Protective effect of viral homologues of bcl-2 on hybridoma cells under apoptosis-inducing conditions.

Targets for metabolic engineering have been identified in a hybridoma cell line to make it more robust in culture toward potential limitations inducing apoptosis. The cells were genetically modified with plasmids harboring endogenous bcl-2 gene and also with viral Bcl-2 homologues, particularly ksbcl-2 and bhrf-1 genes. When cells were exposed to apoptosis-inducing conditions (i.e., glutamine-free medium), the control cells exhibited a decrease in viable cell number within the first 12 h, whereas, for the bcl-2 and ksbcl-2 transfected cell cultures, the viable cell number did not exhibit any clear decrease until after 60 h. Furthermore, hybridoma cells expressing the viral homologue bhrf-1 were even more resistant to cell death, showing a decrease in viability of only 50% at 72 h of culture in glutamine-deprived medium, substantially lower than the 90% viability decrease observed for the control culture. In addition, and most relevant for further bioprocess applications, the cells genetically modified could be brought back to growth conditions even after being exposed to glutamine-deprived conditions during a significant time window, up to 72 h.

The protection of hybridoma cells from apoptosis by caspase inhibition allows culture recovery when exposed to non-inducing conditions.

Programmed cell death (PCD) or apoptosis process in a hybridoma cell line induced by the deprivation of one of the main nutrients, glutamine, has been studied. The use of caspase inhibitors has enabled maintenance of cell viability during a significant period of time, when glutamine depletion was maintained in the culture. Two caspase inhibitors partially suppressed the progress of PCD under glutamine deprivation: Ac-DEVD-cho and z-VAD-fmk. Indeed, as a consequence of this protection, the number of viable cells decreased by 10% (for z-VAD-fmk) and by 80% (for Ac-DEVD-cmk) after 36 h of culture, while it decreased by 90% for a control culture in the absence of protective compounds. However, when the culture was exposed to non-apoptotic conditions after this period of time under apoptosis protection conditions, a normal growth pattern was not recovered. Interestingly, the simultaneous use of both inhibitors made the recovery of the cell culture possible even after a period of 36 h under glutamine depletion, indicating that the inhibition of the effector caspases occurs upstream of the point in which hybridoma cells enter into the commitment step of the death programme.