Glycogen synthase kinase-3β inhibition induces nuclear factor-κB-mediated apoptosis in pediatric acute lymphocyte leukemia cells.
BACKGROUND
Molecular therapies that target genetic abnormalities in leukemic cells and their affected signaling pathways have been emerging in pediatric acute lymphoblastic leukemia (ALL). Glycogen synthase kinase-3β (GSK-3β) has recently been found to positively regulate the activity of nuclear factor-κB (NF-κB). Here, we investigated the relationship between GSK-3β inhibition and NF-κB in apoptosis of pediatric primary leukemia cells obtained from 39 newly diagnosed ALL children in China.


METHODS
Bone marrow mononuclear cells (BMMC) were isolated by density gradient centrifugation from the heparinized aspirates of children with ALL. We used immunofluorescence staining to detect nuclear GSK-3β in these cells. After treatment with chemically distinct GSK-3β inhibitors in vitro, NF-κB transcriptional activity was identified by means of western blotting and electrophoretic mobility shift assay (EMSA). NF-κB-mediated apoptosis was detected by Annexin V-PE/7-AAD double-staining flow cytometry. The expression level of the survivin gene was detected by reverse-transcriptase polymerase chain reaction (RT-PCR).


RESULTS
GSK-3β significantly accumulates in the nuclei of ALL cells than in the nuclei of control cells. Cell death induced by GSK-3β inhibition in ALL cells was mediated by a downregulation of NF-κB p65 transcriptional activity. GSK-3β inhibition significantly decreased the expression of the NF-κB target gene survivin.


CONCLUSIONS
These results indicate that inhibition of GSK-3β downregulates the NF-κB activation pathway, leading to suppression of the expression of an NF-κB-regulated gene and promotion of apoptosis in ALL cells in vitro. Furthermore, our findings suggest that GSK-3β or NF-κB is a potential therapeutic target in the treatment of pediatric ALL.
Laboratory of Oncology, Affiliated Children's Hospital, Chongqing Medical University, No,136, Zhongshan 2nd Road, Yuzhong District, Chongqing 86 400014, China.
Yanni
Y
Hu
Xiaoyan
X
Gu
Ruiyan
R
Li
Qing
Q
Luo
Youhua
Y
Xu
D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002469:Cell Separation; D002478:Cells, Cultured; D002648:Child; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis; D016328:NF-kappa B / Q000378:metabolism*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000378:metabolism* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D000077022:Survivin
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D016328:NF-kappa B; D000077022:Survivin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3