3174

Overexpression of the anti-apoptotic protein Bcl-2 is thought to allow cells that are genetically unstable to avoid apoptosis and become tumorigenic. In addition to its importance in cancer development, high bcl-2 expression in hematological tumors is frequently an obstacle to cancer chemotherapy, since bcl-2 overexpression has been shown to confer cellular resistance to a variety of anticancer drugs. In previous studies we have demonstrated that in HL60 leukemia cells and in chronic lymphocytic leukemia cells from patients, overexpression of the Bcl-2 proto-oncoprotein is the result, at least in part, of increased stability of bcl-2 mRNA. The 3'-untranslated region of bcl-2 mRNA contains an AU-rich element which appears to play a role in the regulation of bcl-2 mRNA half-life. The phospho-protein nucleolin has previously been identified as a trans-acting factor that interacts with the bcl-2 ARE and stabilizes bcl-2 mRNA in HL60 and CLL cells. In an effort to identify additional bcl-2 ARE binding factors, RNA supershift assays were performed with antibodies to known ARE binding factors. These assays identified the mRNA stabilizing factor HuR and the destabilizing factors TTP and AUF1 as potential bcl-2 ARE binding proteins in HL60 cytoplasmic cell extracts. Gel shift assays demonstrated that purified recombinant GST-HuR binds bcl-2 ARE RNA in the absence of other factors. To determine the functional significance of these interactions, we examined whether HuR binds to bcl-2 mRNA in vivo. HL60 cells were treated with formaldehyde to induce covalent protein-nucleic acid cross-links. Extracts of cross-linked cells were precipitated with anti-HuR antibody and precipitated protein was examined for the presence of bcl-2 mRNA by RT-PCR. Bcl-2 PCR products were observed in reactions containing anti-HuR antibody but not in samples precipitated with anti-IgG antibody. Immuno-precipitation assays also demonstrated that nucleolin is co-precipitated with HuR in HL60 extracts, suggesting the two proteins bind bcl-2 mRNA simultaneously. Additionally, we observed that taxol treatment of HL60 cells leads to proteolysis of cytoplasmic HuR, in a similar time frame as bcl-2 mRNA is downregulated. Collectively, these studies suggest that HuR plays a role in stabilization of bcl-2 mRNA, which contributes to the overexpression of Bcl-2 protein in human leukemia cells.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA