Hu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:154 http://www.jeccr.com/content/29/1/154 Page 2 of 8 suggested that inhibition of GSK-3b abrogates NF- B binding to its target gene promoters through an epige- netic mechanism and enhances apoptosis in chronic lymphocytic leukemia (CLL) B cells ex vivo [9]. Conse- quently, inhibition of GSK-3b activity has been proposed to play a role in the regulation of the NF- B signaling pathway that elicits cellular survival responses. However, little is currently known about the signifi- cance of GSK-3b to pediatric ALL cell survival. ALL initiates and progresses in the bone marrow (BM). In the present study, we demonstrated that GSK-3b accu- mulates in the nuclei of primitive pediatric ALL cells from the BM. GSK-3b inhibition leads to suppression of NF- B transcriptional activity and induces apoptosis through the transcriptional downregulation of the survi- vin 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, cytoge- netic, and molecular classification. The informed con- sent 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 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 nonmalig- nant cells with immunomagnetic beads [10]. Reagents and antibodies The GSK-3b 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 con- centrations of 5 and 10 mM. The high-quality fetal bovine serum and RPMI 1640 medium were products 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 follow- ing suppliers: GSK-3b and NF- B p65 from Cell Signal- ing Technology, USA; survivin, b-actin, histone, and goat anti-rabbit IgG-horseradish peroxidase (HRP) from Santa Cruz Biotechnology, CA. Analysis of GSK-3b expression in ALL cells by immunofluorescence microscopy BMMC that had been attached to glass slides by cyto- centrifugation (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. The cells were subsequently subjected to immunofluorescence staining to detect GSK-3b expression with the same primary antibody (diluted 1:200) used for immunoblotting. After a rinse in PBS, cells were incubated with secondary DyLight 549- conjugated goat anti-rabbit IgG antibody. Nuclei were counterstained with Hoechst 33342. SlowFade mounting medium was used. Images were acquired using the Leica Application Suite on a fluorescence microscope (Olym- pus, Japan) equipped with a 40 ×/0.75 oil DIC objective. Western blotting Leukemic cells (1 × 107) undergoing different treatments were rinsed with PBS and lysed in buffer. Nuclear/Cyto- solic fractionation was performed using nuclear-cytosol extraction kit (KENGEN Biotechnology, Nanjing, China) according to the manufacturer’s instructions. Protein sample concentration was quantified by the BCA method and an equal amount (30 μg of cytosolic or nuclear protein extract) of proteins was loaded in each well of a 10% SDS polyacrylamide gel. Cell extracts were separated by polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene difluoride membrane (PVDF). Primary antibodies against GSK-3b, NF- B p65, survivin, b-actin, and histone were used. HRP-conju- gated anti-IgG was used as the secondary antibody. Western blot band intensities were quantified using Quantity One software (Bio-Rad Laboratories, Inc., USA). Electrophoretic mobility shift assays (EMSA) for NF- B Nuclear lysates were prepared and protein concentra- tions were measured by the BCA protein assay accord- ing to the manufacturer’s manual. Equivalent amounts of nuclear extract proteins (2 μg) were preincubated in 1 μl of binding buffer for 20 min at room temperature. Then, a biotin-labeled oligonucleotide probe was added,