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. 2006 Jul;27(7):1316-22.
doi: 10.1093/carcin/bgi373. Epub 2006 Mar 2.

Epigenetic alterations in RASSF1A in human aberrant crypt foci

Emily J Greenspan  1 , Melissa A Jablonski, Thiruchandurai V Rajan, Joel Levine, Glenn S Belinsky, Daniel W Rosenberg
Affiliations

"V体育2025版" Epigenetic alterations in RASSF1A in human aberrant crypt foci

Emily J Greenspan et al. Carcinogenesis. 2006 Jul.

Abstract (V体育安卓版)

CpG island methylation (CIM) is an epigenetic mechanism for transcriptional silencing that occurs at various stages of colon tumorigenesis. CIM has been found in serrated adenomas and hyperplastic polyps VSports手机版. There is also evidence for hypermethylation in aberrant crypt foci (ACF) that are found in resected colons from cancer patients. Our study addresses promoter methylation of a tumor suppressor gene, RASSF1A, within the colonic epithelium of subjects undergoing screening colonoscopies in the absence of synchronous tumors. Patients included in this study were at elevated risk for colorectal cancer (CRC) based on family history, but without a previously occurring or synchronous colon carcinoma. ACF were identified using close-focus magnifying chromendoscopy and collected by biopsy in situ. We isolated ACF and adjacent normal colonic epithelium by laser capture microdissection (LCM) and studied methylation of the RASSF1A promoter region in ACF and in adjacent normal mucosa. Expression of RASSF1A was verified using quantitative real-time polymerase chain reaction (QRT-PCR). We found that 8. 6% (3 out of 35) of ACF had K-ras mutations and 24% (6 out of 25) had RASSF1A hypermethylation. Our results demonstrate that RASSF1A hypermethylation and K-ras mutations are not mutually exclusive and are present in patients at elevated risk of CRC. Importantly, CIM of RASSF1A is an early epigenetic aberration, occurring in the absence of synchronous colon tumors and is not accompanied by field effects into the surrounding epithelium. .

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Conflict of interest statement

Conflict of Interest Statement: None declared.

"V体育ios版" Figures

Fig. 1
Fig. 1
Macroscopic and histological analysis of ACF. (A) Gross views of ACF visualized through the Olympus prototype close-focus endoscope at a magnification of ×60. The colon epithelium was stained with 0.5% methylene blue. (B) H&E stained frozen sections of hyperplastic ACF, cross-sectional (left, ×200) and longitudinal (right, ×400) views. (C) H&E-stained frozen section of hyperplastic ACF with adjacent normal colonic mucosa indicated by the red arrows (×200). (D) H&E-stained frozen section of dysplastic ACF, cross-sectional view (×400). The inset in the upper left shows a region of surface dysplasia from the same lesion.
Fig. 2
Fig. 2
Representative LCM of a hyperplastic ACF and adjacent normal colonic epithelium. LCM was used to isolate aberrant crypts in the hyperplastic ACF (right side, indicated by arrows) from the ANM (left side). The lower images show two populations of microdissected crypts isolated separately from the surrounding stroma. All images are at ×200 magnification.
Fig. 3
Fig. 3
The RASSF1A locus and MSP analysis. (A) Map of the RASSF1 locus and CpG island A that is used for analysis of RASSF1A promoter methylation by MSP. The three major isoforms (RASSF1A, RASSF1C and RASSF1F) are made by alternative splicing and promoter usage. RASSF1A is formed from exons 1α, 2αβ, 3, 4, 5, 6. The promoter region, which is located within CpG island A, is indicated by a black arrow. CpG island A contains 16 CpG dinucleotides (green circles). The flanking PCR reaction, indicated by ‘external for’ and ‘external rev’ primers, amplifies a 144 bp product that is used as a template for the RASSF1A MSP, indicated by the red boxes labeled ‘MSP for primers’ and ‘MSP rev primers’. (B) Representative MSP analysis of the promoter of RASSF1A in cell lines and in microdissected tissue. DNA was analyzed by a chemical modification with sodium bisulfite, and a subsequent flanking PCR reaction amplified all bisulfite-treated DNA followed by RASSF1A MSP, as described under Materials and methods. The 76 bp methylated product and 81 bp unmethylated product were run on a 2% agarose gel and visualized under UV. A549 and HEK293 cells were used as a control for methylated RASSF1A. HCT116 cells were used as a control for unmethylated RASSF1A. ANM = adjacent normal mucosa. All MSP reactions were also run with a H2O control in the flanking PCR and in the RASSF1A MSP. The presence of an unmethylated product in the ACF that are methylated is probably the result of normal contaminating tissue.
Fig. 4
Fig. 4
QRT–PCR analysis of RASSF1A expression in cell lines and microdissected tissue. mRNA levels were quantified as described under Materials and methods. Concentrations were normalized to huHPRT1 to control for differences in cDNA input amounts. Fold differences were determined by using HCT116 as the calibrator sample (expression arbitrarily normalized to 1). (Fold difference = normalized quantity sample/normalized quantity HCT116). HCT116 cells were used as a positive control for RASSF1A mRNA expression, and HEK293 and A549 cells were used as a negative control. A H2O negative control was also included. 032904-230-2, 121103-1100-3 and 032904-230-5 were hyperplastic ACF that had methylated RASSF1A. 042004-1130-3 and 121103-1100-1 were hyperplastic ACF that had unmethylated RASSF1A. The ANM in all samples was unmethylated. All samples were assayed in duplicate.
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