Hu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:154 http://www.jeccr.com/content/29/1/154 Page 5 of 8 Figure 3 Effects of GSK-3b inhibitors on DNA binding activity of NF- B in nuclear extracts of ALL cells. After 48 h of treatment with GSK-3b inhibitors, ALL cells nuclear extracts were prepared and assayed for NF- B activation by EMSA as described under “Methods.” GSK-3b inhibitors resulted in a reduction in NF- B DNA binding activity when compared to control condition (untreated ALL cells). The data shown are representative of 3 independent experiments. 1: negative control; 2: positive control; 3: untreated ALL cells; 4: ALL cells treated with LiCl (10 mM); 5: ALL cells treated with SB216763 (10 μM). target gene survivin in ALL cells relative to control cells (Figure 5). After completion of these experiments, we summarized the data and represented it as a mean value (Figure 5 legend). SB216763 (10 μM) and LiCl (10 mM) treatment resulted in a 47.7% and 25% reduction in sur- vivin mRNA levels, respectively. Moreover, the levels of survivin mRNA decreased dose-dependently after treat- ment with both LiCl and SB216763. These results indi- cate that the inhibition of GSK-3b does not affect the nuclear accumulation of NF- B p65 but might alter the ability of NF- B to regulate target gene promoters in ALL cells. Discussion GSK-3b has recently been shown to be a crucial enzy- matic regulator of cancer cell survival in human tumori- genesis [14,15]. GSK-3b protein is known to be overexpressed in human prostate [16], pancreatic [17], and colon [14] carcinomas. Normally, GSK-3b is expressed in the cytoplasm of cells. Recent studies have shown that GSK-3b could shuttle from the cytoplasm to the nucleus in pancreatic cancer cell lines and in most poorly differentiated human pancreatic adenocarcinomas [17], and in human CLL B cells [9]. In this study, we found aberrant nuclear accumulation of GSK-3b in cells obtained from children with ALL, whereas GSK-3b was not detected in the nucleus of control cells. GSK-3b transposition was thought to participate in the regulation of gene transcription through the phos- phorylation of transcription factors [18]. NF- B, an important transcription factor also involved in the reg- ulation of cell proliferation, differentiation, and apop- tosis, is deregulated in many human tumors [19,20]. Previous studies have suggested that NF- B transcrip- tional activity is regulated by GSK-3b [7]. Genetic depletion of GSK-3b by RNA interference suppresses basal NF- B transcriptional activity, leading to decreased pancreatic cancer cell proliferation and sur- vival [8]. Recently, it has been demonstrated that GSK- 3b positively regulates NF- B-mediated drug resistance in acute myeloid leukemia (AML) [21]. In this study, we tested ex vivo the effect of 2 chemically distinct small-molecule inhibitors of GSK-3b at subtoxic con- centrations: LiCl, a well-known GSK-3b inhibitor, and SB216763, a widely used maleimide-containing GSK-3b inhibitor. Using the pharmacological inhibitors of GSK-3b, we estimated the level of GSK-3b inhibition by detecting the protein levels of GSK-3b in cytosolic and nuclear extracts through western blot assay. In ALL cells, we found that both inhibitors led to deple- tion of the nuclear pool of GSK-3b, whereas no change was found in the cytoplasm extract. Moreover, we found that GSK-3b inhibition in ALL cells did not pre- vent NF- B relocation from the cytoplasm to the nucleus, but the inhibition affected the transcriptional repression of NF- B, as shown by EMSA analysis. Similar to previous studies [7], our studies on pediatric ALL cells show that NF- B can be regulated by GSK- 3b at the level of the nuclear transcriptional complex. The exact mechanism by which GSK-3b affects NF- B transcriptional activity is still unknown. GSK-3b influ- ences NF- B-mediated gene transcription in pancreatic cancer cells at a point distal to the I B kinase complex [7]. Recent data have demonstrated that GSK-3b may contribute to p65/p50 binding to the promoters and transcriptional activation of NF- B in CLL cells by regu- lating histone modification [6]. However, the underlying mechanism by which GSK-3b regulates p65 NF- B binding to target gene promoters has not been defined. NF- B is known as an important factor of cancer cell survival in human tumorigenesis [22]. In this report, we found that GSK-3b suppression sensitized ALL cells to NF- B-mediated apoptosis. Both SB216763 and LiCl have been shown to induce ALL cells apoptosis. In addi- tion, we observed that there is no significant effect on apoptosis induced by GSK-3b inhibitors in control BMMC. A relevant finding was that GSK-3b was not detected in the nucleus of control BMMC but was detected in the nuclei of ALL cells. Taken together, our