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

Fingerprint

Sindbis Virus
Cell death
Cell culture
Viruses
Cells
Apoptosis
Cell Line
Virus Diseases
Cell Death
Cell Culture Techniques
Proteins
Serum
Ladders
Bioreactors
Processing
Cricetulus
Cricetinae
Chromatin
Condensation
Ovary

Keywords

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

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology

Cite this

Comparison of Bcl-2 to a Bcl-2 deletion mutant for mammalian cells exposed to culture insults. / Figueroa, Bruno; Sauerwald, Tina M.; Mastrangelo, Alison J.; Hardwick, J Marie; Betenbaugh, Michael J.

In: Biotechnology and Bioengineering, Vol. 73, No. 3, 05.05.2001, p. 211-222.

Research output: Contribution to journalArticle

Figueroa, Bruno ; Sauerwald, Tina M. ; Mastrangelo, Alison J. ; Hardwick, J Marie ; Betenbaugh, Michael J. / Comparison of Bcl-2 to a Bcl-2 deletion mutant for mammalian cells exposed to culture insults. In: Biotechnology and Bioengineering. 2001 ; Vol. 73, No. 3. pp. 211-222.
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