Translation inhibition during cell cycle arrest and apoptosis: Mcl-1 is a novel target for RNA binding protein CUGBP2.
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.
Subramaniam
Dharmalingam
D
Dept. of Medicine, Univ. of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Natarajan
Gopalan
G
Ramalingam
Satish
S
Ramachandran
Ilangovan
I
May
Randal
R
Queimado
Lurdes
L
Houchen
Courtney W
CW
Anant
Shrikant
S
D020413:3' Untranslated Regions; D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D016203:CDC2 Protein Kinase; D000067878:CELF Proteins; D002453:Cell Cycle / Q000235:genetics; D049109:Cell Proliferation; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D018844:Cyclin-Dependent Kinases; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D045325:HCT116 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008938:Mitosis; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D020413:3' Untranslated Regions; C494826:BAX protein, human; C534699:CCNB1 protein, human; D000067878:CELF Proteins; C117363:CELF2 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D006657:Histones; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014404:Tubulin; D051028:bcl-2-Associated X Protein; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases