancer patients.

Breast cancer remains the most common cancer affecting women in the Western world. Although most breast cancers are sporadic, ϳ10% are inheritable and associated with mutations in at least two loci, BRCA1 and BRCA2. The BRCA1 gene is located at position 17q21 of the human genome, and mutations in this gene are associated with an increased risk of development of breast and ovarian cancer (1). The BRCA1 gene encodes a protein of 1864 amino acid residues that is primarily located in the nucleus (2). The BRCA1 gene product contains several domains that may effect its interaction with many other cellular proteins. The N terminus includes a domain presumably involved in the formation of homodimers and heterodimers (with the BARD1 protein) and a ring finger domain that is involved in interaction with BAP1 and E2F-1; the middle portion of BRCA1 contains a nuclear localization signal and domains that can bind to c-Myc, p53, pRB, and the DNA repair proteins RAD50 and RAD51; the C terminus includes two BRCT domains that interact with multiple factors involved in transcriptional regulation, including CtIP, p300, p53, pRB, CBP, BRCA2, RNA polymerase II, and RNA helicase A (3, 4). Recent studies suggest that the BRCA1 protein may be involved in regulating numerous cellular functions including DNA damage repair, gene transcription, chromosome segregation, cell cycle arrest, and apoptosis (4).Several studies suggest a role for BRCA1 in DNA repair. First, BRCA1 interacts with RAD51, a human homologue of the yeast RecA protein involved in double-stranded DNA break repair (5, 6). In vitro, BRCA1 can associate with the RAD50-MRE11-NBS1 complex, a functional unit implicated in homologous recombination, non-homologous end joining, and meiotic recombination. In irradiated cells, BRCA1 is recruited to this complex, where it likely plays a role in DNA repair (7). Moreover, ectopic expression of BRCA1 decreases cellular sensitivity to radiation and increases the efficiency of DNA break repair (8). Second, BRCA1 interacts with proteins involved in mismatch repair, mainly MSH2, MSH3, and MSH6. Furthermore, the association of BRCA1 with MSH2 and MSH6 was found to be essential for transcription-coupled DNA repair (9). A recent study by Wang et al. (9) provides evidence for the existence of a large BRCA1-containing complex that incorporates factors involved in various types of DNA repair. The identified complex was found to contain BRCA1 and the DNA repair factors MSH2, MSH6, MLH1, ATM, BLM, RAD50, MRE11, NBS1, and replication factor C (9). These results suggest that BRCA1 might provide a scaffold that functions in coordinating multiple activities required for the maintenance of genomic integrity and the fidelity of DNA replication (9).Besides DNA repair function, numerous studies also suggest that BRCA1 might play an important role in transcriptional regulation. BRCA1 physically interacts with key enzymes involved in transcription, mainly RNA polymerase II and RNA helicase A, suggesting that it might be a component of the transcriptional ma...

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.

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