Over expression of bcl-2 is an important component in the development of B-cell lymphomas and some B-cell leukemia as well as in the emergence of cellular resistance to certain anticancer drugs. The 3'-untranslated region (3'-UTR) of bcl-2 mRNA contains several AU-rich elements (AREs) that promote mRNA destabilization. We have observed that the protein, nucleolin, binds to ARE-1 in bcl-2 mRNA, potentially protecting this mRNA from nuclease degradation. Accordingly, the central hypothesis of this proposal is that nucleolin stabilizes bcl-2 mRNA through interaction with AREs in the 3'-UTR, and that ATRA, taxol and okadiac acid destabilize bcl-2 mRNA by inducing down regulation or inactivation of nucleolin. The studies proposed in the first specific aim will evaluate the effects of bcl-2 ARE's 1-4 on bcl-2 mRNA stability in extracts of HL-60, MCF-7 and purified B cells from normal volunteers and patients with B-cell chronic lymphocytic leukemia (CLL). This will be followed by testing the effects of taxol and ATRA on bcl-2 ARE mRNA stability in cell extracts.
Aim 2 is to determine whether functional nucleolin-bcl-2 mRNA interactions occur in intact cells. The ability of recombinant nucleolin to stabilize the bcl-2 mRNAs containing destabilizing AREs (Specific Aim 1) will be tested using transfection assays. We will also determine if exogenous over expression of nucleolin in parental HL-60 cells or MCF-7 cells can confer resistance to ATRA (HL-60) or taxol (HL-60 and MCF-7) concomitant with increased bcl-2 mRNA and protein. Additional experiments will reveal if knockdown of nucleolin expression with siRNAs leads to destabilization of bcl-2 mRNA. The third specific aim is to identify the RNA binding domains of nucleolin that are involved in binding to bcl-2 ARE mRNA. Finally, the information generated in Aims 1-3 will guide the development of high-throughput, robotic screening of diverse chemical libraries to identify small molecules that specifically inhibit nucleolin binding to AREs in bcl-2 mRNA.
| Ishimaru, Daniella; Zuraw, Lisa; Ramalingam, Sivakumar et al. (2010) Mechanism of regulation of bcl-2 mRNA by nucleolin and A+U-rich element-binding factor 1 (AUF1). J Biol Chem 285:27182-91 |
| Gattoni-Celli, Sebastiano; Buckner, Carl L; Lazarchick, John et al. (2009) Overexpression of nucleolin in engrafted acute myelogenous leukemia cells. Am J Hematol 84:535-8 |
| Ishimaru, Daniella; Ramalingam, Sivakumar; Sengupta, Tapas K et al. (2009) Regulation of Bcl-2 expression by HuR in HL60 leukemia cells and A431 carcinoma cells. Mol Cancer Res 7:1354-66 |
| Soundararajan, Sridharan; Wang, Li; Sridharan, Vijayalakshmi et al. (2009) Plasma membrane nucleolin is a receptor for the anticancer aptamer AS1411 in MV4-11 leukemia cells. Mol Pharmacol 76:984-91 |
| Soundararajan, Sridharan; Chen, Weiwei; Spicer, Eleanor K et al. (2008) The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells. Cancer Res 68:2358-65 |
| Otake, Yoko; Soundararajan, Sridharan; Sengupta, Tapas K et al. (2007) Overexpression of nucleolin in chronic lymphocytic leukemia cells induces stabilization of bcl2 mRNA. Blood 109:3069-75 |
