Volume 131, Issue 6, Pages 1835-1843 (December 2006) Correlation Between the Single-Site CpG Methylation and Expression Silencing of the XAF1 Gene in Human Gastric and Colon Cancers  Bing Zou, Chor Sang Chim, Hui Zeng, Suet Yi Leung, Yi Yang, Shui Ping Tu, Marie C.M. Lin, Jide Wang, Hua He, Shi Hu Jiang, Yun Wei Sun, Li Fen Yu, Siu Tsan Yuen, Hsiang Fu Kung, Benjamin C.Y. Wong  Gastroenterology  Volume 131, Issue 6, Pages 1835-1843 (December 2006) DOI: 10.1053/j.gastro.2006.09.050 Copyright © 2006 AGA Institute Terms and Conditions

Figure 1 Demethylation up-regulated the expression of XAF1 in gastric and colon cancer cell lines. Expression of XAF1 was up-regulated in (A) 6 of 8 human colon cancer cell lines and (B) 3 of 4 gastric cancer cell lines after treatment with 5′-AZA for 48 hours. (C) The change of protein levels of XAF1 treated by 5′-AZA (1 and 5 μmol/L), TSA (500 nmol/L), and a combination for 48 hours in LoVo cells. GAPDH and actin were used as internal control. C, control; A, 5′-AZA treatment. Gastroenterology 2006 131, 1835-1843DOI: (10.1053/j.gastro.2006.09.050) Copyright © 2006 AGA Institute Terms and Conditions

Figure 2 Putative promoter activities of 4 fragments around the transcription start site and exon 1 in the XAF1 gene. (A) The map of start and stop sites of 4 fragments based on the state of CpGs around the transcription start site and exon 1 in XAF1 genomic DNA sequence. The thick line shows the exon 1 area, and open ovals denote the CpG sites. (B) The putative promoter activities of these fragments in 2 colon cancer cell lines of LoVo and DLD1. The thick lines on the left show the simulated insert fragments we subcloned into pGL-3 basic plasmid. (C) Putative promoter fragment of F291 was treated by Sss I methylase and 5′-AZA in LoVo and DLD1 cells. Gastroenterology 2006 131, 1835-1843DOI: (10.1053/j.gastro.2006.09.050) Copyright © 2006 AGA Institute Terms and Conditions

Figure 3 Bisulfite sequencing analysis of 8 colon cancer cell lines and 4 gastric cancer cell lines. We amplified the 253–base pair fragment around the transcription start site of the XAF1 gene and used the bisulfite-treated genomic DNA as template. The left image shows the original results of cell lines, and the right image shows the results after treatment with 5′-AZA. We selected 8–10 white clones in each cancer cell line and normal gastric mucosa (results not shown here) for sequencing. Black, gray, and white squares represent complete methylation (70%–100%), partial methylation (30%–69%), and unmethylation on each site, respectively. Gastroenterology 2006 131, 1835-1843DOI: (10.1053/j.gastro.2006.09.050) Copyright © 2006 AGA Institute Terms and Conditions

Figure 4 Methylation status of gastric cancer and normal tissues. (A) MSP results of human gastric mucosa biopsy specimens obtained from patients with no gastric cancers diagnosed by gastroscopy. (B) MSP results of human adjacent uninvolved tissue samples. (C) MSP results of human gastric cancer tissues. U, unmethylated; M, methylated result. Gastroenterology 2006 131, 1835-1843DOI: (10.1053/j.gastro.2006.09.050) Copyright © 2006 AGA Institute Terms and Conditions

Figure 5 Change of putative promoter activity by mutation of CpG sites in fragment F291. Site-directed mutation of CpG changed the putative promoter activity of fragment F291 in LoVo cells. Each bar represents the mean ± SD of at least 3 separate experiments. P value was accepted versus the activity of unmutated F291 fragment. Gastroenterology 2006 131, 1835-1843DOI: (10.1053/j.gastro.2006.09.050) Copyright © 2006 AGA Institute Terms and Conditions