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Translation inhibition during cell cycle arrest and apoptosis: Mcl-1 is a novel target for RNA binding protein CUGBP2
Dharmalingam Subramaniam1, Gopalan Natarajan, Satish Ramalingam
1Dept. of Medicine, Univ. of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
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.
Insights
CUGBP2 expression triggers colon cancer cell death by inhibiting Mcl-1 translation, leading to apoptosis during the G2-M phase. This discovery offers new insights into colon cancer therapy.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- CUGBP2 (CUG triplet repeat, RNA-binding protein A) is a translation inhibitor that induces apoptosis in colon cancer cells.
- Mcl-1, an antiapoptotic protein, prevents apoptosis by interfering with mitochondrial activation.
- Understanding the interplay between CUGBP2 and Mcl-1 is crucial for developing targeted colon cancer therapies.
Purpose of the Study:
- To investigate the effect of CUGBP2 expression on Mcl-1 expression in colon cancer cells.
- To elucidate the mechanism by which CUGBP2 influences Mcl-1 levels and induces apoptosis.
- To determine the role of Mcl-1 translation inhibition in CUGBP2-mediated G2-M phase cell cycle arrest and apoptosis.
Main Methods:
- Development of a stable HCUG2 cell line expressing CUGBP2 in HCT-116 colon cancer cells.
- Flow cytometry and Western blot analyses to assess cell proliferation, apoptosis, cell cycle phase distribution, and protein/mRNA expression.
- Immunocytochemistry and TUNEL assays to evaluate protein localization and DNA damage.
- In vitro and cellular assays to determine CUGBP2 binding to Mcl-1 3'-UTR and its effect on mRNA stability and translation.
Main Results:
- HCUG2 cells exhibited decreased proliferation, increased apoptosis, and accumulation in the G2-M phase compared to HCT-116 cells.
- CUGBP2 expression led to reduced levels of Bcl-2 and Mcl-1 proteins, but increased Bax, cyclin B1, and Cdc2 expression.
- CUGBP2 directly binds to the Mcl-1 3'-UTR, inhibiting Mcl-1 mRNA translation without affecting its stability.
- Microtubule disorganization and nuclear accumulation of cyclin B1/Cdc2 were observed, correlating with apoptosis during the G2-M phase.
Conclusions:
- CUGBP2 expression induces apoptosis in colon cancer cells during the G2-M phase of the cell cycle.
- CUGBP2 inhibits Mcl-1 protein expression by suppressing its translation, mediated by binding to the Mcl-1 3'-UTR.
- These findings reveal a novel mechanism of CUGBP2-driven apoptosis via Mcl-1 translation inhibition, highlighting CUGBP2 as a potential therapeutic target in colon cancer.
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