Abstract #3257: Identification of interaction of anti-apoptotic protein Bcl-2 with the small GTPase Rac1: A possible BH3 domain-mediated binding and regulation at the mitochondria

We previously reported that over-expression of Bcl-2 in chronic myeloid leukemia cells resulted in a slightly elevated constitutive levels of superoxide (O2.-) anion conferring resistance to chemotherapy1. The major goal of this project was to understand the molecular basis for Bcl-2 mediated pro-oxidant state using leukemia and lymphoma cells as models. To that end, we investigated the effect of gene silencing as well as functional inhibition of the small GTPase on Bcl-2-induced pro-oxidant state. Results show that silencing Rac1 or inhibition of its activity upon transfection with a dominant negative form of Rac (RacN17) decreases intra-mitochondrial O2.- levels in Bcl-2 over-expressing cells, hence identifying Rac1 as an important functional partner of Bcl-2 in maintaining an oxidative milieu in tumor cells. Furthermore, using co-immunoprecipitation (co-IP), confocal microscopic analysis and GST-fusion proteins (Rac1 and Bcl-2), we identified an interaction between Bcl-2 (but not Bax or BclxL) and GTPase Rac1. A detailed analysis of the subcellular localization of these proteins demonstrates the increased association of Bcl-2 and mitochondrial Rac1 in Bcl-2 overexpressing cells. This interaction can be blocked in vivo and in vitro with the use of BH3 mimetics such as HA14-I, BH3-I and the use of BH3 domain peptides. Furthermore, blocking this interaction with BH3 domain specific peptides decreases intra-mitochondrial O2.- levels and sensitizes Bcl-2 overexpressing cells to drug-induced apoptosis. Lastly, with the use of co-IP and immunofluorescence, increased Bcl-2 and Rac1 interaction was observed in biopsy materials obtained form patients diagnosed with B-cell lymphoma as compared with normal peripheral blood lymphocytes and benign lymph nodes, thus indicating a clinical functional role of this complex in oncogenesis. 1Clement, M-V., Hirpara, J.L., and Pervaiz, S. Decrease in intracellular superoxide sensitizes Bcl-2 overexpressing tumor cells to receptor- and drug-induced apoptosis independent of the mitochondria. Cell Death Diff. 10(11):1273-85, 2003

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3257.