Induction of Apoptosis in Nutrient-deprived Cultures of Hybridoma and Myeloma Cells

In the present study, cell death was investigated in cultures of NS/0 myelomas and SP2/0-derived D5 hybridomas through morphological examination of cells stained with acridine orange and ethidium bromide. The relative contribution of elevated levels of lactic acid and ammonia, as well as deprivation of glutamine, cystine, and glucose on the induction of necrosis or apoptosis, was investigated. In batch culture of D5 hybridoma cells, induction of apoptotic cell death correlated with the exhaustion of glutamine, while in the case of NS/0 myelomas, it coincided with exhaustion of cystine. To determine whether limiting nutrients were the actual triggering factors for apoptosis in batch culture, exponentially growing cells were resuspended in glutamine or cystine-free media. Within 30 to 40 h, viability decreased to 50% and the nonviable cell population displayed typical apoptotic morphology, with crescents of condensed chromatin around the periphery of the nucleus, or with the entire nucleus present as one or a group of featureless, brightly staining spherical beads. Similarly, D5 hybridomas and NS/0 myelomas cultivated in glucose-free medium died mainly from apoptosis. Cells were also cultivated in fresh medium supplemented with elevated concentrations of ammonia (3.0 mM) and/or lactate (35 mM, 50 mM). This resulted in decreased viabilities and necrotic death in both cell lines. From these results, we conclude that D5 hybridomas and NS/0 myelomas deprived of essential nutrients die by apoptosis, whereas incubation in the presence of elevated levels of metabolic byproducts such as ammonia and lactate will induce necrotic cell death in these cells.