Delta-like 3 is silenced by methylation and induces apoptosis in human hepatocellular carcinoma

Abstract

The genetic and epigenetic events of hepato-carcinogenesis are relatively poorly understood. By analyzing genes from human hepatocellular carcinoma (HCC) with restriction landmark genomic scanning, several aberrantly methylated genes, including Delta-like 3 (DLL3), have been isolated. In this study, we investigated the function of DLL3 in hepatocarcinogenesis. Methylation of the DLL3 gene in HCC cell lines was investigated with methylation-specific PCR and expression of DLL3 mRNA in HCC cells was examined by RT-PCR. Reactivation of DLL3 expression by treatment with a demethylating agent was examined in methylation-silenced HuH2 cells. Human DLL3 cDNA was cloned and DLL3 function was examined by restoring DLL3 expression in HuH2 cells. The effects of DLL3 on cell growth were evaluated by colony formation assay. Induction of cell death by overexpression of DLL3 was examined by flow cytometric assay using Annexin V and PI. Apoptotic cells were detected by TUNEL staining and the amount of single-stranded DNA was measured by ELISA. As a result, the promoter region of the DLL3 gene was methylated in four of ten HCC cell lines. This aberrant methyl-ation correlated well with the suppression of RNA expression and a demethylating agent reactivated DLL3 expression in methylation-silenced HCC cells. Interestingly, the restoration of DLL3 in the methylation-silenced HuH2 cells led to growth suppression on colony formation assay. Flow cytometric assay with Annexin V and PI showed that this growth suppression by DLL3 expression is associated with the induction of apoptosis. Furthermore, these apoptotic effects were confirmed by TUNEL staining and measurement of single-stranded DNA. These results suggest that DLL3 was silenced by methylation in human HCC and that it negatively regulates the growth of HCC cells.

Figure 1

DLL3 mRNA expression and aberrant methylation of DLL3 gene. (A) RT-PCR analysis of DLL3 mRNA from 10 HCC cell lines. No mRNA was detected in HuH1, HuH2, HiuH4, Alex or Kim-1 cells. (B) Methylation of DLL3 was analyzed with MSP using the primer set around the translation start site in 10 HCC cell lines, a non-tumorous liver and lymphocytes. Visible bands in M-lanes (arrows) are methylated PCR products with methylation-specific primers. Visible bands in U-lanes are unmethylated PCR products with unmethylation-specific primers. (C) Three methylated cell lines (HuH2, Alex and Kim1) were treated with or without 5-Aza-2′-deoxycytidine (5-Aza-dC) and DLL3 mRNA expression was analyzed by RT-PCR.

Figure 2

Growth suppression by DLL3. Methylation-silenced cells (HuH2 and Kim1) were transfected with either DLL3 expression vector or backbone vector and selected for 4 weeks with G418.

Figure 3

Induction of cell death by DLL3. Methylation-silenced HuH2 cells were transiently transfected with either (A) backbone vector or (B) DLL3 expression vector and cell death was detected by flow cytometry using PI and Annexin V staining. (C) Apoptotic cells were detected with the TUNEL method in HuH2 cells transfected with either backbone vector or DLL3 expression vector and numbers of TUNEL-positive cells were counted under microscopy. (D) Amount of single-stranded DNA was compared between control and DLL3-transfected cells by ELISA.

Figure 4

Notch activation by DLL3. Methylation-silenced HuH2 cells were transiently transfected with either DLL3 expression vector or backbone vector and activation of Notch1 was detected by western blot analysis.