Ammonium ions improve the survival of glutamine-starved hybridoma cells

Abstract

Background: As a consequence of a reprogrammed metabolism, cancer cells are dependent on the amino acid l-glutamine for their survival, a phenomenon that currently forms the basis for the generation of new, cancer-specific therapies. In this paper, we report on the role which ammonium ions, a product of glutaminolysis, play on the survival of l-glutamine-deprived Sp2/0-Ag14 mouse hybridoma cells.

Results: The supplementation of l-glutamine-starved Sp2/0-Ag14 cell cultures with either ammonium acetate or ammonium chloride resulted in a significant increase in viability. This effect did not depend on the ability of cells to synthesize l-glutamine, and was not affected by the co-supplementation with α-ketoglutarate. When we examined the effect of ammonium acetate and ammonium chloride on the induction of apoptosis by glutamine deprivation, we found that ammonium salts did not prevent caspase-3 activation or cytochrome c leakage, indicating that they did not act by modulating core apoptotic processes. However, both ammonium acetate and ammonium chloride caused a significant reduction in the number of l-glutamine-starved cells exhibiting apoptotic nuclear fragmentation and/or condensation.

Conclusion: All together, our results show that ammonium ions promote the survival of l-glutamine-deprived Sp2/0-Ag14 cells and modulate late-apoptotic events. These findings highlight the complexity of the modulation of cell survival by l-glutamine, and suggest that targeting survival-signaling pathways modulated by ammonium ions should be examined as a potential anti-cancer strategy.

Keywords: Ammonium; Apoptosis; Caspase; Glutamine; Glutaminolysis; Hybridoma.

Fig. 1

Effect of ammonium salts on the morphology of glutamine-starved Sp2/0-Ag14 cells. Cells were deprived of l-glutamine (−Gln) for 3 h in the presence of 5 mM ammonium salt/sodium salt or an equivalent volume of PBS. Cultures in which Gln was added at the start of the experiment (+Gln) were included as controls. magnification: ×400

Fig. 2

Ammonium salts improve the viability of glutamine-starved Sp2/0-Ag14 cells—trypan blue exclusion assay. Cells were deprived of l-glutamine for 3 h in the presence of 5 mM ammonium salt/sodium salt/methylamine chloride or an equivalent volume of PBS. Cell viability was determined by trypan blue exclusion. Cultures in which Gln was added at the start of the experiment were included as controls. *p < 0.05 vs corresponding sodium salt-supplemented control

Fig. 3

Ammonium salts improve the viability of glutamine-starved Sp2/0-Ag14 cells—clonogenic and flow cytometry assays. Cells were deprived of l-glutamine for 3 h in the presence of 5 mM ammonium salt/sodium salt or an equivalent volume of PBS. Panels a and b, clonogenic assay. Panel c, Annexin-V/propidium iodide assay. Cultures in which Gln was added at the start of the experiment were included as controls. *p < 0.05 vs corresponding sodium salt-supplemented control

Fig. 4

Effect of ammonium salts on the viability of Gln-starved Sp2/0-Ag14 cells does not depend on glutamine synthesis. a Ramos B cells were deprived of l-glutamine for 24 h in the presence or absence of methionine sulfoximine (MSO). Cell viability was then determined using the trypan blue assay. *p < 0.05 vs corresponding control cultured in the presence of Gln ŧ < 0.05 vs corresponding PBS control. b Sp2/0-Ag14 cells were deprived of glutamine for 3 h in the presence of MSO and of 5 mM ammonium salt/sodium salt or an equivalent volume of PBS. Gln (4 mM) was then added and culture was resumed for 24 h. Cell viability was determined by trypan blue exclusion. Gln-deprived, PBS-supplemented cultures in which MSO was omitted were used as controls. For both panels, cultures in which Gln was added at the start of the experiment were included as controls. *p < 0.05 vs corresponding sodium salt-supplemented control

Fig. 5

Non-additive effect of ammonium salts and α-ketoglutarate on the viability of Gln-starved Sp2/0-Ag14 cells. Cells were deprived of l-glutamine for 3 h in the presence of 2 mM dimethyl α-ketoglutarate (KG) and 5 mM ammonium salt/sodium salt or an equivalent volume of PBS. Cultures in which ethanol (Eth) was added in place of KG were included as controls. Gln (4 mM) was then added and culture was resumed for 24 h. Cell viability was determined by trypan blue exclusion. Cultures in which Gln was added at the start of the experiment were included as controls. *p < 0.05 vs corresponding ethanol-supplemented control. ŧ < 0.05 vs corresponding sodium salt-supplemented control

Fig. 6

Ammonium salts do not interfere with the core apoptotic machinery. Sp2/0-Ag14 cells were deprived of l-glutamine (Gln) for 3 h in the presence of 5 mM ammonium salt/sodium salt or an equivalent volume of PBS. a Caspase-3 activity assay. b Caspase-3 processing. The arrow and the star indicate the unprocessed, inactive form and the cleaved, active form of caspase-3, respectively. c cleavage of PARP and Lamin A/C. The arrow and the star respectively indicate the full length and the caspase-3 cleaved forms of PARP and Lamin. Ponceau staining of the membrane was used as a loading control. d DNA fragmentation analysis. e cytosolic cytochrome c release. In both panels b and e, actin was used as a gel loading control. SC sodium chloride, SA sodium acetate, AC ammonium chloride. AA ammonium acetate

Fig. 7

Ammonium salts interfere with apoptotic nuclear condensation and fragmentation. Sp2/0-Ag14 cells were deprived of l-glutamine (Gln) for 3 h in the presence of 5 mM ammonium salt/sodium salt or an equivalent volume of PBS. Hoechst 33342 was added to the cultures 30 min before the end of the experiment. The cells were then processed for fluorescence microscopy. a fluorescence microphotographs (magnification: ×400). b enumeration of cells showing apoptotic nuclear condensation/fragmentation. At least 200 nuclei were enumerated for each group. *p < 0.05 vs corresponding PBS-supplemented control. ŧ < 0.05 vs corresponding sodium salt-supplemented control