Glutamine removal inhibits mTORC1 activity and induces apoptosis in AML cells. (A) The MOLM-14 and OCI-AML3 cell lines, and primary AML cells from a representative patient, were cultured for 6 or 24 hours with or without 4 mM glutamine (Gln) and/or 0.4 mM leucine (Leu). Cells were lysed in Laemmli buffer and western blotting was performed to analyze the p70S6K T389 and 4E-BP1 S65 phosphorylation status. (B) MOLM-14, OCI-AML3, MV4-11, and HL-60 cell lines were cultured with or without Gln for 24 hours and apoptosis was quantified by flow cytometry analysis of Annexin-V binding. (C) The indicated cell lines were cultured for 24 hours with or without glutamine and then assessed for caspase-3 and PARP cleavage by western blotting. (D) Normal CD34⁺ cell were obtained from 3 healthy allogeneic transplant donors after positive sorting and primary AML cells from 8 AML patients were also tested. Cells were cultured 48 hours with or without glutamine (4 mM), and the apoptotic responses were quantified by flow cytometry analysis of Annexin-V binding.