Glutamine Synthetase activity is required in both MSCs and leukemia cells for optimal protection from l-asparaginase. (A) ALL-MSCs were transfected with scramble (kept at 1) or GLUL-siRNA (see Materials and Methods), and the expression of GLUL mRNA was evaluated after 48 hours. Means ± SD of 4 independent experiments performed with MSCs from 4 patients (UPN#12-14-15-16, supplemental Table 2A) are shown. ***P < .001. (B-D) Scramble-transfected or GLUL-silenced ALL-MSCs were treated for 72 hours with l-asparaginase (lane A) or with l-asparaginase + methionine-l-sulfoximine (lane AM). (B) Expression of GS protein. A representative experiment is shown with actin used for loading control. (C) Cell viability. Data are expressed as percentage of the viability of untreated scramble-transfected cells and are means ± SD of 12 independent experiments performed with MSCs from 2 patients (UPN#14-15, supplemental Table 2A). ***P < .001, scramble transfected cells treated with l-asparaginase vs scramble transfected cells treated with l-asparaginase + methionine-l-sulfoximine; ###P < .001 l-asparaginase-treated GLUL-silenced cells vs control GLUL-silenced cells; $$$P < .001 vs l-asparaginase-treated scramble transfected cells. (D) Representative images of cell cultures treated as in panel C (bar = 100 μm). (E) RS4;11 cells were treated for 48 hours with l-asparaginase (ASNase,1 U/ml) in monoculture or in direct contact with scramble-transfected or GLUL-silenced MSCs in the absence or in the presence of methionine-l-sulfoximine (MSO), and the protection index was calculated. Data are means ± SD of 4 independent experiments performed with MSCs from 2 different patients (UPN#12-16, supplemental Table 2A). *P < .05, **P < .01 vs scramble-transfected cells treated with ASNase alone; $$P < .01 GLUL-silenced cells treated with ASNase alone vs GLUL-silenced cells treated with ASNase + MSO. For panels A, C, and E, in each panel different symbol identifies MSCs from an individual donor.