Figure 4.
Leukemia cells release neo-synthetized Gln and MSCs use the amino acid to synthetize and secrete Asn. (A) A working model of the amino acid trade-off between ALL blasts and MSCs in the bone marrow niche is shown. Upon l-asparaginase treatment, asparagine (Asn) and glutamine (Gln) are hydrolyzed in the bulk phase, promoting GS expression in MSCs and ALL blasts. Asparagine Synthetase (ASNS)-negative ALL blasts synthetize Gln that is provided to MSCs for the synthesis of Asn, which is then extruded to supply ALL blasts, allowing their survival. (B) RS4;11 cells were incubated for 48 hours in the absence or presence of Gln (2 mM), and the exchange rate of Gln was evaluated. Data are means ± SD of 6 independent experiments. (C) RS4;11 cells were transfected with scramble or GLUL-siRNA (see Materials and Methods), and the expression of GLUL mRNA was evaluated after 24 hours. ***P < .001. (D) Expression of GS protein was evaluated in scramble-transfected or GLUL-silenced RS4;11 cells after 48 hours of incubation in the absence or in the presence of Gln (2 mM). Tubulin was used for loading control. A representative experiment is shown. (E) Scramble-transfected or GLUL-silenced RS4;11 cells were incubated for 48 hours in the absence of Gln, and Gln secretion was evaluated. Data are means ± SD of 6 independent experiments. ***P < .001 (F) After a 24-hour incubation in standard growth medium in the presence or in the absence of 1 mM methionine-l-sulfoximine (MSO), RS4;11 cells were incubated in the absence of Gln for further 24 hours. During this period, cells preincubated in the absence of MSO were maintained without the inhibitor (Control), while cells preincubated in the presence of MSO were maintained with the inhibitor (+MSO) or incubated in the absence of the inhibitor (MSO pretreatment). Data are expressed as % of Gln secretion in Control cells and are means ± SD of 4 independent experiments. **P < .01; ***P < .001 vs Control. (G) Exchange rates of Asn and Gln in 13C5-Gln-labeled ALL-MSCs incubated for 48 hours in Plasmax medium (Gln = 0.6 mM) in the absence or presence of Asn (41 μM) were determined. Data are means ± SD of 4 independent experiments with MSCs from 4 different patients (UPN#6-17-18-19, supplemental Table 2A). On the right, a schematic representation of labeling of intracellular metabolites upon incubation with 13C-Gln is shown.

Leukemia cells release neo-synthetized Gln and MSCs use the amino acid to synthetize and secrete Asn. (A) A working model of the amino acid trade-off between ALL blasts and MSCs in the bone marrow niche is shown. Upon l-asparaginase treatment, asparagine (Asn) and glutamine (Gln) are hydrolyzed in the bulk phase, promoting GS expression in MSCs and ALL blasts. Asparagine Synthetase (ASNS)-negative ALL blasts synthetize Gln that is provided to MSCs for the synthesis of Asn, which is then extruded to supply ALL blasts, allowing their survival. (B) RS4;11 cells were incubated for 48 hours in the absence or presence of Gln (2 mM), and the exchange rate of Gln was evaluated. Data are means ± SD of 6 independent experiments. (C) RS4;11 cells were transfected with scramble or GLUL-siRNA (see Materials and Methods), and the expression of GLUL mRNA was evaluated after 24 hours. ***P < .001. (D) Expression of GS protein was evaluated in scramble-transfected or GLUL-silenced RS4;11 cells after 48 hours of incubation in the absence or in the presence of Gln (2 mM). Tubulin was used for loading control. A representative experiment is shown. (E) Scramble-transfected or GLUL-silenced RS4;11 cells were incubated for 48 hours in the absence of Gln, and Gln secretion was evaluated. Data are means ± SD of 6 independent experiments. ***P < .001 (F) After a 24-hour incubation in standard growth medium in the presence or in the absence of 1 mM methionine-l-sulfoximine (MSO), RS4;11 cells were incubated in the absence of Gln for further 24 hours. During this period, cells preincubated in the absence of MSO were maintained without the inhibitor (Control), while cells preincubated in the presence of MSO were maintained with the inhibitor (+MSO) or incubated in the absence of the inhibitor (MSO pretreatment). Data are expressed as % of Gln secretion in Control cells and are means ± SD of 4 independent experiments. **P < .01; ***P < .001 vs Control. (G) Exchange rates of Asn and Gln in 13C5-Gln-labeled ALL-MSCs incubated for 48 hours in Plasmax medium (Gln = 0.6 mM) in the absence or presence of Asn (41 μM) were determined. Data are means ± SD of 4 independent experiments with MSCs from 4 different patients (UPN#6-17-18-19, supplemental Table 2A). On the right, a schematic representation of labeling of intracellular metabolites upon incubation with 13C-Gln is shown.

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