BHRF1 exerts an antiapoptotic effect and cell cycle arrest via Bcl-2 in murine hybridomas

• Published in: Journal of biotechnology 2015-09, Vol.209, p.58-67
• Main Authors: Milián, Ernest, Prats, Eva, Cairó, Jordi J., Gòdia, Francesc, Vives, Joaquim
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis; Apoptosis - drug effects; Batch Cell Culture Techniques; Bcl-2; Benzopyrans - pharmacology; BHRF1; Biotechnology; Cell Cycle Checkpoints; Cell Line; Delay; Gene expression; Genes; Glutamine; HA14-1; Hybridoma; Hybridomas - cytology; Hybridomas - drug effects; Inhibitors; Membrane Potential, Mitochondrial; Mice; Mitochondria - metabolism; Nitriles - pharmacology; Proteins; Proto-Oncogene Proteins c-bcl-2 - metabolism; Thiazoles - pharmacology; Transfection; Up-Regulation; Viral Proteins - genetics; Viral Proteins - metabolism; YC-137
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2015.06.379

•BHRF1 is an Epstein–Barr anti-apoptotic gene with potential applications in cell culture processes.•BHRF1 protein locates in the mitochondria in normal growing conditions and under apoptosis-triggering conditions.•BHRF1 can exert its antiapoptotic and cell cycle arrest activities through upregulation of endogenous Bcl-2.•BHRF1 binds to Bim, but not to Bcl-2, Bax and Bak. Apoptosis has been widely studied in order to find methods to increase the life-span and production performance in large-scale animal cell cultures. The use of anti-apoptotic genes has emerged as an efficient method to reduce apoptosis in a variety of biotechnological relevant cell lines, including CHO and hybridomas, alternatively to small molecule inhibitors. It is already known that expression of BHRF1, an Epstein–Barr virus-encoded early protein homologous to the anti-apoptotic protein Bcl-2, protects hybridoma cells from apoptosis in batch and continuous operation modes resulting in a delay in the cell death process under glutamine starvation conditions. In the present study, the mechanism of action of BHRF1 was investigated in a murine hybridoma cell line. BHRF1 protein was found in the mitochondrial cell fraction both under normal growing conditions and apoptosis-inducing conditions. Remarkably, the expression of the anti-apoptotic gene bcl2 in BHRF1-expressing cells was up-regulated 25-fold compared to mock-transfected controls under apoptosis triggering conditions and its expression correlated with survival of transgenic cultures and cell cycle arrest in G1. Bcl-2 activity was revealed to be crucial for the BHRF1-mediated effect since the addition of specific inhibitors of Bcl-2 (namely HA14-1 and YC-137) resulted in a loss of function of BHRF1-expressing cells under glutamine starvation conditions. Moreover, the interaction of BHRF1 with the pro-apoptotic BH3-only Bim conferred mitochondrial stability to BHRF1 expressing cells under apoptosis-triggering conditions.