- What was tested: 5 mM ammonium acetate or ammonium chloride, products of glutamine breakdown (glutaminolysis), added to Sp2/0-Ag14 mouse hybridoma cells starved of glutamine for 3 hours [1].
- Main result: viability, measured 24 hours after the cells were returned to complete medium, rose from 30% without ammonium to 54% with ammonium chloride and 72% with ammonium acetate. Glutamine-fed controls were above 90% [1].
- Mechanism clue: ammonium did not reduce caspase-3 activation or cytochrome c release, but it reduced apoptotic nuclear condensation and fragmentation [1].
- Limits: one mouse cell line in culture. The rescue was partial, and the mechanism was not identified [1].
Why this matters
As cancer cells reprogram their metabolism, many become dependent on glutamine, a dependence called glutamine addiction that underlies several new therapeutic strategies [1]. For background, see the earlier explainer on glutamine addiction.
Breaking down glutamine produces two end products: α-ketoglutarate and ammonium ions [1]. Ammonium is generally considered harmful to cells, yet some studies have shown that it can prevent apoptosis. That raised a question: when glutamine runs out, could changes in ammonium metabolism help decide whether a cell dies [1]? The same cell line was used in the group’s earlier work on MCL-1 and obatoclax [2].
The study
Sp2/0-Ag14 (Sp2/0) mouse hybridoma cells undergo apoptosis through the intrinsic pathway within minutes of glutamine deprivation [1]. The researchers placed the cells in glutamine-free medium for 3 hours, with 5 mM ammonium acetate (AA) or ammonium chloride (AC) added to some cultures. Controls received PBS, sodium acetate or sodium chloride, and glutamine-fed cultures served as a reference [1].
The team measured viability by trypan blue exclusion (24 hours after the cells were returned to complete medium), colony formation, and Annexin V and propidium iodide flow cytometry. They also measured caspase-3 activity and processing, cleavage of Lamin A/C and PARP, DNA fragmentation, cytochrome c release, and nuclear shape with Hoechst staining. Statistics were two-tailed Student’s t tests, with p < 0.05 as the threshold [1].
What the researchers found
| Question | Result |
|---|---|
| Do ammonium salts improve viability? | Yes. Viability was 30% with PBS, 54% with AC and 72% with AA. Sodium salts gave results similar to PBS, methylamine chloride did not help, and glutamine-fed controls were above 90% [1]. |
| Do other assays agree? | Yes. Colony formation and Annexin V and propidium iodide staining also showed significantly more surviving cells with AA or AC [1]. |
| Is new glutamine synthesis needed? | No. The glutamine synthetase inhibitor MSO did not block the benefit [1]. |
| Does α-ketoglutarate add to it? | Not additively. Dimethyl-αKG alone raised viability (43% versus 20%), but gave no extra protection when combined with ammonium [1]. |
| Is core apoptosis blocked? | No. Caspase-3 activity and processing, Lamin A/C and PARP cleavage, DNA fragmentation and cytochrome c release were not reduced [1]. |
| What did ammonium change? | It significantly reduced the share of starved cells with apoptotic nuclear condensation and fragmentation [1]. |
Ammonium can supply amine groups for glutamine synthesis through glutamate dehydrogenase and glutamine synthetase. MSO (10 mM) sensitized the human Ramos B-cell line to starvation but did not lower Sp2/0 viability further, which the authors link to low glutamine synthetase activity in mouse hybridomas, and it did not block the ammonium effect. Dimethyl-αKG was limited to 2 mM because higher doses were toxic to Sp2/0 cells, which may explain the lack of an additive effect [1].
What this does not show
- A partial rescue. Ammonium was not sufficient to stop the death program completely. The authors say other factors are required, possibly additional glutamine-derived metabolites [1].
- One mouse cell line in culture. Whether similar effects occur in human cells or in tumors was not tested; the human Ramos line was used only as an inhibitor control [1].
- An open mechanism. The authors discuss possibilities drawn from other studies, such as effects on phospholipase A2, autophagy, protein glycosylation and potassium transport. These were not tested here [1].
- One dose. All experiments used 5 mM ammonium salts. The study does not address ammonium levels in tumors [1].
What happens next
The authors conclude that ammonium, a major product of glutaminolysis, should be considered a potential target of anti-cancer strategies, and that survival-signaling pathways modulated by ammonium ions should be examined [1]. The group’s earlier study found that MCL-1 falls in glutamine-starved Sp2/0 cells and that obatoclax sensitizes them to apoptosis [2].
- Paper
- Abusneina A, Gauthier ER. Cell & Bioscience 6:23, 2016. doi:10.1186/s13578-016-0092-8
- PubMed
- PMID 27087916
- Full text
- Free on PubMed Central; the full text was used for this summary
- Disclosure
- The author of this post is the first author of the paper.
- Funding
- Listed in the paper.
This post summarizes a published study for general information.
References
- A. Abusneina and E. R. Gauthier, “Ammonium ions improve the survival of glutamine-starved hybridoma cells,” Cell Biosci., vol. 6, art. 23, Apr. 2016. doi: 10.1186/s13578-016-0092-8. PMID: 27087916. Full text: PMC4832542.
- C. C. Harnett, A. Abusneina, J. Clément, and E. R. Gauthier, “Inhibition of MCL-1 by obatoclax sensitizes Sp2/0-Ag14 hybridoma cells to glutamine deprivation-induced apoptosis,” Cell Biochem. Funct., vol. 33, no. 5, pp. 334–340, Jul. 2015. doi: 10.1002/cbf.3121. PMID: 26178811.
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Department of Molecular Diagnostics, Faculty of Biomedical Sciences, University of Benghazi, Libya
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