Hu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:154 http://www.jeccr.com/content/29/1/154 Page 3 of 8 and the reaction mixture was incubated for 20 min at room temperature. For reactions involving competitor oligonucleotides, the unlabeled competitor and the labeled probes were premixed before addition to the reaction mixture. The samples were analyzed on 6.5% acrylamide gels and electrophoresis was carried out at 180 V for 70 min. Gel contents were transferred to binding-membrane, dried, incubated with streptavidin- HRP, and exposed with an intensifying screen. Reverse-transcriptase polymerase chain reaction analysis (RT-PCR) Total RNAs were extracted according to the manufac- turer’s instructions and were reverse-transcribed using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). Of a 20 μl cDNA reaction, 5 μl was used as template for ampli- fication with the following specific primers. For human survivin forward: 5’-TCCACTGCCCCACTGAGAAC-3’ and reverse 5’-TGGCTCCCAGCCTTCCA-3’; for human GAPDH forward: 5’-CAGCGACACCCACTCCTC-3’ and reverse 5’-TGAGGTCCACCACCCTGT-3’. The PCR was performed with the first denaturation step at 94°C for 5 min, and 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 30 s, and extension at 72°C for 1 min. The PCR reaction products were detected with gel electrophoresis and ultraviolet transillumination. Apoptosis assay BMMC (1 × 106 cells) were exposed to GSK-3b inhibitors or a matched concentration of diluents. After the treat- ment, cells were rinsed twice in cold PBS, resuspended in binding buffer, and then analyzed for apoptosis level by a PE-labeled Annexin-V/7-AAD assay. These cells were directly analyzed in a FACScan (BD FACS Calibur Co., USA) with a sample size of at least 10,000 cells gated on the basis of forward and side scatter. Storing and proces- sing of data were accomplished using FACScan software. Statistical analysis Results are expressed as mean ± standard deviation. Sta- tistical analysis was conducted using SPSS 15.0 software. Differences between groups were examined for statistical significance using a one-way analysis of variance and Student’s t-test; P values less than 0.05 were considered statistically significant. Results GSK-3b accumulated in the nucleus of primary ALL cells Using immunofluorescence staining, we identified the localization of GSK-3b in ALL BMMC in 8 children with ALL. As shown in Figure 1, we found nuclear accumulation of GSK-3b in 6 primary pediatric ALL BMMC samples, whereas it was not detected in the nucleus of control BMMC. Inhibition of GSK-3b suppressed the binding of NF- B to the DNA in ALL cells GSK-3b has been shown to play a critical role in NF- B-mediated survival of cancer cells. The aberrant accu- mulation of GSK-3b in nuclei of ALL cells prompted us to examine the effect of GSK-3b inhibition on NF- B activity. Using primary ALL cells, we tested ex vivo the effect of 2 chemically distinct small-molecule inhibitors of GSK-3b: SB216763 (ATP-competitive, arylindolema- leimide) [11], and LiCl (non-ATP-competitive) [12]. Forty-eight hours after GSK-3b inhibitors treatment, we estimated the level of GSK-3b inhibition by detection of the cytosolic/nuclear level of GSK-3b by western blot. We found that both the distinct GSK-3b inhibitors can decrease the level of GSK-3b in nuclear extracts of ALL cells (Figure 2). With the same treatments, nuclear levels of NF- B p65 in ALL cells were not significantly chan- ged (Figure 2). To further investigate the role of GSK- 3b in the regulation of NF- B activity, we detected NF- B DNA binding activity by EMSA. The data show that GSK-3b inhibition in ALL cells decreased the binding of NF- B p65 to its target gene promoter (Figure 3). Taken together, these results suggest that GSK-3b affects NF- B activity at the transcriptional level in pediatric ALL cells. Pharmacologic inhibition of GSK-3b induced apoptosis in ALL cells Since NF- B is a potential target of GSK3b-dependent cell survival pathway, we detected apoptotic cells as an Annexin-V+/7-AAD+ population within DMSO or SB216763-treated malignant cells cultured ex vivo from each of the 11 patients with ALL by using Annexin-V staining and flow cytometry. Although the mean num- ber of apoptotic cells was 12% in DMSO-treated ALL cells, the apoptotic cell fraction in the SB216763-treated cells was significantly higher; the mean number of apop- totic cells reached 36% (SB216763, 5 μM), 52% (SB216763, 10 μM) and 70% (SB216763, 15 μM) after 48 h of exposure (Figure 4A, B; P < 0.001). It demon- strated that the number of apoptotic cells dose-depen- dently increased with SB216763 treatment. We also evaluated the apoptotic effect of LiCl, another GSK-3b inhibitor, on ALL cells. LiCl, at subtoxic concentrations, induced NF- B-mediated apoptosis in a dose-dependent manner (Figure 4C; P < 0.05). These results confirmed that GSK-3b suppression leads to ALL apoptosis. GSK-3b inhibitors had no significant effect on cell apoptosis in normal BMMC To further evaluate whether GSK-3b inhibition specifi- cally induced apoptosis in ALL cells, we examined the effect of GSK-3b inhibitors on normal BMMC. GSK-3b inhibition was previously shown to preserve umbilical