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 G₂-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 α-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 G₂-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 G₂ phase of the cell cycle. [ABSTRACT FROM AUTHOR]