Figure 2.
ALL-MSCs adapt to l-asparaginase through increased Glutamine Synthetase activity. (A) ALL-MSCs were incubated for 48 hours with the indicated doses of l-asparaginase (ASNase) in the absence or in the presence of methionine-l-sulfoximine (MSO, 1 mM), and viability was assessed with the resazurin method. Data are means ± SD of 9 independent experiments performed on MSCs derived from 3 distinct patients (UPN#12-15-16, supplemental Table 2A). Nonlinear regression analysis was used to obtain dose response curves. (B) ALL-MSCs were incubated in the absence (Control) or in the presence of ASNase (1 U/ml), MSO (1 mM), or ASNase + MSO, and cell growth was evaluated at the indicated times. Data are expressed as Log2 of the ratio between viable cells at the experimental time and viable cells at time 0 and are means ± SD of 7 independent experiments performed with cells from 3 ALL patients (UPN#14-15-16, supplemental Table 2A). *** P < .001 vs control cells at 6d; ### P < .001 cells treated with ASNase vs cells treated with ASNase + MSO. (C-E) ALL-MSCs were incubated for the indicated times in normal growth medium (Control) or in the presence of ASNase (A) or ASNase + MSO (AM) and then extracted for mRNA (C), protein (D), or amino acid (E) analysis. (C) The relative expression of DDIT3 and GLUL was determined, normalizing data for RPL-15 mRNA and expressing the results as folds of the mean value obtained in control cells, kept at 1. Data are means ± SD of 3 independent experiments performed with MSCs from 3 different patients (UPN#14-15-16, supplemental Table 2A). *P < .05, **P < .01, ***P < .001 vs control cells. (D) A representative western blot of GS is shown. Actin was used for loading control. The experiment was performed with ALL-MSCs from 4 different patients (UPN#11-12-13-16, supplemental Table 2A) with comparable results. (E) The intracellular content of Gln was determined at the indicated experimental times. Data are means ± SD of 9 independent experiments performed with MSCs from 3 different patients (UPN#12-14-15, supplemental Table 2A). **P < .01 vs cells undergoing the same treatment of 6 hours, ## P < .01 cells treated with l-asparaginase vs cells treated with ASNase + MSO for 48 hours. (F) ALL-MSCs were incubated for 48 hours in a Gln- and Asn-free medium in the absence or in the presence of MSO (1 mM) or in normal growth medium (Control). At the indicated times, intracellular Gln was determined. Data are means ± SD of 11 independent experiments performed with MSCs from 4 patients (UPN#11-12-13-16, supplemental Table 2A). ***P < .001 vs cells undergoing the same treatment of 6 hours; ###P < .001, cells incubated in the absence of MSO vs cells incubated in the presence of MSO for 48 hours. For panels A, B, C, E, and F, in each panel different symbol identifies MSCs from an individual donor.

ALL-MSCs adapt to l-asparaginase through increased Glutamine Synthetase activity. (A) ALL-MSCs were incubated for 48 hours with the indicated doses of l-asparaginase (ASNase) in the absence or in the presence of methionine-l-sulfoximine (MSO, 1 mM), and viability was assessed with the resazurin method. Data are means ± SD of 9 independent experiments performed on MSCs derived from 3 distinct patients (UPN#12-15-16, supplemental Table 2A). Nonlinear regression analysis was used to obtain dose response curves. (B) ALL-MSCs were incubated in the absence (Control) or in the presence of ASNase (1 U/ml), MSO (1 mM), or ASNase + MSO, and cell growth was evaluated at the indicated times. Data are expressed as Log2 of the ratio between viable cells at the experimental time and viable cells at time 0 and are means ± SD of 7 independent experiments performed with cells from 3 ALL patients (UPN#14-15-16, supplemental Table 2A). *** P < .001 vs control cells at 6d; ### P < .001 cells treated with ASNase vs cells treated with ASNase + MSO. (C-E) ALL-MSCs were incubated for the indicated times in normal growth medium (Control) or in the presence of ASNase (A) or ASNase + MSO (AM) and then extracted for mRNA (C), protein (D), or amino acid (E) analysis. (C) The relative expression of DDIT3 and GLUL was determined, normalizing data for RPL-15 mRNA and expressing the results as folds of the mean value obtained in control cells, kept at 1. Data are means ± SD of 3 independent experiments performed with MSCs from 3 different patients (UPN#14-15-16, supplemental Table 2A). *P < .05, **P < .01, ***P < .001 vs control cells. (D) A representative western blot of GS is shown. Actin was used for loading control. The experiment was performed with ALL-MSCs from 4 different patients (UPN#11-12-13-16, supplemental Table 2A) with comparable results. (E) The intracellular content of Gln was determined at the indicated experimental times. Data are means ± SD of 9 independent experiments performed with MSCs from 3 different patients (UPN#12-14-15, supplemental Table 2A). **P < .01 vs cells undergoing the same treatment of 6 hours, ## P < .01 cells treated with l-asparaginase vs cells treated with ASNase + MSO for 48 hours. (F) ALL-MSCs were incubated for 48 hours in a Gln- and Asn-free medium in the absence or in the presence of MSO (1 mM) or in normal growth medium (Control). At the indicated times, intracellular Gln was determined. Data are means ± SD of 11 independent experiments performed with MSCs from 4 patients (UPN#11-12-13-16, supplemental Table 2A). ***P < .001 vs cells undergoing the same treatment of 6 hours; ###P < .001, cells incubated in the absence of MSO vs cells incubated in the presence of MSO for 48 hours. For panels A, B, C, E, and F, in each panel different symbol identifies MSCs from an individual donor.

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