title: BHRF1 exerts an antiapoptotic effect and cell cycle arrest via Bcl-2 in murine hybridomas.
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.
authors.affiliation: Department d'Enginyeria Química, Escola d'Enginyeria, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain. Electronic address: ernest.miliangonzalez@mcgill.ca.
authors.forename: Ernest
authors.initials: E
authors.lastname: Milián
authors.affiliation: Centre d'Investigació i Desenvolupament, CID-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: Prats
authors.affiliation: Department d'Enginyeria Química, Escola d'Enginyeria, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain.
authors.forename: Jordi J
authors.initials: JJ
authors.lastname: Cairó
authors.affiliation: Department d'Enginyeria Química, Escola d'Enginyeria, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain.
authors.forename: Francesc
authors.initials: F
authors.lastname: Gòdia
authors.affiliation: Department d'Enginyeria Química, Escola d'Enginyeria, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain; Divisió de Teràpies Avançades/XCELIA, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, 116, 08005 Barcelona, Spain. Electronic address: jvives@bst.cat.
authors.forename: Joaquim
authors.initials: J
authors.lastname: Vives
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D061249:Batch Cell Culture Techniques; D001578:Benzopyrans / Q000494:pharmacology; D059447:Cell Cycle Checkpoints; D002460:Cell Line; D006825:Hybridomas / Q000166:cytology* / Q000187:drug effects; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology; D014162:Transfection; D015854:Up-Regulation; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: C083278:BHRF1 protein, Human herpesvirus 4; D001578:Benzopyrans; D009570:Nitriles; D019253:Proto-Oncogene Proteins c-bcl-2; D013844:Thiazoles; D014764:Viral Proteins; C493272:YC137; C456800:ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate
keywords: Apoptosis; BHRF1; Bcl-2; HA14-1; Hybridoma; YC-137