Biochemical and Biophysical Research Communications
Down-regulation of Bcl-2-interacting protein BAG-1 confers resistance to anti-cancer drugs☆
Section snippets
Materials and methods
Antibodies. The monoclonal antibody against BAG-1 was provided by S. Takayama (The Burnham Institute, CA). The antibodies against apoptosis-related genes such as Bcl-2 and p53 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Hsp70 and anti-Hsp90 monoclonal antibodies were obtained from Transduction Laboratories (Lexington, KY). Phospho-ERK monoclonal antibody and ERK polyclonal antibody were obtained from Cell Signaling Technology (Beverly, MA).
Anti-cancer and ER stress drugs.
SiRNA B246 effectively down-regulates BAG-1 expression
In order to find appropriate sites on the BAG-1 mRNA for siRNA in down-regulating BAG-1, three different potential target sites were selected, as depicted in Fig. 1A and in Table 1, and 21mer siRNAs were synthesized by in vitro transcription, as described in Materials and methods. Each siRNA was transfected into human cervical carcinoma line HeLa cells, and the cells were harvested two and three days after transfection. Transfected cells were split into two at day three, and one aliquot was
Discussion
BAG-1 is a multi-functional anti-apoptotic protein recently identified as a Bcl-2 interacting protein [1]. BAG-1 enhanced the anti-apoptotic function of Bcl-2 when the two genes were co-transfected into a human lymphoid cell line, Jurkat cells [1]. Furthermore, it was reported that cells overexpressing BAG-1 exhibit resistance against a variety of external stresses. Overexpression of BAG-1 protects rat liver cells from ischemia/reperfusion injury [27]. Several human cervical cancer cell lines
Acknowledgements
We thank S. Takayama for BAG-1 antibody and members of our laboratory for critical discussion.
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Abbreviations: RNAi, RNA interference; siRNA, small interfering RNA; Hsp, heat-shock protein; IC50, concentration inhibiting 50%; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.