1. Volume 124, Issue 3, Pages 626-633 (March 2003)
p14 methylation in human colon cancer is associated with microsatellite instability and wild-type p53 
Lanlan Shen, Yutaka Kondo, Stanley R. Hamilton, Asif Rashid, Jean–Pierre J. Issa 
Gastroenterology 
Volume 124, Issue 3, Pages (March 2003)
DOI: /gast
Copyright © 2003 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Hypermethylation and silencing of p14 in colon cancer. (A) On the top is shown a diagram of the CpG island of p14. Each vertical line represents a single CpG site. The location of p14 exon 1 is shown. The arrow indicates putative transcription initiation sites. Thick bars on top indicate the location of MSP and COBRA primers. (B) Methylation of p14 in colon cancer cell lines by COBRA. The restriction enzymes FNU4HI (F) or αTaqI (T) were used to determine the methylation status of this region whereas (B) BstUI, which has no cutting site in this fragment, was used as a negative control. Arrows point to digested bands, which indicate methylated alleles. Note that Caco2 is unmethylated, HCT116 is partially methylated, and RKO and SW48 are highly methylated. (C) Correlation between p14 methylation and gene expression. Shown are the results of reverse-transcription PCR studies using limited cycle numbers for p14 or glyceraldehyde-3-phosphate-dehydrogenase H (used as an RNA integrity control). C, control; D, DAC treatment; T, TSA treatment; DT, DAC + TSA treatment. Note silencing in SW48 and RKO, moderate induction with DAC but not TSA alone, and strong induction with the combination of DAC and TSA. (D) p14 methylation (by COBRA) in primary colorectal cancer. Methylation was measured as in (B), using αTaqI. RKO was included as a positive control. Normal mucosa for all these patients had no methylation using this assay (data not shown). An arrowhead indicates the methylated alleles, present in T8-T12 but absent in T1-T7.
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

3. Fig. 2
p14 methylation and CIMP, microsatellite instability, and p53 mutations in colon cancer. (A) Bar graph of p14 methylation in subgroups of colon cancer that are CIMP+ or CIMP−, and MSI-H positive, or negative (MSS). Gray bars indicate the frequency of p14 methylation. The actual number of methylated cases in each group is shown below the bars. For comparison, black bars indicate p53 mutations in each group. One cancer sample was missing MSI data, and 3 were missing p53 data. (B) Inverse correlation between p14 methylation and p53 mutations in colon cancer.
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions

4. Fig. 3
p14 methylation in colon mucosa. (A) p14 methylation analysis in colonic mucosa using MSP with 40 cycles of amplification. M, PCR products amplified by methylated-specific primers; U, PCR products amplified by unmethylated-specific primers. The age of each patient is shown on the top, and the percentage of methylation is shown below each lane. IBD, inflammatory bowel disease. Note that the 4 patients under age 50 years had no methylation (top), whereas patients over age 80 years had substantial methylation by this sensitive assay (middle). Two patients with inflammatory bowel disease also had substantial methylation despite their young age (bottom). (B) p14 methylation vs. age in colonic mucosa. The percentage of methylation in each mucosa was measured by the density of the methylated band divided by the sum of the methylated band and unmethylated band. This % methylation is plotted against the age of each patient. Circles indicate the unaffected mucosa of 28 patients with colorectal cancer, and squares indicate the mucosa of 17 control patients who had no evidence of colon cancer. Note a general increase in % methylation with age. This assay is very sensitive and overestimates the degree of methylation. Thus, although the actual % number appears high, the relative number of affected cells is actually very low (based on quantitative assays).
Gastroenterology  , DOI: ( /gast )
Copyright © 2003 American Gastroenterological Association Terms and Conditions