Dose-dependent reduction of apoptosis in nutrient-limited cultures of NS/0 myeloma cells transfected with the E1B-19K adenoviral gene.
It is now well documented that apoptosis represents the prevalent mode of death in lymphoid cultures and occurs spontaneously in late-exponential phase of batch cultures following nutrient exhaustion. In an attempt to enhance the cell survival of these cell lines, we have initially engineered nonproducing NS/0 myeloma cells with a vector expressing the adenoviral E1B-19K protein. NS/0 cells transfected with E1B-19K were found to be more resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. In this study, we have characterised a number of NS/0 subclones constitutively expressing different levels of E1B-19K, as well as several subclones in which the expression of E1B-19K was regulated by a tetracycline-controllable gene switch. We have found that a threshold E1B-19K level was required in order to achieve protection against apoptosis. The extent of resistance against cell death induced by nutrient deprivation in glutamine-free medium and in the late phase of batch cultures correlated with the level of E1B-19K expression up to an optimal level where further increases in E1B-19K levels did not result in significant additional protection. To assess the effects of E1B-19K on antibody productivity, an apoptosis-resistant NS/0 clone was then transfected with a chimeric antibody construct. Despite their improved viability, the antibody productivity of E1B-19K clones in batch culture was not significantly improved. Moreover, while the use of E1B-19K considerably delayed cell death, cells eventually died by apoptosis. Surprisingly, E1B-19K had no beneficial effect on the efficiency of fusion of NS/0 myelomas and splenocytes for the generation of hybridoma cells. Furthermore, the resulting hybridomas, although expressing E1B-19K at levels comparable to the myeloma parent, were no longer resistant to apoptosis. This indicates that the ability of E1B-19K to prevent apoptosis is not only dose-dependent but also seems to be cell-type dependent.
Groupe d'Ingénierie des Cellules Animales, Institut de Recherche en Biotechnologie, Conseil National de Recherches du Canada, 6100 Avenue Royalmount, Montréal, PQ, Canada, H4P 2R2.
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D017501:Adenovirus E1B Proteins / Q000235:genetics*; D000911:Antibodies, Monoclonal / Q000235:genetics; D017209:Apoptosis / Q000235:genetics*; D002459:Cell Fusion; D002999:Clone Cells; D003470:Culture Media; D005822:Genetic Vectors; D005973:Glutamine; D018869:Heat-Shock Response; D006801:Humans; D006825:Hybridomas; D009101:Multiple Myeloma / Q000235:genetics / Q000473:pathology*; D013154:Spleen / Q000166:cytology; D014162:Transfection; D014407:Tumor Cells, Cultured
D017501:Adenovirus E1B Proteins; D000911:Antibodies, Monoclonal; D003470:Culture Media; D005973:Glutamine