Expression of the breast cancer susceptibility gene, BRCA1, is induced by 17-b estradiol (E 2 ) in estrogen receptor containing breast cancer cell lines. Our previous studies have shown that BRCA1 transcription is also regulated with the cell cycle, reaching maximal levels just before the onset of DNA synthesis. In this study, we have examined whether the estrogen induction of BRCA1 is direct or is a result of the mitogenic activity of the hormone. Four lines of evidence lead us to conclude that E 2 induces BRCA1 primarily through an increase in DNA synthesis: (1) The kinetics and magnitude of induction are di erent from the directly E 2 inducible gene, pS2; (2) Induction of BRCA1, but not pS2, is blocked by cycloheximide indicating that de novo protein synthesis is required; (3) Other hormonal and growth factor treatments that induce DNA synthesis have a similar e ect, including IGF-1, EGF and DNA synthetic ares induced by tamoxifen and retinoic acid; (4) BRCA1 genomic fragments near the 5' end of the gene containing putative estrogen response elements fail to respond to E 2 when transfected into breast cancer cell lines. The most consistent explanation for these ®ndings and other published studies is that BRCA1 transcription is induced as a result of the mitogenic activity of E 2 in estrogen receptor positive cells.

The hereditary breast cancer susceptibility gene, BRCA2, is considered to be a tumor suppressor gene that may be involved in the cellular response to DNA damage. The transcript for this gene is cell cycle regulated with mRNA levels reaching a peak just before the onset of DNA synthesis. In order to de®ne the mechanisms by which BRCA2 is transcriptionally regulated, we have begun to study upstream regulatory sequences. In this report, we de®ne a minimal promoter region that has strong activity in human breast epithelial cells. Deletions of this sequence narrowed the strong basal activity to a region extending from 766 to +129 with respect to the BRCA2 transcriptional start site. This sequence demonstrated cell cycle regulated activity with kinetics similar to the endogenous transcript. Examination of the sequence revealed several consensus binding sites for transcription factors including an E-box, E2F and Ets recognition motifs. Electrohoretic mobility shift assays revealed speci®c protein binding to two sequences upstream of the start site; the palindromic Ebox and an Ets/E2F site. Site-directed mutagenesis of either of these sites reduced both the basal activity in log phase cells and the cell cycle regulated activity of the promoter. Mutational inactivation of both sites within the same construct eectively eliminated promoter activity. Antibodies to candidate transcription factors used in super shift experiments revealed speci®c interactions between the BRCA2 promoter and the basic region/helix ± loop ± helix containing USF-1 and 2 proteins and Elf-1, an Ets domain protein. Binding of these factors depended upon the presence of intact recognition sequences. The USF factors were shown to bind predominantly as a heterodimeric complex of USF-1 and 2 while Elf-1 bound the promoter when it was not occupied by USF. Co-transfection studies with USF proteins and the varicella zoster IE62 protein provide evidence for the involvement of endogenous and exogenous USF in the activation of the BRCA2 promoter. We propose that interactions between USF-1, USF-2 and Elf-1 play an important role in the transcriptional regulation of the BRCA2 gene.

A causal role has been inferred for ERBB2 overexpression in the etiology of breast cancer and other epithelial malignancies. The development of therapeutics that inhibit this tyrosine kinase cell surface receptor remains a high priority. This report describes the specific downregulation of ERBB2 protein and mRNA in the breast cancer cell line SK-BR-3 by using antisense DNA phosphorothioates. An approach was developed to examine antisense effects which allows simultaneous measurements ofantisense dose and gene specific regulation on a per cell basis. A fluorescein isothiocyanate end-labeled tracer oligonucleotide was codelivered with antisense DNA followed by immunofluorescent staining for ERBB2 protein expression. Two-color flow cytometry measured the amount of both intracellular oligonucleotide and ERBB2 protein. In addition, populations of cells that received various doses of nucleic acids were physically separated and studied. In any given transfection, a 100-fold variation in oligonucleotide dosage was found. ERBB2 protein expression was reduced greater than 50%1, but only in cells within a relatively narrow uptake range. Steady-state ERBB2 mRNA levels were selectively diminished, indicating a specific antisense-effect. Cells receiving the optimal antisense dose were sorted and analyzed for cell cycle changes. After 2 days of ERBB2 suppression, breast cancer cells showed an accumulation in the G1 phase of the cell cycle.Overexpression of the ERBB2 tyrosine kinase receptor is a common feature of human breast cancer (1-3). High level expression is associated with increased transcription, frequently due to gene amplification (4, 5). Both ERBB2 gene amplification and high level expression appear to be early events in the progression of breast cancer, and these properties are maintained during metastatic spread of the disease (6, 7). The oncogenic capacity of ERBB2 can be directly demonstrated by introducing high-level expression vectors into nontransformed fibroblasts, mammary epithelial cells, and transgenic mice, which results in morphologic and growth changes characteristic of neoplasia (8-10). In addition, ERBB2 has a relatively restricted pattern of expression in normal adult tissues (11). These properties make the ERBB2 gene and protein product appealing targets for immune and gene-based therapies for breast cancer.Decreased expression of ERBB2 has been achieved by using monoclonal antibodies specific for the extracellular domain of the protein (12)(13)(14). This has resulted in growth inhibition and, in some cases, differentiation of breast cancer cell lines. Antibodies have been shown to decrease cell surface expression of the ERBB2 receptor via internalization (15). These antibodies may also be acting as receptor agonists, mimicking ligand binding. Ligands for ERBB3 and ERBB4 have similar differentiating effects on breast epithelial cell lines (16)(17)(18)(19)(20).Antibody treatment can be equally effective on cell lines with both normal and high level ERBB2 expression (14), further obscuring th...