Role of Bovine Bcl2A1 Gene in Staurosporine-Induced Apoptotic Cell Lines
Bcl2A1 (B-cell lymphoma 2 - related protein A1) is a member of a gene set that plays a critical role in regulating apoptosis of mammalian cells. The family is divided into 2 groups: (1) anti-apoptotic genes Bcl-2, Bcl-xL, Bcl2A1 and (2) pro-apoptotic genes Bid, Bax, Bak. Apoptosis in cells is regulated by a balanced expression of these groups. In this study, the ORF encoding the 175 amino acid bovine Bcl2A1 protein was amplified by reverse transcription polymerase chain reaction (RT-PCR), using specific primers deduced from the bovine Bcl2A1 mRNA sequence (www//ncbi.nlm.nih.org. Acc: AB195549) and which also contained appropriate restriction endonuclease cleavage sites for cloning. The product was then cloned into plasmid pcDNA3+ to construct the eucaryotic expression plasmid pcDNA3.Bcl2A1. Hela, L11, Vero and WSL cell lines were used to investigate apoptosis induced by staurosporine. Monitoring of cellular DNA fragmentation revealed that incubation with staurosporine at a concentration of 2 µM results in a sufficient level of apoptosis induction in Hela, L11, WSL cell cultures after 6 hours and in Vero cell cultures after 12 hours. Transfection of these cell lines with pcDNA3.Bcl2A1, using pseudorabies virus Us3 protein kinase (PrVUs3) which prevents staurosporine induced apoptosis by interacting with the pro-apoptotic proteins Bid and Bad as a control, proved that expression of the bovine Bcl2A1 gene blocks staurosporine-induced apoptosis in Hela and L11 cell lines. However, this activity was not observed in WSL and Vero cell cultures.
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