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.

Bcl‐xL mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells

“…Results from studies with different hosts and experimental designs, though extensive have met with varied success and the merits of bcl-2 modified cell lines remain inconclusive. Similar, but less widespread work, has focused on the alternate bcl-2 family member, bcl-x L (Charbonneau et al, 2000(Charbonneau et al, , 2002Figueroa et al, 2003Figueroa et al, , 2004Fussenegger et al, 1998;Jung et al, 2002;Mastrangelo et al, 2000a,b;Meents et al, 2002). When directly compared to bcl-2 in rCHO cells, the overexpression of bcl-x L was shown to be more efficient in enhancing production Mastrangelo et al, 2000;Meents et al, 2002).…”

“…Overexpression of anti-apoptotic genes towards delaying the triggering of programmed cell death has been attempted in several biotechnologically relevant hosts including hybridoma, NS0 myeloma, baby hamster kidney (BHK) and CHO cells (Charbonneau et al, 2003;Fassnacht et al, 1999;Figueroa et al, 2001Figueroa et al, , 2003Figueroa et al, , 2004Goswami et al, 1999;Jung et al, 2002;Mastrangelo et al, 2000a,b;Mercille and Massie, 1999;Simpson et al, 1998;Tey et al, 2000a,b). The bcl-2 gene was the first to be recognized as a survival gene involved in the apoptotic pathway and as a consequence is the predominant gene selected for cellular augmentation to prolong cell survival.…”

Regulating apoptosis in mammalian cell cultures

“…This gene has been expressed in a wide range of mammalian cell lines exposed to various stresses including nutrient limitation, serum deprivation, accumulation of spent medium, exposure to toxins, irradiation, and viral infections (Charbonneau et al 2003;Chung et al 1998;Cory et al 2003;Figueroa et al 2003;Jung et al 2002;Kim and Lee 2002b;Mastrangelo et al 2000a;Mastrangelo et al 2000b;Meents et al 2002;Perani 1998;Simpson et al 1999;Vives et al 2003). The expression of anti-apoptotic members of the Bcl-2 family has been shown to effectively increase cellular viabilities and in some cases enhance protein production levels as described below.…”

“…The expression of anti-apoptotic members of the Bcl-2 family has been shown to effectively increase cellular viabilities and in some cases enhance protein production levels as described below. Various anti-apoptotic Bcl-2 family members including Bcl-x L , Bcl-w, and viral homologues including E1B-19K from Adenovirus, KsBcl-2 from Karposi's sarcoma-associated herpesvirus and BHFR-1 from Epstein-Barr virus have been expressed and shown to inhibit programmed cell death in mammalian cell culture (Jung et al 2002). The expression of the Adenoviral Bcl-2 homologue, E1B-19K, in NS0 mouse myeloma cells increased viabilities and chimeric antibody production in perfusion culture (Mercille et al 1999).…”

Inhibiting the apoptosis pathway using MDM2 in mammalian cell cultures

“…Bcl-2 family members act by directly interacting with pro-apoptotic molecules and by modifying mitochondrial membrane permeability (Mastrangelo et al, 2000b;Sauerwald et al, 2003). Consequently, these genes have been expressed in a wide range of mammalian cell lines exposed to various stresses including nutrient, cytokine, and serum deprivation, accumulation of spent medium, exposure to toxins and drugs, irradiation, and viral infections (Charbonneau et al, 2003;Chung et al, 1998;Cory et al, 2003;Figueroa et al, 2003;Jung et al, 2002;Kim and Lee, 2002;Mastrangelo et al, 2000a,b;Meents et al, 2002;Perani, 1998;Simpson et al, 1999;Vives et al, 2003). While these methods offer protection against cell death, they block death at a point downstream of the initial signaling event.…”