Transcriptional silencing of the DLC-1 tumor suppressor gene by epigenetic mechanism in gastric cancer cells

Abstract

Southern analysis showed that seven of nine human gastric cancer cell lines did not express DLC-1 mRNA, but contained the DLC-1 gene. To identify the mechanism of the loss of DLC-1 mRNA expression in these cell lines, we investigated the methylation status of DLC-1 gene by using methylation-specific PCR (MSP) and Southern blot, and found that five of seven DLC-1 nonexpressing gastric cancer cell lines were methylated in the DLC-1 CpG island. Treatment with 5-aza-2 0 -deoxycytidine (5-Aza-dC) induced DLC-1 mRNA expression in the gastric cancer cell lines that have the methylated alleles. Studies using SNU-601 cell line with methylated DLC-1 alleles revealed that nearly all CpG sites within DLC-1 CpG island were methylated, and that the in vitro methylation of the DLC-1 promoter region is enough to repress DLC-1 mRNA expression, regardless of the presence of transcription factors capable of inducing this gene. In all, 29 of 97 (30%) primary gastric cancers were also shown to be methylated, demonstrating that methylation of the DLC-1 CpG island is not uncommon in gastric cancer. In addition, we demonstrated that DLC-1 mRNA expression was induced, and an increase in the level of acetylated H3 and H4 was detected by the treatment with trichostatin A (TSA) in two DLC-1 nonexpressing cell lines that have the unmethylated alleles. Taken together, the results of our study suggest that the transcriptional silencing of DLC-1, by epigenetic mechanism, may be involved in gastric carcinogenesis.

The major 8p22 tumor suppressor DLC1 is frequently silenced by methylation in both endemic and sporadic nasopharyngeal, esophageal, and cervical carcinomas, and inhibits tumor cell colony formation

“…We also showed that DLC1 functions as a TSG in NPC and esophageal carcinoma cells. As DLC1 methylation has not been reported in these three tumors before, our results add more candidates to the reported list of cancers (liver, breast, colon, gastric, lung and prostate) with epigenetic abnormalities of this TSG (Kim et al, 2003;Plaumann et al, 2003;Wong et al, 2003;Yuan et al, 2003aYuan et al, , b, 2004. Interestingly, in one of our independent studies screening for downregulated genes in NPC cell lines using whole-genomic microarray expression profiling combined with pharmacologic demethylation, DLC1 was also identified as one of the downregulated target genes (Tao et al, manuscript in preparation).…”

“…DLC1 was reported to be a functional TSG frequently inactivated by promoter methylation in multiple carcinomas (Kim et al, 2003;Plaumann et al, 2003;Wong et al, 2003;Yuan et al, 2003aYuan et al, , b, 2004. DLC1 is located at 8p22 -a region where we also detected heterozygous deletion in some esophageal cell lines by conventional CGH (Tang et al, 2001), but no study of DLC1 has been performed in esophageal carcinoma yet.…”

“…It has high sequence similarity (86%) with the rat p122RhoGAP, a GTPase-activating protein for the Rho family of proteins involved in cell cytoskeleton organization and other functions (Homma and Emori, 1995;Etienne-Manneville and Hall, 2002;Moon and Zheng, 2003). More recent studies have demonstrated that DLC1 is an important TSG, being inactivated by aberrant promoter methylation in multiple carcinomas, including liver, colon, gastric, lung, breast and prostate carcinomas (Kim et al, 2003;Plaumann et al, 2003;Wong et al, 2003;Yuan et al, 2003aYuan et al, , b, 2004, while no study has been performed for NPC yet. Here, we report on the epigenetic alterations of DLC1 in NPC and other tumors (esophageal and cervical carcinomas) which have not been studied for this gene.…”

Aberrant methylation of integrin α4 gene in human gastric cancer cells

“…In addition, DNA methylation and the chromatin condensation of integrin aL were detected in fibroblasts, and were suggested to contribute to the tissue-specific expression of integrin aL (Akama et al, 1997;Lu et al, 2002). Previously, we reported that DNA methylation is one of the predominant mechanisms for inactivating various genes, like, TGF-b type I receptor, TIMP-3, p16, COX-2 and DLC-1, during gastric carcinogenesis (Kang et al, 1999;Kang et al, 2000;Song et al, 2000Song et al, , 2001Kim et al, 2003). These findings led us to investigate whether integrin a4 expression could be regulated by DNA methylation, because integrin a4 expression is very low in our gastric carcinoma cells (Figure 1a).…”

Gene silencing of TSPYL5 mediated by aberrant promoter methylation in gastric cancers

“…5 The promoter hypermethylation of multiple tumor suppressor genes resulting in gene silencing has been recognized as an important mechanism in gastrointestinal carcinogenesis. [6][7][8][9][10][11][12][13][14][15] In a previous study, TSPYL5 was identified as one of the genes induced after treatment with DNA methylation and histone deacetylation inhibitors in glioma cell lines using microarray analysis. 16 The TSPYL5 showed a high frequency of DNA methylation-mediated silencing in both glioma cell lines and primary glial tumors.…”