Comparison of Bcl-2 to a Bcl-2 deletion mutant for mammalian cells exposed to culture insults

Apoptosis has been found to occur in bioreactors as a result of environmental stresses. The overexpression of bcl‐2 is a widely used strategy to limit the induction of apoptosis in mammalian cell cultures. In this study, the effectiveness of wild‐type Bcl‐2 was compared to a Bcl‐2 mutant lacking the...

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Veröffentlicht in:Biotechnology and bioengineering 2001-05, Vol.73 (3), p.211-222
Hauptverfasser: Figueroa Jr, Bruno, Sauerwald, Tina M., Mastrangelo, Alison J., Hardwick, J. Marie, Betenbaugh, Michael J.
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container_issue 3
container_start_page 211
container_title Biotechnology and bioengineering
container_volume 73
creator Figueroa Jr, Bruno
Sauerwald, Tina M.
Mastrangelo, Alison J.
Hardwick, J. Marie
Betenbaugh, Michael J.
description Apoptosis has been found to occur in bioreactors as a result of environmental stresses. The overexpression of bcl‐2 is a widely used strategy to limit the induction of apoptosis in mammalian cell cultures. In this study, the effectiveness of wild‐type Bcl‐2 was compared to a Bcl‐2 mutant lacking the nonstructured loop domain in two commercially prominent cell lines, Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells. The generation of a DNA “ladder” and condensation of chromatin indicated that apoptosis occurred in these cell lines following Sindbis virus infection and serum deprivation. When cells were engineered to overexpress the bcl‐2 mutant, cell death due to Sindbis virus was inhibited in a concentration‐dependent manner. Furthermore, the Bcl‐2 mutant provided increased protection as compared to wild‐type Bcl‐2 following two model insults, Sindbis virus infection and serum deprivation. Total production for a heterologous protein encoded on the Sindbis virus was increased in cell lines expressing the Bcl‐2 variants compared to the parental cell line. In order to understand the reasons for the improved anti‐apoptosis properties of the mutant, wild‐type Bcl‐2 and mutant Bcl‐2 were examined by Western blot following each model insult. Wild‐type Bcl‐2 was observed to degrade into a 23 kDa fragment following both Sindbis virus infection and serum withdrawal in both cell lines, while the mutant Bcl‐2 protein was not degraded during the same period. The processing of Bcl‐2 was found to correlate with reduced cell viabilities following the two external insults to suggest that Bcl‐2 degradation may limit its ability to inhibit apoptosis. These studies indicate that the cells regulate anti‐apoptosis protein levels and these processing events can limit the effectiveness of cell death inhibition strategies in mammalian cell culture systems. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 211–222, 2001.
doi_str_mv 10.1002/bit.1053
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Marie</creatorcontrib><creatorcontrib>Betenbaugh, Michael J.</creatorcontrib><title>Comparison of Bcl-2 to a Bcl-2 deletion mutant for mammalian cells exposed to culture insults</title><title>Biotechnology and bioengineering</title><addtitle>Biotechnol. Bioeng</addtitle><description>Apoptosis has been found to occur in bioreactors as a result of environmental stresses. The overexpression of bcl‐2 is a widely used strategy to limit the induction of apoptosis in mammalian cell cultures. In this study, the effectiveness of wild‐type Bcl‐2 was compared to a Bcl‐2 mutant lacking the nonstructured loop domain in two commercially prominent cell lines, Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells. The generation of a DNA “ladder” and condensation of chromatin indicated that apoptosis occurred in these cell lines following Sindbis virus infection and serum deprivation. When cells were engineered to overexpress the bcl‐2 mutant, cell death due to Sindbis virus was inhibited in a concentration‐dependent manner. Furthermore, the Bcl‐2 mutant provided increased protection as compared to wild‐type Bcl‐2 following two model insults, Sindbis virus infection and serum deprivation. Total production for a heterologous protein encoded on the Sindbis virus was increased in cell lines expressing the Bcl‐2 variants compared to the parental cell line. In order to understand the reasons for the improved anti‐apoptosis properties of the mutant, wild‐type Bcl‐2 and mutant Bcl‐2 were examined by Western blot following each model insult. Wild‐type Bcl‐2 was observed to degrade into a 23 kDa fragment following both Sindbis virus infection and serum withdrawal in both cell lines, while the mutant Bcl‐2 protein was not degraded during the same period. 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source Wiley Online Library - AutoHoldings Journals; MEDLINE
subjects Animal cells
Animals
apoptosis
Apoptosis - physiology
baby hamster kidney cells
Bcl-2 protein
Biological and medical sciences
Biotechnology
Cells, Cultured - pathology
Cells, Cultured - virology
Chinese hamster ovary cells
chloramphenicol acetyltransferase
CHO Cells
Cricetinae
Culture Media, Serum-Free - pharmacology
Establishment of new cell lines, improvement of cultural methods, mass cultures
Eukaryotic cell cultures
Fundamental and applied biological sciences. Psychology
Humans
mammalian cell culture
Methods. Procedures. Technologies
Mutation
Proto-Oncogene Proteins c-bcl-2 - deficiency
Proto-Oncogene Proteins c-bcl-2 - genetics
Proto-Oncogene Proteins c-bcl-2 - metabolism
Proto-Oncogene Proteins c-bcl-2 - physiology
serum deprivation
Sindbis virus
Sindbis Virus - growth & development
Transfection
title Comparison of Bcl-2 to a Bcl-2 deletion mutant for mammalian cells exposed to culture insults
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