Cells

The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Tokai University School of Medicine (number 20I-34). Written informed consent was obtained from all patients with BCR/ABL⁺ leukemia and umbilical cord blood (CB) donors prior to sample collection. BMMNCs were isolated from the samples using Lymphoprep (StemCell Technologies) density gradient centrifugation. The CB-CD34⁺ cell fraction was prepared using the CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec). BMMNCs from patients with leukemia and CB-CD34⁺ cells from healthy donors were frozen in a medium supplemented with dimethyl sulfoxide (DMSO) and fetal calf serum using a step-down freezing procedure, and stored in liquid nitrogen. Aliquots of frozen samples were thawed immediately before use. The interleukin-3-dependent mouse hematopoietic cell line, 32D, and human leukemic cell lines—K562 (blast crisis of chronic myeloid leukemia), KCL22 (Philadelphia chromosome-positive human acute lymphoblastic leukemia), HL60 (promyelocytic leukemia), Jurkat (T-cell lymphoblastic leukemia), and Raji (B-cell lymphoblastic leukemia)—were purchased from the American Type Culture Collection. The MOLM-14 (human monocytic leukemia) cell line was kindly provided by the Cell Biology Institute, Research Center, Hayashibara Biochemical Laboratories. K562 cells stably expressing luciferase (Luc-K562 cells) were obtained by transduction of lentivirus containing the luciferase-enhanced green fluorescent protein gene (CSII-CMV-Luciferase2-EGFP). The 32D-p210^BCR/ABL and T315I-32D-p210^BCR/ABL cell lines were established by infection of 32D cells with MSCV-BCR-ABL-ires-GFP and MSCV-BCR-ABL1-T315I mutant-ires-GFP, respectively, as previously described⁵¹. The cells were maintained in RPMI 1640 medium supplemented with 10% (20% for KCL22 cells) heat-inactivated fetal bovine serum and antibiotics (100 U penicillin/mL and 100 μg streptomycin/mL) at 37 °C in a humidified 5% CO₂ atmosphere.