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.