1. Translation inhibition during cell cycle arrest and apoptosis: Mcl-1 is a novel target for RNA binding protein CUGBP2.
- Author
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Subramaniam D, Natarajan G, Ramalingam S, Ramachandran I, May R, Queimado L, Houchen CW, and Anant S
- Subjects
- 3' Untranslated Regions, Blotting, Western, CDC2 Protein Kinase, CELF Proteins, Cell Proliferation, Colonic Neoplasms enzymology, Colonic Neoplasms genetics, Colonic Neoplasms pathology, Cyclin B metabolism, Cyclin B1, Cyclin-Dependent Kinases, Flow Cytometry, Genes, Reporter, HCT116 Cells, Histones metabolism, Humans, Immunohistochemistry, Mitosis, Myeloid Cell Leukemia Sequence 1 Protein, Neoplasm Proteins genetics, Nerve Tissue Proteins genetics, Phosphorylation, Protein Binding, Proto-Oncogene Proteins c-bcl-2 genetics, RNA Processing, Post-Transcriptional, RNA Stability, RNA, Messenger metabolism, RNA-Binding Proteins genetics, Transfection, Tubulin metabolism, bcl-2-Associated X Protein metabolism, Apoptosis genetics, Cell Cycle genetics, Colonic Neoplasms metabolism, Gene Expression Regulation, Neoplastic, Neoplasm Proteins metabolism, Nerve Tissue Proteins metabolism, Protein Biosynthesis, Proto-Oncogene Proteins c-bcl-2 metabolism, RNA-Binding Proteins metabolism
- Abstract
CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mcl-1, an antiapoptotic Bcl-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mcl-1 expression. We developed a HCUG2 cell line by stably expressing CUGBP2 in the HCT-116 colon cancer cells. HCUG2 cells demonstrate decreased levels of proliferation and increased apoptosis, compared with HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in the G(2)-M phase. Western blot analyses demonstrated that there was decreased Bcl-2 and Mcl-1 protein but increased expression of Bax, cyclin B1, and Cdc2. Immunocytochemistry also demonstrated increased levels of cyclin B1 and Cdc2 in the nucleus of HCUG2 cells. However, there was colocalization of phosphorylated histone H3 with transferase-mediated dUTP nick-end labeling (TUNEL). Furthermore, immunostaining for alpha-tubulin demonstrated that there was disorganization of microtubules. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during the G(2)-M phase of the cell cycle. We next determined the mechanism of CUGBP2-mediated reduction in Mcl-1 expression. Mcl-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mcl-1 3'-untranslated region (3'-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mcl-1 and luciferase mRNA containing the Mcl-1 3'-UTR. However, luciferase protein expression from the luciferase-Mcl-1 3'-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mcl-1 expression by inhibiting Mcl-1 mRNA translation, resulting in driving the cells to apoptosis during the G(2) phase of the cell cycle.
- Published
- 2008
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