Submitted on December 24, 2007
Accepted on February 20, 2008
Translation inhibition during cell cycle arrest and apoptosis: Mcl-1 is a novel target for RNA binding protein CUGBP2
Dharmalingam Subramaniam1, Gopalan Natarajan2, Satish Ramalingam3, Ilangovan Ramachandran4, Randal May3, Lurdes Queimado5, Courtney W Houchen3, and Shrikant Anant6*
1 Oklahoma City, Oklahoma, United States; Medicine/Section of Digestive Diseases and Nutrition, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
2 Medicine/Section of Digestive Diseases and Nutrition, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States; Oklahoma City, Oklahoma, United States
3 Medicine/Section of Digestive Diseases and Nutrition, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
4 Otorhinolaryngology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
5 Cell Biology, University of Oklahoma Health Sciences Center, United States; Otorhinolaryngology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
6 Medicine/Section of Digestive Diseases and Nutrition, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States; Cell Biology, University of Oklahoma Health Sciences Center, United States
* To whom correspondence should be addressed. E-mail: shrikant-anant{at}ouhsc.edu.
CUGBP2, a translation inhibitor induces colon cancer cells to undergo apoptosis. Mcl-1, a 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 levels of apoptosis, when compared to HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in G2-M phase. Western blot analyses demonstrated decreased levels of Bcl2 and Mcl-1 but increased levels of Bax, cyclin B1 and Cdc2 proteins. Immunocytochemistry also demonstrated increased nuclear levels of cyclin B1 and Cdc2 in HCUG2 cells. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during G2-M phase of cell cycle. We next determined the mechanism of CUGBP2-mediated reduction in Mcl-1 expression. Although Mcl-1 protein was lower in HCUG2 cells, Mcl-1 mRNA levels were increased, suggesting translation inhibition. RNA binding studies demonstrated that CUGBP2 binds to Mcl-1 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, the half-life increasing from 30 min in HCT-116 cells to 3 h in HCUG2 cells. 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 G2 phase of cell cycle.
