Inducible expression of Bcl‐X_L restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures

Abstract

B-cell hybridomas are widely used to produce monoclonal antibodies via large-scale cell culture. Unfortunately, these cells are highly sensitive to apoptotic death under conditions of nutrient deprivation observed at the plateau phase of batch cultures. Previous work has indicated that constitutive high-level expression of antiapoptotic genes in hybridoma cells could delay apoptosis, resulting in higher cell densities and prolonged viability. However, the constitutive high-level expression of antiapoptotic genes has been shown to have detrimental effects on genomic stability of other types of cultured cells. Inducible gene expression may be used to avoid this problem. In the present study, we first constructed an expression vector in which the promoter of a mammalian metallothionein (MT) gene drives the expression of bcl-XL in response to metal exposure. The vector was then used to exogenously control the expression of bcl-XL in D5 hybridoma cells. Our data show that stably transfected D5 cells (4G1.D9) expressed high levels of Bcl-X(L) following overnight exposure to ZnSO(4) concentrations (50 to 100 microM) that did not affect control cells. The level of Bcl-X(L) expressed after ZnSO(4) induction was sufficient to prevent apoptosis experimentally induced by cycloheximide and allowed 4G1.D9 cells to grow at higher densities and remain viable for prolonged periods in suboptimal culture conditions. The use of inducible bcl-XL expression permits extension of the viability of cultured B-cell hybridomas during the antibody secretion phase without the adverse genetic effects associated with constitutive long-term bcl-XL expression.