title: 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.
authors: Department of Anatomy and Cell Biology, Osaka Medical College, Takatsuki, Osaka, Japan. an2011@art.osaka-med.ac.jp
authors: Kentaro
authors: K
authors: Maemura
authors: Hirohide
authors: H
authors: Yoshikawa
authors: Kazutake
authors: K
authors: Yokoyama
authors: Teruo
authors: T
authors: Ueno
authors: Hitomi
authors: H
authors: Kurose
authors: Kazuhisa
authors: K
authors: Uchiyama
authors: Yoshinori
authors: Y
authors: Otsuki
mesh_terms: D017209:Apoptosis; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004789:Enzyme Activation; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051881:Receptor, Notch1 / Q000378:metabolism
chemical_list: C496666:DLL3 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; D012333:RNA, Messenger; D051881:Receptor, Notch1