Inhibition of apoptosis in Glutamine‐starved Mouse Hybridoma Cells by Ammonium Ions and DON, a Glutamine Analog
Notice bibliographique
Résumé
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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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
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Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,001 | 0,000 |
| Méta-épidémiologie (sens large) | 0,001 | 0,001 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».