High expression of the anti-apoptotic protein Bcl-2 in malignant cells has been associated with anti-cancer drug resistance. Our preliminary studies showed that taxol-induced apoptosis in human myeloid leukemia cells (HL6O) was preceded by bcl-2 mRNA destabilization. This suggests that factors which influence bcl-2 mRNA stability may be important determinants of cellular sensitivity to anti-cancer drugs. Preliminary data for this project suggests that in HL6O cells the down-regulation of bcl-2 expression induced by taxol and okadaic acid is controlled through post-transcriptional processes. The experiments proposed here will focus on elucidating the detailed molecular interactions involved in this process. Specifically, the proposed studies will test the hypothesis that apoptosis of HL6O cells induced by chemotherapeutic agents such as taxol is intimately related to accelerated bcl2 mRNA decay and that this mRNA destabilization involves specific regulatory sequences, AU-rich elements, (AREs), in bcl-2 mRNA. Accordingly, our objectives are to identify the sequences in bcl-2 mRNA that determines its stability, and to identify and characterize the trans-acting factor(s) that interacts with these sequences. To achieve these objectives, we have the following specific aims: 1) To define the AREs that regulate bcl-2 mRNA destabilization in response to chemotherapeutic agents; 2) To identify the ARE-binding protein (ARE-BP) that stabilizes bcl-2 mRNA and clone the gene coding for ARE-BP; 3) To measure ARE-BP protein and ARE RNA binding activity in taxol-treated HL6O cells to determine if taxol modulates ARE-BP function; and 4) To determine the abundance and activity of ARE-3P in chemoresistant cells and determine if synthetic oligoribonucleotides can competitively inhibit ARE-BP in vitro. Currently, little is known about post-transcriptional regulation of bcl-2 expression. The studies proposed in this application will address this deficiency in our knowledge and will provide important insights into the mechanisms by which anti-cancer drugs induce apoptosis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA087553-03
Application #
6758511
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Forry, Suzanne L
Project Start
2002-07-01
Project End
2006-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
3
Fiscal Year
2004
Total Cost
$208,050
Indirect Cost
Name
Medical University of South Carolina
Department
Biochemistry
Type
Schools of Medicine
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
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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
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Sengupta, Tapas K; Bandyopadhyay, Sumita; Fernandes, Daniel J et al. (2004) Identification of nucleolin as an AU-rich element binding protein involved in bcl-2 mRNA stabilization. J Biol Chem 279:10855-63
Bandyopadhyay, Sumita; Sengupta, Tapas K; Fernandes, Daniel J et al. (2003) Taxol- and okadaic acid-induced destabilization of bcl-2 mRNA is associated with decreased binding of proteins to a bcl-2 instability element. Biochem Pharmacol 66:1151-62