Inhibition of apoptosis in Glutamine‐starved Mouse Hybridoma Cells by Ammonium Ions and DON, a Glutamine Analog
Authors: Gauthier Eric R. , Zhou Catherine , Abusneina Abdelmuhsen 
Published: November 2023

Actively proliferating cells are dependent on the amino acid L‐glutamine for their growth and survival, a phenomenon referred to as “glutamine addiction”. This cellular dependency toward glutamine has led to the development of “glutamine inhibitors” to target cancer cells. In preliminary experiments, we noticed that one such inhibitor, the glutamine analog 6‐diazo‐5‐oxo‐L‐norleucine (DON), caused a small but significant increase in the viability of glutamine‐starved cells. We also recently showed that supplementing glutamine‐starved cells with ammonium ions (a product of glutaminolysis) increased cell survival without interfering with the core apoptotic machinery (Cell & Bioscience. 2016. 6:23). As part of our efforts to elucidate the molecular and cellular basis of glutamine addiction, we tested the effect of the combination of ammonium salts and DON on the viability of glutamine‐starved Sp2/0‐Ag14 (Sp2/0) mouse hybridoma cells. This cell line is acutely sensitive to glutamine deprivation, with biochemical hallmarks typical of intrinsic apoptosis being detected as early as 30 min after glutamine withdrawal. Our data showed that the combination of DON and either ammonium acetate or ammonium chloride resulted in a significant increase in the viability of Sp2/0 cells starved of glutamine for 3 hours (as determined by a Trypan Blue dye exclusion assay). The effect of ammonium salts/DON on cell viability was greater than when either chemical was used on its own. In fact, when ammonium acetate was used in combination with DON, the viability of glutamine‐starved Sp2/0 cells was not statistically different than the glutamine‐supplemented control. Examination of cell morphology revealed that, while glutamine starvation triggered apoptosis, glutamine‐deprived cells treated with ammonium salts/DON exhibited a normal morphology. The inhibition of apoptosis was confirmed by flow cytometry analysis (Annexin V/propidium iodide staining). The effect of ammonium salts/DON on caspase‐3 activation was also tested. Glutamine‐deprived Sp2/0 cells treated with ammonium salts/DON showed a significant reduction in caspase‐3 enzymatic activity. This was confirmed in Western blot experiments, where ammonium salts/DON supplementation led to a decrease in caspase‐3 processing, and a reduction in the cleavage of lamin A/C, a caspase‐3 substrate. All together, these results demonstrate that the combination of ammonium salts and the glutamine analog DON is sufficient to protect Sp2/0 cells from the induction of apoptosis caused by glutamine withdrawal. The elucidation of the molecular pathways responsible for this phenomenon promises to shed light on the molecular and cellular mechanisms underlying glutamine addiction. Finally, these data show that glutamine analogs can, under some circumstances, promote cancer cell survival.

Support or Funding Information

Financial support for this work: Discovery Grant from NSERC‐CANADA (to ERG), and a Ph.D. fellowship from MOHE‐Libya (to AA).