Inhibition of apoptosis in Glutamine‐starved Mouse Hybridoma Cells by Ammonium Ions and DON, a Glutamine Analog
Bibliographic record
Abstract
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).
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".