Role of HuR in the regulation of bcl‐2 mRNA stability in human HL60 leukemia cells

Abstract

High bcl‐2 expression in hematological tumors is frequently an obstacle to cancer chemotherapy. We have demonstrated that Bcl‐2 overexpression in some leukemia cells is a consequence of abnormal bcl‐2 mRNA stability. Previously, nucleolin was identified as a factor that stabilizes bcl‐2 mRNA in human HL60 leukemia cells. This interaction occurs via an AU‐rich element (ARE) in the 3’‐untranslated region of bcl‐2 mRNA. Our current study demonstrated that HuR also plays a role in the regulation of bcl‐2 mRNA stability in HL60 cells. Using EMSA we observed that HuR in HL60 cell extracts binds to the bcl‐2 mRNA ARE. Also, we found that such interactions occur in vivo. Taxol treatment induced proteolysis of cytoplasmic HuR, suggesting that modulation of HuR may play a role in the downregulation of bcl‐2 that precedes taxol‐induced apoptosis. Pull‐down assays indicate that nucleolin and HuR co‐immunoprecipitate in an RNA‐dependent manner, suggesting the two proteins are part of common bcl‐2 mRNP complexes. Moreover, nucleolin and HuR can bind independently and concurrently to bcl‐2 ARE RNA in vitro. This suggests HuR and nucleolin bind to separate, non‐overlapping sites on the bcl‐2 ARE. Sh‐RNA‐mediated knockdown of HuR in A431 epithelial cancer cells leads to reduced levels of bcl‐2 mRNA and protein. Taken together, our studies suggest that both HuR and nucleolin play a role in stabilization of bcl‐2 mRNA, which contributes to the overexpression of Bcl‐2 protein in some types of leukemia cells. Supported by NCI R01CA87553 (EKS) and Leukemia and Lymphoma Society Grant 6006‐06 (DJF).