ABSTRACT
The antiapoptotic BCL-2 family protein BCL-W is often overexpressed in colorectal carcinoma (CRC) where it correlates with advanced stage and expression of p53. In this work we have analysed the Bcl-w promoter to identify potential regulators of BCL-W expression in CRC cells. The Bcl-w promoter was highly active in cell lines derived from CRC as well as other cancer types. Although expression of p53 and BCL-W correlate in CRC, overexpression of wild type or mutant p53 did not significantly alter Bcl-w promoter activity, and deletion of endogenous p53 did not alter the expression of Bcl-w RNA in HCT116 cells. Promoter deletion analysis lead to the identification of a potential binding site for TCF/LEF factors, obligate binding partners for beta-catenin, a downstream target of the WNT signalling pathway. TCF4 and beta-catenin interacted with the Bcl-w promoter in intact HCT116 cells and mutation of this site significantly decreased promoter activity. The activity of the Bcl-w promoter was increased or decreased, respectively, by overexpression of beta-catenin or dominant negative TCF4. beta-catenin is activated in the majority of CRC and these results suggest that BCL-W may function as a downstream effector of inappropriate WNT/beta-catenin signalling.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Colorectal Neoplasms/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic , Promoter Regions, Genetic , Transcription Factors/metabolism , beta Catenin/metabolism , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Binding Sites , Cell Line, Tumor , Chromatin Immunoprecipitation , Cloning, Molecular , Computational Biology , Humans , Protein Binding , RNA, Neoplasm/metabolism , Sequence Deletion , Transcription Factor 4 , Tumor Suppressor Protein p53/metabolismABSTRACT
Cobalt promotes apoptosis in multiple cell systems, however, the molecular mechanisms that influence cobalt-induced apoptosis are not fully understood. We investigated mechanisms of cobalt chloride induced apoptosis in HCT116 colorectal cancer cells. Cobalt chloride induced dose dependent apoptosis in HCT116 cells (250-750 muM) which, at higher concentrations (500-750 muM), was associated with an increase in the expression of the Bcl-2-related Mcl-1 survival protein. Cobalt chloride caused the accumulation of higher molecular weight ubiquitin-conjugates of Mcl-1 in intact HCT116 cells and inhibited the activity of the trypsin-like site of the 20S proteasome in an in vitro assay. Although siRNA-mediated knockdown of Mcl-1 increased apoptosis in HCT116 cells, the combination of Mcl-1 siRNA and cobalt chloride induced very high levels of cell killing. Therefore, inhibition of the proteasome by cobalt chloride leads to the accumulation of Mcl-1 which acts to limit cobalt chloride induced apoptosis.
Subject(s)
Apoptosis/physiology , Carcinoma/metabolism , Cobalt/pharmacology , Colorectal Neoplasms/metabolism , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Antimutagenic Agents/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/drug effects , Apoptosis Regulatory Proteins/metabolism , Binding Sites/drug effects , Binding Sites/physiology , Carcinoma/drug therapy , Carcinoma/physiopathology , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/physiopathology , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Down-Regulation/genetics , Feedback, Physiological/drug effects , Feedback, Physiological/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Proteasome Endopeptidase Complex/drug effects , Proto-Oncogene Proteins c-bcl-2/drug effects , Proto-Oncogene Proteins c-bcl-2/genetics , RNA Interference/physiology , RNA, Small Interfering , Ubiquitination/drug effects , Ubiquitination/physiologyABSTRACT
Murine Nramp1 encodes a divalent cation transporter that is expressed in late endosomes/lysosomes of macrophages, and the transported cations facilitate intracellular pathogen growth control. The Nramp1 promoter is TATA box-deficient, has two initiator elements, and is repressed by c-Myc, in accordance with the notion that genes that deplete the iron content of the cell cytosol antagonize cell growth. Repression via c-Myc occurs at the initiator elements, whereas a c-Myc-interacting protein (Miz-1) stimulates transcription. Here we demonstrate that a non-canonical E box (CAACTG) inhibits basal promoter activity and activation by Miz-1. A consensus Sp1-binding site or GC box is also necessary for Miz-1-dependent transactivation, but not repression. Repression occurs by c-Myc competing with p300/CBP for binding Miz-1. Our results show that an Sp1 site mutant inhibits coactivation by p300 and that the murine Nramp1 promoter is preferentially expressed within macrophages (relative to a beta-actin control) compared with non-macrophage cells. The effect of the Sp1 site mutation on promoter function shows cell-type specificity: stimulation in COS-1 and inhibition in RAW264.7 cells. Miz-1-directed RNA interference confirms a stimulatory role for Miz-1 in Nramp1 promoter function. c-Myc, Miz-1, and Sp1 were identified as binding to the Nramp1 core promoter in control cells and following acute stimulation with interferon-gamma and lipopolysaccharide. These results provide a description of sites that modulate the activity of the initiator-binding protein Miz-1 and indicate a stimulatory role for GC box-binding factors in macrophages and a inhibitory role for E box elements in proliferating cells.
