Translation inhibition during cell cycle arrest and apoptosis: Mc1-1 is a novel target for RNA binding protein CUGBP2

CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mc1-1, an antiapoptotic Bc1-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mc1-1 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.sub.2]-M phase. Western blot analyses demonstrated that there was decreased Bc1-2 and Mc1-1 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.sub.2]-M phase of the cell cycle. We next determined the mechanism of CUGBP2mediated reduction in Mc1-1 expression. Mc1-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mc1-1 3'-untranslated region (3'-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mc1-1 and luciferase mRNA containing the Mc1-1 3'-UTR. However, luciferase protein expression from the luciferase-Mc1-1 3'-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mc1-1 expression by inhibiting Mc1-1 mRNA translation, resulting in driving the cells to apoptosis during the [G.sub.2] phase of the cell cycle. Bc1-2 family member; [G.sub.2]-M arrest; checkpoint kinases; RNA stability