BHRF1 exerts an antiapoptotic effect and cell cycle arrest via Bcl-2 in murine hybridomas
Ernest Milián1, Eva Prats2, Jordi J Cairó1
1Department d'Enginyeria Química, Escola d'Enginyeria, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain.
Abstract:
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.
Insights
Epstein-Barr virus protein BHRF1 enhances hybridoma cell survival by upregulating Bcl-2 expression and interacting with Bim, delaying apoptosis during glutamine starvation. This offers a new strategy for improving cell culture lifespan and production.
Area of Science:
- Biotechnology
- Cell Biology
- Virology
Background:
- Apoptosis (programmed cell death) reduction is key for enhancing animal cell culture lifespan and productivity.
- Anti-apoptotic genes, like Epstein-Barr virus's BHRF1 (homologous to Bcl-2), are effective alternatives to small molecule inhibitors for controlling apoptosis in cell lines.
- BHRF1 has previously shown to protect hybridoma cells from apoptosis, delaying cell death under glutamine starvation.
Purpose of the Study:
- To investigate the precise mechanism of action of the anti-apoptotic BHRF1 protein in a murine hybridoma cell line.
- To elucidate how BHRF1 influences Bcl-2 expression and interacts with other apoptotic regulators.
- To determine the role of Bcl-2 activity in BHRF1-mediated cell survival.
Main Methods:
- Subcellular fractionation to localize BHRF1 protein.
- Quantitative analysis of Bcl-2 gene expression in BHRF1-expressing cells under apoptosis-inducing conditions.
- Treatment with specific Bcl-2 inhibitors (HA14-1, YC-137) to assess functional impact.
- Investigation of BHRF1 interaction with the pro-apoptotic protein Bim.
Main Results:
- BHRF1 protein was localized to the mitochondrial fraction under both normal and apoptotic conditions.
- Bcl-2 gene expression was significantly upregulated (25-fold) in BHRF1-expressing cells during apoptosis induction, correlating with cell survival and G1 cell cycle arrest.
- Inhibition of Bcl-2 activity abolished the protective effect of BHRF1 under glutamine starvation.
- BHRF1 was found to interact with Bim, contributing to mitochondrial stability during apoptosis induction.
Conclusions:
- BHRF1 confers resistance to apoptosis in hybridoma cells, primarily by upregulating Bcl-2 expression and activity.
- The interaction between BHRF1 and Bim is crucial for maintaining mitochondrial stability and cell survival.
- Understanding BHRF1's mechanism provides insights for optimizing cell culture processes in biotechnology.
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