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Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

Tardito S, Oudin A, Ahmed SU, Fack F, Keunen O, Zheng L, Miletic H, Sakariassen PØ, Weinstock A, Wagner A, Lindsay SL, Hock AK, Barnett SC, Ruppin E, Mørkve SH, Lund-Johansen M, Chalmers AJ, Bjerkvig R, Niclou SP, Gottlieb E - Nat. Cell Biol. (2015)

Bottom Line: However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation.Moreover, Gln-starved cells are not rescued by TCA cycle replenishment.In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons.

View Article: PubMed Central - PubMed

Affiliation: Cancer Metabolism Research Unit, Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

ABSTRACT
L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

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GS activity regulates cell growth and purine availability under Gln starvation. (a) Top: LN18 clones stably expressing iRFP or iRFP-GS were incubated +/− Gln for 5 days and protein expression assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. Bottom: For each clone growth was determined from the protein amount and referred as % of respective control. Dashed lines show the mean % values obtained - Gln. (b) iRFP4 and iRFP-GS5 cells were incubated for the indicated times in medium +/− Gln and MSO (1mM), and counted. (c) iRFP4 and iRFP-GS5 cells were incubated +/− Gln and MSO (1mM) for 21 days, and colonies in representative wells are shown. Data derive from one experiment performed twice. (d-j) iRFP4 and iRFP-GS5 cells were incubated +/− Gln for 24h in medium supplemented with 0.8 mM . The intracellular isotopologues of Gln, UMP, AICAR, IMP, AMP, ATP, and GTP are shown as % of values obtained for the 15N0 metabolites in iRFP4 cells in the presence of Gln. (k) Cell lines were incubated +/− Gln for 24h and the relative amount of intracellular IMP is shown. Mean ± S.E.M. n=3 independent experiments. (l) Scatter plot of the changes in intracellular IMP levels in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (m-n) iRFP4 (m) and iRFP-GS5 (n) cells were incubated for 72h +/− Gln in medium containing adenosine (A) guanosine (G) cytidine (C) thymidine (T) uridine (U), each at 0.2mM, or in combination (AGCTU) at 0.2 mM each, Glu (4mM), MSO (1mM), as indicated. Cells numbers are shown as % of untreated control. Dashed lines show percentage values obtained in the absence of Gln without any further supplementation. Mean ± S.E.M. n=3 independent experiments. (a, b, d, e, f, g, h, i, j) Data derive from one experiment performed once (a, b) or twice (d-j). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
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Figure 4: GS activity regulates cell growth and purine availability under Gln starvation. (a) Top: LN18 clones stably expressing iRFP or iRFP-GS were incubated +/− Gln for 5 days and protein expression assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. Bottom: For each clone growth was determined from the protein amount and referred as % of respective control. Dashed lines show the mean % values obtained - Gln. (b) iRFP4 and iRFP-GS5 cells were incubated for the indicated times in medium +/− Gln and MSO (1mM), and counted. (c) iRFP4 and iRFP-GS5 cells were incubated +/− Gln and MSO (1mM) for 21 days, and colonies in representative wells are shown. Data derive from one experiment performed twice. (d-j) iRFP4 and iRFP-GS5 cells were incubated +/− Gln for 24h in medium supplemented with 0.8 mM . The intracellular isotopologues of Gln, UMP, AICAR, IMP, AMP, ATP, and GTP are shown as % of values obtained for the 15N0 metabolites in iRFP4 cells in the presence of Gln. (k) Cell lines were incubated +/− Gln for 24h and the relative amount of intracellular IMP is shown. Mean ± S.E.M. n=3 independent experiments. (l) Scatter plot of the changes in intracellular IMP levels in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (m-n) iRFP4 (m) and iRFP-GS5 (n) cells were incubated for 72h +/− Gln in medium containing adenosine (A) guanosine (G) cytidine (C) thymidine (T) uridine (U), each at 0.2mM, or in combination (AGCTU) at 0.2 mM each, Glu (4mM), MSO (1mM), as indicated. Cells numbers are shown as % of untreated control. Dashed lines show percentage values obtained in the absence of Gln without any further supplementation. Mean ± S.E.M. n=3 independent experiments. (a, b, d, e, f, g, h, i, j) Data derive from one experiment performed once (a, b) or twice (d-j). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Mentions: To corroborate the causal link between Gln biosynthesis and Gln-dependency, GS was overexpressed in LN18 cells, which display low GS levels and high sensitivity to Gln deprivation. To this end, LN18-derived clones stably expressing infra-Red Fluorescent Protein only (iRFP)26,27 or iRFP and GS were established. To eliminate intrinsic clonal variability, GS expression and its effect on growth under Gln starvation were evaluated in multiple clones (6 iRFP and 9 iRFP-GS). After five days of starvation, growth of iRFP control cells reached, on average, 16% ± 5 % of control Gln-fed cells, while iRFP-GS clones reached on average, 54% ± 12 % (Fig. 4a). Under Gln-supplementation, the iRFP-GS5 clone proliferated slower than iRFP controls. This was not rectified by GS inhibition with MSO (Fig. 5b), consistent with a reported non-metabolic, anti-proliferative role of GS28. Nevertheless, iRFP-GS5, but not control iRFP4 cells, proliferated and formed colonies in Gln-free medium and this growth advantage was blocked by MSO (Fig. 4b-c). These results imply that under Gln starvation the amidation of Glu via GS sustains cell growth. In line with this, when supplemented with 15N1-ammonia, GS-expressing cells displayed 15N incorporation into ~50% of the total Gln pool, even when Gln fed (Fig 4d). When incubated with 15N1-ammonia without Gln, residual intracellular Gln was higher in iRFP-GS5 cells compared to control iRFP4 cells, and produced entirely by GS as judged by 15N incorporation (Fig. 4d).


Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma.

Tardito S, Oudin A, Ahmed SU, Fack F, Keunen O, Zheng L, Miletic H, Sakariassen PØ, Weinstock A, Wagner A, Lindsay SL, Hock AK, Barnett SC, Ruppin E, Mørkve SH, Lund-Johansen M, Chalmers AJ, Bjerkvig R, Niclou SP, Gottlieb E - Nat. Cell Biol. (2015)

GS activity regulates cell growth and purine availability under Gln starvation. (a) Top: LN18 clones stably expressing iRFP or iRFP-GS were incubated +/− Gln for 5 days and protein expression assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. Bottom: For each clone growth was determined from the protein amount and referred as % of respective control. Dashed lines show the mean % values obtained - Gln. (b) iRFP4 and iRFP-GS5 cells were incubated for the indicated times in medium +/− Gln and MSO (1mM), and counted. (c) iRFP4 and iRFP-GS5 cells were incubated +/− Gln and MSO (1mM) for 21 days, and colonies in representative wells are shown. Data derive from one experiment performed twice. (d-j) iRFP4 and iRFP-GS5 cells were incubated +/− Gln for 24h in medium supplemented with 0.8 mM . The intracellular isotopologues of Gln, UMP, AICAR, IMP, AMP, ATP, and GTP are shown as % of values obtained for the 15N0 metabolites in iRFP4 cells in the presence of Gln. (k) Cell lines were incubated +/− Gln for 24h and the relative amount of intracellular IMP is shown. Mean ± S.E.M. n=3 independent experiments. (l) Scatter plot of the changes in intracellular IMP levels in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (m-n) iRFP4 (m) and iRFP-GS5 (n) cells were incubated for 72h +/− Gln in medium containing adenosine (A) guanosine (G) cytidine (C) thymidine (T) uridine (U), each at 0.2mM, or in combination (AGCTU) at 0.2 mM each, Glu (4mM), MSO (1mM), as indicated. Cells numbers are shown as % of untreated control. Dashed lines show percentage values obtained in the absence of Gln without any further supplementation. Mean ± S.E.M. n=3 independent experiments. (a, b, d, e, f, g, h, i, j) Data derive from one experiment performed once (a, b) or twice (d-j). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
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Figure 4: GS activity regulates cell growth and purine availability under Gln starvation. (a) Top: LN18 clones stably expressing iRFP or iRFP-GS were incubated +/− Gln for 5 days and protein expression assessed. Unprocessed scans of western blots are shown in Supplementary Figure 8. Bottom: For each clone growth was determined from the protein amount and referred as % of respective control. Dashed lines show the mean % values obtained - Gln. (b) iRFP4 and iRFP-GS5 cells were incubated for the indicated times in medium +/− Gln and MSO (1mM), and counted. (c) iRFP4 and iRFP-GS5 cells were incubated +/− Gln and MSO (1mM) for 21 days, and colonies in representative wells are shown. Data derive from one experiment performed twice. (d-j) iRFP4 and iRFP-GS5 cells were incubated +/− Gln for 24h in medium supplemented with 0.8 mM . The intracellular isotopologues of Gln, UMP, AICAR, IMP, AMP, ATP, and GTP are shown as % of values obtained for the 15N0 metabolites in iRFP4 cells in the presence of Gln. (k) Cell lines were incubated +/− Gln for 24h and the relative amount of intracellular IMP is shown. Mean ± S.E.M. n=3 independent experiments. (l) Scatter plot of the changes in intracellular IMP levels in relation to the growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (m-n) iRFP4 (m) and iRFP-GS5 (n) cells were incubated for 72h +/− Gln in medium containing adenosine (A) guanosine (G) cytidine (C) thymidine (T) uridine (U), each at 0.2mM, or in combination (AGCTU) at 0.2 mM each, Glu (4mM), MSO (1mM), as indicated. Cells numbers are shown as % of untreated control. Dashed lines show percentage values obtained in the absence of Gln without any further supplementation. Mean ± S.E.M. n=3 independent experiments. (a, b, d, e, f, g, h, i, j) Data derive from one experiment performed once (a, b) or twice (d-j). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
Mentions: To corroborate the causal link between Gln biosynthesis and Gln-dependency, GS was overexpressed in LN18 cells, which display low GS levels and high sensitivity to Gln deprivation. To this end, LN18-derived clones stably expressing infra-Red Fluorescent Protein only (iRFP)26,27 or iRFP and GS were established. To eliminate intrinsic clonal variability, GS expression and its effect on growth under Gln starvation were evaluated in multiple clones (6 iRFP and 9 iRFP-GS). After five days of starvation, growth of iRFP control cells reached, on average, 16% ± 5 % of control Gln-fed cells, while iRFP-GS clones reached on average, 54% ± 12 % (Fig. 4a). Under Gln-supplementation, the iRFP-GS5 clone proliferated slower than iRFP controls. This was not rectified by GS inhibition with MSO (Fig. 5b), consistent with a reported non-metabolic, anti-proliferative role of GS28. Nevertheless, iRFP-GS5, but not control iRFP4 cells, proliferated and formed colonies in Gln-free medium and this growth advantage was blocked by MSO (Fig. 4b-c). These results imply that under Gln starvation the amidation of Glu via GS sustains cell growth. In line with this, when supplemented with 15N1-ammonia, GS-expressing cells displayed 15N incorporation into ~50% of the total Gln pool, even when Gln fed (Fig 4d). When incubated with 15N1-ammonia without Gln, residual intracellular Gln was higher in iRFP-GS5 cells compared to control iRFP4 cells, and produced entirely by GS as judged by 15N incorporation (Fig. 4d).

Bottom Line: However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation.Moreover, Gln-starved cells are not rescued by TCA cycle replenishment.In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons.

View Article: PubMed Central - PubMed

Affiliation: Cancer Metabolism Research Unit, Cancer Research UK, Beatson Institute, Switchback Road, Glasgow G61 1BD, UK.

ABSTRACT
L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

Show MeSH
Related in: MedlinePlus