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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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Gln starvation reduces GBM cell proliferation. (a) Dose-response curves for cell lines incubated for 3 days in DMEM or SMEM with the indicated concentrations of Gln. (b) Cells were incubated for the indicated times in SMEM +/− Gln. (c) Cells were incubated for 72 hours in SMEM +/− Gln or Glucose as indicated. Each dot represents the number of dead cells (with plasma membrane integrity loss) normalized over a confluence index. The resulting cell death index was assessed every hour. (d) Cell cycle distribution of cell lines incubated for 3 days +/− Gln. Mean of 3 independent experiments is shown in Supplementary Figure 1b. (e) Growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (f) Scatter plot of the doubling time obtained for the cell lines in Gln-fed conditions, in relation to growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (a-d) Data derive from one experiment performed twice (a, b, c), or three times (d). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
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Figure 1: Gln starvation reduces GBM cell proliferation. (a) Dose-response curves for cell lines incubated for 3 days in DMEM or SMEM with the indicated concentrations of Gln. (b) Cells were incubated for the indicated times in SMEM +/− Gln. (c) Cells were incubated for 72 hours in SMEM +/− Gln or Glucose as indicated. Each dot represents the number of dead cells (with plasma membrane integrity loss) normalized over a confluence index. The resulting cell death index was assessed every hour. (d) Cell cycle distribution of cell lines incubated for 3 days +/− Gln. Mean of 3 independent experiments is shown in Supplementary Figure 1b. (e) Growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (f) Scatter plot of the doubling time obtained for the cell lines in Gln-fed conditions, in relation to growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (a-d) Data derive from one experiment performed twice (a, b, c), or three times (d). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.

Mentions: To explore their growth response to different nutrient supplies, GBM cells were incubated either in DMEM containing supra-physiological concentrations of glucose and lacking some of the non-essential amino acids, or in a newly-formulated SMEM, containing nutrient concentrations comparable to human serum (Supplementary Table 1). Both media were supplemented with various concentrations of Gln (Fig. 1a). In serum-like medium, all cell lines grew comparably to or faster than cells cultured in DMEM. In both media, the minimal Gln concentration required for maximal growth was below 0.65 mM, hereafter used as the control concentration. In the absence of Gln, cells grew faster in SMEM, demonstrating that medium formulation affects the response to Gln deprivation. Gln starvation hindered proliferation to different extents (Fig. 1b and Supplementary Fig. 1a) without inducing cell death, contrary to previous reports3,4, and to their response to glucose withdrawal (Fig. 1c). DNA flow-cytometry analysis showed that Gln starvation did not cause cell cycle arrest at any particular phase (Fig. 1d and Supplementary Fig. 1b). Overall, Gln withdrawal resulted in cell line-specific growth inhibition, ranging from 20% for U251 and SF188, to 80% for LN18 cells (Fig. 1e), independently of the initial proliferation rate (Fig. 1f).


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)

Gln starvation reduces GBM cell proliferation. (a) Dose-response curves for cell lines incubated for 3 days in DMEM or SMEM with the indicated concentrations of Gln. (b) Cells were incubated for the indicated times in SMEM +/− Gln. (c) Cells were incubated for 72 hours in SMEM +/− Gln or Glucose as indicated. Each dot represents the number of dead cells (with plasma membrane integrity loss) normalized over a confluence index. The resulting cell death index was assessed every hour. (d) Cell cycle distribution of cell lines incubated for 3 days +/− Gln. Mean of 3 independent experiments is shown in Supplementary Figure 1b. (e) Growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (f) Scatter plot of the doubling time obtained for the cell lines in Gln-fed conditions, in relation to growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (a-d) Data derive from one experiment performed twice (a, b, c), or three times (d). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
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Related In: Results  -  Collection

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Figure 1: Gln starvation reduces GBM cell proliferation. (a) Dose-response curves for cell lines incubated for 3 days in DMEM or SMEM with the indicated concentrations of Gln. (b) Cells were incubated for the indicated times in SMEM +/− Gln. (c) Cells were incubated for 72 hours in SMEM +/− Gln or Glucose as indicated. Each dot represents the number of dead cells (with plasma membrane integrity loss) normalized over a confluence index. The resulting cell death index was assessed every hour. (d) Cell cycle distribution of cell lines incubated for 3 days +/− Gln. Mean of 3 independent experiments is shown in Supplementary Figure 1b. (e) Growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (f) Scatter plot of the doubling time obtained for the cell lines in Gln-fed conditions, in relation to growth inhibition caused by Gln starvation. Mean ± S.E.M. n=3 independent experiments. (a-d) Data derive from one experiment performed twice (a, b, c), or three times (d). Raw data of independent repeats are provided in the statistics source data Supplementary Table 5.
Mentions: To explore their growth response to different nutrient supplies, GBM cells were incubated either in DMEM containing supra-physiological concentrations of glucose and lacking some of the non-essential amino acids, or in a newly-formulated SMEM, containing nutrient concentrations comparable to human serum (Supplementary Table 1). Both media were supplemented with various concentrations of Gln (Fig. 1a). In serum-like medium, all cell lines grew comparably to or faster than cells cultured in DMEM. In both media, the minimal Gln concentration required for maximal growth was below 0.65 mM, hereafter used as the control concentration. In the absence of Gln, cells grew faster in SMEM, demonstrating that medium formulation affects the response to Gln deprivation. Gln starvation hindered proliferation to different extents (Fig. 1b and Supplementary Fig. 1a) without inducing cell death, contrary to previous reports3,4, and to their response to glucose withdrawal (Fig. 1c). DNA flow-cytometry analysis showed that Gln starvation did not cause cell cycle arrest at any particular phase (Fig. 1d and Supplementary Fig. 1b). Overall, Gln withdrawal resulted in cell line-specific growth inhibition, ranging from 20% for U251 and SF188, to 80% for LN18 cells (Fig. 1e), independently of the initial proliferation rate (Fig. 1f).

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