However, little is currently known about the HSP inhibitor significance of GSK-3β to pediatric ALL cell survival. ALL initiates and progresses in the bone marrow (BM). In the present study, we demonstrated that GSK-3β accumulates in the nuclei of primitive pediatric ALL cells from the BM. GSK-3β inhibition leads
to suppression of NF-κB transcriptional activity and induces apoptosis through the transcriptional downregulation of the survivin gene. Methods Primary cells Fresh ALL samples were obtained from 39 children with newly diagnosed acute lymphoblastic leukemia, with 11 normal BM samples as control, in Affiliated Children’s Hospital, Chongqing Medical University. The diagnosis of ALL was based on morphology, immunology, cytogenetic,
and molecular classification. The informed consent was obtained from parents, guardians, or patients (as appropriate). Isolation of leukemia cells and cell culture Bone marrow mononuclear cells (BMMC) were isolated from heparinized aspirates by Ficoll-Hypaque density gradient centrifugation within 24 h after sampling. To remove adherent cells, BMMC were suspended in RPMI 1640 medium supplemented with 20% fetal calf serum (FCS) and incubated in plastic dishes Citarinostat at 37°C for 24 h before collection of nonadherent cells. These ALL cells were then either used immediately for the laboratory studies described below or cryopreserved in RPMI 1640 medium with 20% FCS and 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until use. If necessary, leukemic samples were further enriched to more than 90% leukemic blasts by removing nonmalignant cells with immunomagnetic beads . Reagents and antibodies The GSK-3β inhibitors SB216763, and lithium chloride (LiCl) were obtained from Sigma, USA. A 20 mg/ml solution of SB216763
was prepared in dimethyl sulfoxide (DMSO), stored in small aliquots at -20°C, and then thawed and diluted in cell-culture medium as required. LiCl was dissolved in RPMI 1640 and used at final concentrations of 5 and 10 mM. The high-quality fetal bovine serum and RPMI 1640 medium were products Montelukast Sodium of Gibco Company, USA. RNAiso Plus, Reverse Transcription PCR kits, and primers were products of TaKaRa Biotechnology, Dalian, China. DyLight 549-conjugated goat anti-rabbit IgG and Hoechst 33342 were obtained from CWBio, Beijing, China. Antibodies for immunoblot analysis were obtained from the following suppliers: GSK-3β and NF-κB p65 from Cell Signaling Technology, USA; survivin, β-actin, histone, and goat anti-rabbit IgG-horseradish peroxidase (HRP) from Santa Cruz Biotechnology, CA. Analysis of GSK-3β expression in ALL cells by immunofluorescence microscopy BMMC that had been attached to glass slides by cytocentrifugation (StatSpin InC, USA) were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.3% Triton X-100 for 10 min at room temperature, and blocked with 3% bovine serum albumin (BSA) for 30 min.