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 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.
Figueroa
B
B
Department of Chemical Engineering, The Johns Hopkins University, 3400 North Charles St., Baltimore, Maryland 21218, USA.
Sauerwald
T M
TM
Mastrangelo
A J
AJ
Hardwick
J M
JM
Betenbaugh
M J
MJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D016466:CHO Cells; D002478:Cells, Cultured / Q000473:pathology / Q000821:virology; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012845:Sindbis Virus / Q000254:growth & development; D014162:Transfection
D016895:Culture Media, Serum-Free; D019253:Proto-Oncogene Proteins c-bcl-2