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

Bruno Figueroa, Tina M. Sauerwald, Alison J. Mastrangelo, J. Marie Hardwick, Michael J. Betenbaugh

Research output: Contribution to journalArticle

Abstract

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 x depriited 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 sysrems.

Original languageEnglish (US)
Pages (from-to)211-222
Number of pages12
JournalBiotechnology and bioengineering
Volume73
Issue number3
DOIs
StatePublished - May 5 2001

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Keywords

  • Apoptosis
  • Baby hamster kidney cells
  • Chinese hamster ovary cells
  • Chloramphenicol acetyltransferase
  • Mammalian cell culture
  • Serum deprivation
  • Sindbis virus

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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