BRCA1 negatively regulates IGF-1 expression through an estrogen-responsive element-like site.

The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway is critical for both normal mammary gland development and malignant transformation. It has been reported that the IGF-1 stimulates breast cancer cell proliferation and is upregulated in tumors with BRCA1/2 mutations. We report here that IGF-1 is negatively regulated by BRCA1 at the transcriptional level in human breast cancer cells. BRCA1 knockdown (BRCA1-KD) induces the expression of IGF-1 mRNA in MCF7 cells in an estrogen receptor α (ERα)-dependent manner. We found that both BRCA1 and ERα bind to the endogenous IGF-1 promoter region containing an estrogen-responsive element-like (EREL) site. BRCA1-KD does not significantly affect ERα binding on the IGF-1 promoter. Reporter analysis demonstrates that BRCA1 could regulate IGF-1 transcripts via this EREL site. In addition, enzyme-linked immunosorbent assay revealed that de-repression of IGF-1 transcription by BRCA1-KD increases the level of extracellular IGF-1 protein, and secreted IGF-1 seems to increase the phospho-IGF-1Rß and activate its downstream signaling pathway. Blocking the IGF-1/IGF-1R/phosphoinositide 3-kinase (PI3K)/AKT pathway either by a neutralizing antibody or by small-molecule inhibitors preferentially reduces the proliferation of BRCA1-KD cells. Furthermore, the IGF-1-EREL-Luc reporter assay demonstrates that various inhibitors, which can inhibit the IGF-1R pathway, can suppress this reporter activity. These findings suggest that BRCA1 defectiveness keeps turning on IGF-1/PI3K/AKT signaling, which significantly contributes to increase cell survival and proliferation.

Hepatitis B virus: DNA polymerase activity of deletion mutants.

The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain.

RNase H activity of human hepatitis B virus polymerase expressed in Escherichia coli.

Human Hepatitis B Virus (HBV) replication is accomplished by its own polymerase. The HBV RNase H domain of HBV polymerase has been expressed in Escherichia coli and purified by affinity column chromatography. The MBP-RNase H fusion protein (43 kDa MBP plus 17 kDa HBV RNase H domain) was proved to be RNase H by in vitro activity assay, inhibitor studies, and mutagenesis. The HBV RNase H domain represented the optimal RNase H activity in the presence of either 8 mM MgCl2 or 16 mM MnCl2. In Tris-Cl buffer, the optimum pH for MBP-RNase H fusion protein is between 7.7 and 8.2. The MBP-RNase H fusion protein required 40 mM monovalent cation for its enzyme activity, whereas it showed lower activity at a salt concentration of more than 100 mM. Ribonucleoside Vanadyl complex (RAV) and 2'-deoxyadenosine 5'-monophosphate (dAMP) inhibited the RNase H activity. Moreover, the mutation of highly conserved amino acids in the HBV RNase H domain diminished the RNase H activity. These results clearly suggest that the RNase H activity is separable from viral HBV polymerase enzymatic activities.