Role of ammonium ions on the modulation of apoptosis in glutamine-starved cells

Abstract

Highly proliferating cells reprogram their metabolism in order to fulfill their requirements in biosynthetic precursors. One consequence of this metabolic adaptation is glutamine (Gln) addiction, a phenomenon whereby cells become dependent on Gln for their survival. How cells sense Gln levels and link this information to the survival machinery remains to be fully understood. Our laboratory previously showed that when Sp2/0- Ag14 (Sp2/0) hybridoma cells were deprived of Gln, apoptosis is triggered within minutes. In this study, I used the Sp2/0 cell line to characterize the role which ammonium ions, a product of glutaminolysis, play in the modulation of cell survival. In a first series of experiments, I demonstrated that, when supplemented with ammonium salts, Gln-deprived Sp2/0 cell cultures showed a significant increase in viability. This effect of ammonium salts was independent of the cells’ ability to synthesize Gln, and was not affected by co-treatment with -ketoglutarate. In agreement with their effect on cell viability, ammonium salts caused a significant reduction in the number of Glnstarved Sp2/0 cells with apoptotic nuclear condensation and fragmentation. Unexpectedly, supplementing Gln-starved Sp2/0 cells with ammonium salts did not alter cytosolic cytochrome c release, caspase-3 activation, DNA fragmentation or nuclear lamin A/C cleavage, indicating that the mechanisms triggering the apoptotic machinery remained intact. In a second set of experiments, I used the Gln antagonists 6-Diazo-5- oxo-L-Norleucine (DON) and azaserine (AZA) to interfere with Gln metabolism in lieu of Gln deprivation. Both antagonists were toxic to Sp2/0 cells, an effect which was attenuated by supplementation with ammonium salts. This indicated that Gln metabolism was required for cell viability, and that the effect of ammonium salts did not require metabolic pathways utilizing Gln. Unexpectedly, the supplementation of DON or AZA to Gln-starved cultured led to a small but significant increase in cell viability. Moreover, the supplementation with ammonium salts of Gln-deprived Sp2/0 cultures treated with DON or AZA caused a marked increase in cell viability. More importantly, the combination of ammonium salts and Gln antagonists resulted in a significant reduction in caspase-3 activation in Gln-starved Sp2/0 cells, indicating that they interfered with the apoptotic process. Altogether, our data demonstrate that ammonium ions trigger a pro-survival cellular response in Gln-deprived Sp2/0 cells.