β-catenin accumulation in nuclei of hepatocellular carcinoma cells up-regulates glutathione-s-transferase M3 mRNA

Abstract

Aim: To identify the differentially over-expressed genes associated with β-catenin accumulation in nuclei of hepatocellular carcinoma (HCC) cells.

Methods: Differentially expressed genes were identified in radiation-induced B6C3 F1 mouse HCC cells by mRNA differential display, Northern blot and RT-PCR, respectively. Total glutathione-s-transferase (GST) activity was measured by GST activity assay and β-catenin localization was detected with immunostaining in radiation-induced mouse HCC cells and in HepG2 cell lines.

Results: Two up-regulated genes, glutamine synthetase and glutathione-s-transferase M3 (GSTM3), were identified in radiation-induced mouse HCC cells. Influence of β-catenin accumulation in nuclei of HCC cells on up-regulation of GSTM3 mRNA was investigated. The nearby upstream domain of GSTM3 contained the β-catenin/Tcf-Lef consensus binding site sequences [5'-(A/T)(A/T) CAAAG-3'], and the total GST activity ratio was considerably higher in B6C3F1 mouse HCC cells with β-catenin accumulation in nuclei of HCC cells than in those without β-catenin accumulation (0.353 ± 0.117 vs. 0.071 ± 0.064, P < 0.001). The TWS119 (a distinct GSK-3β inhibitor)-induced total GST activity was significantly higher in HepG2 cells with β-catenin accumulation than in those without β-catenin accumulation in nuclei of HCC cells. Additionally, the GSTM3 mRNA level was significantly higher at 24 h than at 12 h in TWS119-treated HepG2 cells.

Conclusion: β-catenin accumulation increases GST activity in nuclei of HCC cells, and GSTM3 may be a novel target gene of the β-catenin/Tcf-Lef complex.

Keywords: Differential display analysis; Glutathione-s-transferase M3; Hepatocellular carcinoma; Radiation; β-catenin accumulation.

Figure 1

Identification of differentially displayed cDNA fragments from paired hepatocellular carcinoma cells (T) and nontumorous liver tissues (N). A: Differentially displayed PCR products (“I” and “II”) amplified with AP-10 primer and T₁₂MA. Lanes 1-3 denote the three B6C3 F1 mouse samples, respectively; B: Recovered “I” and “II” from the dried DNA sequencing gel reamplified by PCR. MW lane shows the pUC118 DNA fragments cut by HapII (a restriction enzyme) as molecular weight markers; C: Nucleotide sequences of the bands shown in A. Flanking sequences of T₁₂MA primers are underlined. The two insert-containing fragments were sequenced and identified as gene fragments of GLNS and GSTM3 in comparison with those of nucleotide in GenBank.

Figure 2

Levels of glutamine synthetase and glutathione-s-transferase M3 mRNA and expression of β-catenin. A: T denotes surgically resected B6C3F1 mouse hepatocellular carcinoma (HCC) tissue samples and N denotes matched nontumorous liver tissue samples; B: Representative immunofluorescence photomicrographs for β-catenin (red photomicrographs) in HCC tissue samples. B1 denotes negative β-catenin staining in nuclei of HCC cells, B2 denotes positive β-catenin staining in nuclei of HCC cells, and white arrows indicate β-catenin detected in nuclei of HCC cells. Bars: 50 μm. GLNS: Glutamine synthetase; GSTM3: Glutathione-s-transferase M3.

Figure 3

Identification of β-catenin/Tcf-Lef consensus binding sites with three β-catenin/Tcf-Lef consensus binding site sequences [5'-(A/T)(A/T) CAAAG-3'] located at the nearby upstream domain of mouse GSTM3 by searching GenBank.

Figure 4

Total glutathione-s-transferase activity (A) and glutathione-s-transferase M3 mRNA expression (B) in HepG2 cells. ^aP < 0.05, ^bP < 0.01 vs TWS119 at 24 h by one-way analysis of variance followed by Bonferroni’s test.