Article Figures & Data
Figures
- Figure 1.
Overview of the canonical Wingless-type MMTV integration site family (WNT)/β-catenin signaling. In the absence of WNT signal, β-catenin is captured by adenomatous polyposis coli (APC) and axin within a destruction complex, phosphorylated (P) by casein kinase Iα (CKIα) and glycogen synthase kinase-3β (GSK-3β) and targeted for ubiquitination (Ub) and proteasomal degradation. Transcription by transcription factor/lymphoid-enhancer-binding factor (TCF/LEF) is off due to the binding of repressors. A pool of β-catenin is engaged in the interactions with cadherins for cell–cell adhesion. The binding of WNT3a ligand to frizzled and low-density lipoprotein receptor-related proteins 5/6 (LRP5/6) co-receptors determines the inactivation of the destruction complex. This allows β-catenin to accumulate in the cytoplasm and translocate to the nucleus, where it forms a transcriptionally active complex with TCF/LEF family members.
- Figure 2.
Immunohistochemistry of Wingless-type MMTV integration site family (WNT)/β-catenin signaling pathway. A: Normal cortex. No WNT3a expression, ×200. B: Grade II oligodendroglioma. Moderate WNT3a expression on the cell membranes, ×400. C: Grade II astrocytoma. No WNT3a expression, ×400. D: Glioblastoma. Diffuse cytoplasmic and membranous WNT3a expression, ×400. E: Normal white matter. Slight membranous β-catenin expression, ×200. F: Grade II oligodendroglioma. Diffuse membranous β-catenin expression, ×400. G: Grade III astrocytoma. Moderate cytoplasmic and membranous β-catenin expression, ×400. H: Glioblastoma. High cytoplasmic and membranous β-catenin expression, ×400. I: Glioblastoma. Cytoplasmic and membranous β-catenin expression in proliferative endothelial cells, ×200. J: Glioblastoma. Nuclear and cytoplasmic β-catenin expression in hyperplastic endothelial cells, as indicated by arrows, ×630. K: Grade III astrocytoma. Granular expression of phospho-β-catenin, ×200. L: Glioblastoma. Phospho-β-catenin expression in the cytoplasm of proliferative endothelial cells, ×200. M: Glioblastoma. Positivity for phopsho-GSK-3β, ×200. All specimens stained with 3,3’ diaminobenzidine.
- Figure 3.
Wingless-type MMTV integration site family member 3a (WNT3a) and β-catenin immunohistochemical expression with respect to tumor type and histological malignancy grade.
- Figure 4.
Correlations among transcription factor 4 (TCF4), Ki-67/MIB-1 and cyclin D1 labeling indices (LIs). A: Glioblastoma. High nuclear TCF4 expression in several tumor cells, ×200. B: Grade II oligodendroglioma. TCF4 intensely positive nuclei, ×400. C: Glioblastoma. Several cyclin D1-positive nuclei in the same area as in (A), ×200. D: Several Ki-67/MIB-1-positive nuclei in the same area as in (A), ×200. E: Several Ki-67/MIB-1-positive nuclei in the same area as in (B), ×400. F: Glioblastoma. Nuclei of proliferative endothelial cells with high TCF4 expression, as indicated by arrows, ×400. All stained with 3,3’ diaminobenzidine.
- Figure 5.
Correlation among transcription factor 4 (TCF4), Ki-67/MIB-1 and cyclin D1 labeling indices (LIs). A: Linear regression analysis between TCF4 and Ki-67/MIB-1 LIs (r=0.761, p<0.0001). B: TCF4 and cyclin D1 LIs (r=0.739, p<0.0001). C: Cyclin D1 and KI-67/MIB-1 LIs (r=0.873, p<0.0001).
- Figure 6.
Immunofluorescence (IF) and immunocytochemistry (ICC) of glioblastoma stem cells. IF, Neurospheres: A: High cytoplasmic Wingless-type MMTV integration site family member 3a (WNT3a) expression. B: High cytoplasmic β-catenin expression. C: High nuclear transcription factor 4 (TCF4) expression. D: Cytoplasmic phopsho-glycogen synthase kinase-3β (GSK-3β) expression. IF, Adherent cells: E: Very poor cytoplasmic WNT3a expression. F: Negative β-catenin expression. G: Negative TCF4 expression. H: Positive phopsho-GSK-3β expression. IF, Adherent cells after 50 μM temozolomide treatment for 48 h: I: Positive cytoplasmic WNT3a expression. J: Positive cytoplasmic β-catenin expression. K: Nuclear and cytoplasmic TCF4 expression. L: Poor cytoplasmic phopsho-GSK-3β expression. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole. All ×400 magnification. ICC, Neurospheres: M: Cytoplasmic WNT3a expression. N: Cytoplasmic and nuclear β-catenin expression. O: Nuclear TCF4 expression. ICC, Adherent cells: P: Prevailing nuclear TCF4 expression. All 3,3’ diaminobenzidine, ×400 magnification.
- Figure 7.
Western blot analysis. A: Representative analysis of two independent experiments displaying Wingless-type MMTV integration site family member 3a (WNT3a) and β-catenin expression in whole-cell extracts from glioblastoma cell lines, cultured as neurospheres or adherent cells. B: Quantitative analysis of WNT3a and β-catenin levels normalized to α-tubulin, showing higher amounts of the two proteins in neurospheres than adherent cells. C, D: β-Catenin expression by western blotting analysis in cytosolic and nuclear extracts from glioblastoma cell lines and tumor tissues.
- Figure 8.
WNT3A expression levels by quantitative real-time polymerase chain reaction in gliomas and glioblastoma cell lines. A: Relative WNT3A expression levels in glioma tumor samples of different types and histological malignancy grades. B: Relative WNT3A expression levels in a panel of 24 glioblastoma cell lines, cultured as neurospheres (NS) or adherent cells (AC). Reported values are the average ± standard deviation (SD) of at least two independent experiments in triplicate.
- Figure 9.
Relationship of Wingless-type MMTV integration site family member 3a (WNT3a) and β-catenin expression with survival in glioma patients. Kaplan–Meier survival curves for overall survival (OS) with respect to WNT3a expression (A) and subcellular localization of β-catenin expression (B). M: membranous; C: cytoplasmic; N: nuclear.
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