ABSTRACT
<p><b>OBJECTIVE</b>To construct an ERbeta expression vector and study its expression and function in different cancer cells.</p><p><b>METHODS</b>Standard PCR was used to amplify the full-length coding sequence of ERbeta. The amplified ERbeta gene was cloned into the eukaryotic expression vector pCDNA3, generating pCDNA3-ERbeta. The ERbeta expression was detected by Western blot and in vitro translation. The biological activity of ERbeta was detected by transfecting the pCDNA3-ERbeta into SV40-transformed embryonic kidney cell line 293T,breast cancer cell lines MDA-MB-435, MDA-MB-436, SKBR3, and prostate cancer cell line PC-3, with reporters containing estrogen response elements.</p><p><b>RESULTS</b>The recombinant plasmid pCDNA3-ERbeta was confirmed by restriction analysis to contain the ERbeta gene. The 63 000 ERbeta expression was shown by Western blot and further confirmed by in vitro translation. The ERbeta expression in different cancer cells was demonstrated to stimulate the expression of the reporters containing estrogen response elements, ERE and C3.</p><p><b>CONCLUSION</b>ERbeta protein is successfully expressed and has biological activity, laying solid foundation for further study on its role in cancer cells.</p>
Subject(s)
Female , Humans , Male , Breast Neoplasms , Metabolism , Pathology , Cell Line , Cell Line, Tumor , Embryo, Mammalian , Epithelial Cells , Estrogen Receptor beta , Genetics , Metabolism , Genes, Reporter , Genetics , Genetic Vectors , Kidney , Cell Biology , Plasmids , Prostatic Neoplasms , Metabolism , Pathology , Recombinant Proteins , Genetics , Metabolism , Response Elements , Genetics , TransfectionABSTRACT
Breast cancer susceptibility gene 1(BRCA1) plays an important role in breast cancer development and progression. BRCA1 encodes a 1863-amino acid protein with two BRCA1 C-terminal (BRCT) domains at its C-terminus, BRCT1 and BRCT2. Many cancer-predisposing mutations are located in the BRCT domains, which have been shown to induce chromatin unfolding by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To map the important region of BRCT domain (amino acid residues 1642-1736), six deletion mutant constructs were made. The chromatin structure assay showed that amino acid residues 1691-1721 are involved in the induction of chromatin unfolding. To further localize the critical amino acid residues, ten alanine scanning mutant constructs were made. The chromatin structure assay demonstrated that the 1707IAGGK1711 region is critical for the chromatin unfolding activity. Based on the mapped important region, Blast analysis identified a novel homologous protein. Mapping of the BRCT1 domain may aid in the presymptomatic risk assessment and provide a valuable tool for further study on the BRCT1 structure and function.