AB3579 Sigma-AldrichAnti-BimEL (pS69) human / (pS65) rat Antibody

While glucocorticoids and the liganded glucocorticoid receptor (GR) have a well-established role in the maintenance of differentiation and suppression of apoptosis in breast tissue, the involvement of unliganded GR in cellular processes is less clear. Our previous studies implicated unliganded GR as a positive regulator of the BRCA1 tumour suppressor gene in the absence of glucocorticoid hormone, which suggested it could play a similar role in the regulation of other genes.An shRNA vector directed against GR was used to create mouse mammary cell lines with depleted endogenous levels of this receptor in order to further characterize the role of GR in breast cells. An expression microarray screen for targets of unliganded GR was performed using our GR-depleted cell lines maintained in the absence of glucocorticoids. Candidate genes positively regulated by unliganded GR were identified, classified by Gene Ontology and Ingenuity Pathway Analysis, and validated using quantitative real-time reverse transcriptase PCR. Chromatin immunoprecipitation and dual luciferase expression assays were conducted to further investigate the mechanism through which unliganded GR regulates these genes.Expression microarray analysis revealed 260 targets negatively regulated and 343 targets positively regulated by unliganded GR. A number of the positively regulated targets were involved in pro-apoptotic networks, possibly opposing the activity of liganded GR targets. Validation and further analysis of five candidates from the microarray indicated that two of these, Hsd11b1 and Ch25h, were regulated by unliganded GR in a manner similar to Brca1 during glucocorticoid treatment. Furthermore, GR was shown to interact directly with and upregulate the Ch25h promoter in the absence, but not the presence, of hydrocortisone (HC), confirming our previously described model of gene regulation by unliganded GR.This work presents the first identification of targets of unliganded GR. We propose that the balance between targets of liganded and unliganded GR signaling is responsible for controlling differentiation and apoptosis, respectively, and suggest that gene regulation by unliganded GR may represent a mechanism for reducing the risk of breast tumourigenesis by the elimination of abnormal cells.

The ID family of helix-loop-helix proteins regulates cell proliferation and differentiation in many different developmental pathways, but the functions of ID4 in mammary development are unknown. We report that mouse Id4 is expressed in cap cells, basal cells and in a subset of luminal epithelial cells, and that its targeted deletion impairs ductal expansion and branching morphogenesis as well as cell proliferation induced by estrogen and/or progesterone. We discover that p38MAPK is activated in Id4-null mammary cells. p38MAPK is also activated following siRNA-mediated Id4 knockdown in transformed mammary cells. This p38MAPK activation is required for the reduced proliferation and increased apoptosis in Id4-ablated mammary glands. Therefore, ID4 promotes mammary gland development by suppressing p38MAPK activity.

Bryostatin-1 and related diacylglycerol (DAG) analogues activate RasGRPs in lymphocytes, thereby activating Ras and mimicking some aspects of immune receptor signaling. To define the role of RasGRPs in lymphocyte apoptosis and to identify potential therapeutic uses for DAG analogues in lymphocyte disorders, we characterized the response of lymphoma-derived cell lines to DAG analogues.

We have previously demonstrated a 1000-fold induction of gene expression exclusive to Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) cells using an adenoviral vector (ad5.oriP). This platform allows us to explore tumor-specific gene therapy with BimS (ad5.oriP.BimS), a potent proapoptotic Bcl-2 family member. Ad5.oriP.BimS (25 infectious units (ifu)/cell) reduced C666-1 viability in a time- and dose-dependent manner to 15% survival. The effect was enhanced with radiation (6 Gy). Three days after infection, the proportion of apoptotic cells increased from 3.5% (control) to 47.5% (25 ifu/cell). Confocal microscopy demonstrated Bim colocalization to the mitochondria within 18 h of ad5.oriP.BimS infection. Ad5.oriP.BimS induced a 2.8-, 2.1-, and 1.85-fold increase in caspase-3, -8, and -9 activities, respectively. When C666-1 cells were infected with ad5.oriP.BimS (20 ifu/cell), no tumors formed in 7/9 mice followed for 100 days. Six intratumoral injections of ad5.oriP.BimS (1.5 x 10(9) ifu/dose) in combination with radiation were sufficient to cause almost complete disappearance of established C666-1 or C15 xenograft tumors. Intravenous injections of ad5.oriP.BimS (10(9) ifu) induced mild perturbation in liver function tests, associated with hepatocyte apoptoses and mitoses. This vector appears to be safe and effectively cytotoxic to EBV-positive NPC cells both in vitro and in vivo, mediated primarily through the induction of apoptosis.