1. GPC3 promotes hepatocellular carcinoma growth by stimulating the canonical Wnt pathway (A-B) GPC3 induces the stabilization of cytoplasmic β-catenin.
GPC3 promotes hepatocellular carcinoma growth by stimulating the canonical Wnt pathway (A-B) GPC3 induces the stabilization of cytoplasmic β-catenin. Western blot analysis of cytoplasmic β-catenin in PLC-PRF-5 (A) or HLF (B) cells transfected with GPC3, GPC3ΔGAG (ΔGAG), or vector alone (EF). Actin or CDK4 were used as loading controls. One representative result of three. C-E, soluble GPC3 lacks stimulatory activity. PLC-PRF-5 cells were transfected with expression vectors containing wild-type GPC3 (GPC3) or a mutant GPC3 lacking the GPI-anchoring domain (GPC3ΔGPI) and vector alone (EF). C, total RNA was isolated from these cells and cDNA was synthesized using reverse transcriptase (RT, +). As a negative control, the same RNAs were incubated in the absence of reverse transcriptase (−). PCR was then done using GPC3 or β-actin specific primers. D, proliferation rate of the transfected cells was analyzed and the number of cells counted by quadruplicates at the indicated time points. Representative experiments of three. E, Western blot analysis of cytoplasmic levels of β-catenin in the indicated transfected cells using actin as loading control. One representative result from three independent experiments. F-H, DKK1 inhibits GPC3-induced proliferation. F, proliferation rate of PLC-PRF-5 [EF, GPC3, or ΔGAG-GPC3 (ΔGAG)] was assessed in the presence of conditioned medium from 293T cells transfected with DKK1 (DKK1-CM) or vector control (pCDNA-CM). Cell counts were done by quadruplicates at the indicated time points. The degree of DKK1-induced inhibition of cell proliferation compared with control conditioned medium (as percentage) is indicated in (G). H, Western blot analysis shows the presence of DKK1 in the conditioned medium.
Mariana I. Capurro et al. Cancer Res 2005;65:
©2005 by American Association for Cancer Research