Artificially accumulated beta-catenin inhibits proliferation and induces neurite extension of neuroblastoma cell line NB-1 via up-regulation of trkA.
Recent evidence suggests an association between up-regulation of beta-catenin/Wnt signaling pathway and neuronal differentiation of neuroblastoma. We overexpressed beta-catenin into a human neuroblastoma cell line NB-1 and observed its effect on cellular morphology, growth potential and alteration in a known differentiation related gene, trkA. Expression plasmids containing wild-type and mutated forms of beta-catenin gene were transfected into NB-1 cells, using liposome-based transfection method. The mutated forms were a deletion of three nucleotides of codon 45 and a large deletion involving the whole exon 3. In the transient transfection model, cell viability assay demonstrated significant negative effect of mutated beta-catenin transfection, but not wild-type, on the cell proliferation. To investigate impacts of beta-catenin overexpression in detail, a stable transfection model was established. Clones with comparable expression of beta-catenin at the mRNA level were selected. Only the selected clones with mutated form of beta-catenin exhibited neurite extension pattern and stunned cell proliferation, in association with higher accumulation of total cellular beta-catenin protein as evidenced by Western blot and immunocytochemistry. Cell cycle progression demonstrated significantly higher G0-G1 fraction in each stable cell clone with beta-catenin expression plasmid. In addition, retarded G1/S transition was observed exclusively in the cell clones with mutated form. Concomitantly with overexpressed beta-catenin, up-regulations of trkA and Ha-ras were also identified. Our study suggests a potential availability of beta-catenin/Wnt signaling pathway as a target of molecular manipulation for treatment of high-risk neuroblastoma and a potential association between the pathway and the trkA/neurotrophin cascades.
Sangkhathat
Surasak
S
Department of Pediatric Surgery, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
Nara
Keigo
K
Kusafuka
Takeshi
T
Yoneda
Akihiro
A
Fukuzawa
Masahiro
M
D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D009154:Mutation; D016501:Neurites / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism*; D020917:Receptor, trkA / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D015854:Up-Regulation; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
D051176:beta Catenin; D020917:Receptor, trkA