Comparison of bcl-2 to a bcl-2 deletion mutant for mammalian cells exposedto culture insults

Apoptosis has been found to occur in bioreactors as a result of environment al 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, Chines e hamster ovary (CHO) and baby hamster kidney (BHK) cells. The generation o f a DNA "ladder" and condensation of chromatin indicated that apoptosis occ urred in these cell lines following Sindbis virus infection and serum depri vation. When cells were engineered to overexpress the bcl-2 mutant, cell de ath 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 s erum deprivation. Total production for a heterologous protein encoded on th e Sindbis virus was increased in cell lines expressing the Bcl-2 variants c ompared to the parental cell line. In order to understand the reasons for t he improved anti-apoptosis properties of the mutant, wild-type Bcl-2 and mu tant 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 Si ndbis virus infection and serum withdrawal in both cell lines, white the mu tant 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 abili ty to inhibit apoptosis. These studies indicate that the cells regulate ant i-apoptosis protein levels and these processing events can limit the effect iveness of cell death inhibition strategies in mammalian cell culture syste ms. (C) 2001 John Wiley & Sons, Inc.