The glutaminase activity of l-ase inhibits mTORC1 and protein translation in AML cells. (A) OCI-AML3 cells were cultured for 24 hours with the indicated doses of E colil-ase, and the levels of glutamine and asparagine in the intracellular compartment were quantified by ion exchange chromatography. (B) The OCI-AML3 cell line was cultured for 6 or 24 hours with or without E coli or E chrysanthemil-ases at an equal level of asparaginase activity (0.1-10 UI/mL) and then lysed in Laemmli buffer. Western blotting was performed with anti-phospho-p70S6K T389, anti-phospho-4E-BP1 S65, anti-p70S6K, anti-4E-BP1, and anti-actin antibodies. (C) Cells were treated as indicated in B, and the level of Gln in the extracellular medium was quantified by ion exchange chromatography. (D) Primary bone marrow leukemic cells from AML patients were treated as in A, and the same analysis was assessed by western blotting. The intensities of phospho-p70S6K T389, phospho-4E-BP1 S65, and actin signals were quantified in different AML samples. Ratios of phospho-p70S6K T389 or phospho-4E-BP1 S65 to actin were calculated and assigned a reference value of 1 in the control culture. (E) HEK-293T cells were transfected with a Flag-Raptor-Rheb15 expression vector or a control vector and then cultured for 24 hours with or without glutamine at 4 mM or E colil-ase (10 UI/mL). Western blotting was then performed with anti-flag, anti-actin, or anti-phospho-p70S6K T389 antibodies. (F) [S³⁵] methionine pulses were performed in MOLM-14, OCI-AML3, MV4-11, and HL-60 cells to evaluate the global protein synthesis profile with or without E colil-ase at 10 UI/mL during a 6-hour period. (G) OCI-AML3, MOLM-14, MV4-11, and HL-60 cell lines and a representative AML sample were cultured for 24 hours with or without E colil-ase. The expression of Mcl-1 and c-Myc was then analyzed by western blotting.