Promoter Methylation Status of E-Cadherin, hMLH1, and p16 Genes in Nonneoplastic…

10 Мар 2015 | Author: | No comments yet »
Toyota TAC3


Promoter Methylation Status of . hMLH1 . and p16 Genes in Nonneoplastic Epithelia

Abstract

Silencing of suppressor and tumor-related genes by at promoter CpG islands is one of the major in human tumorigenesis. Promoter is also present in nonneoplastic as an age-related tissue-specific phenomenon precedes the development of neoplasia. To the significance of promoter methylation in gastric epithelia as a precancerous we investigated promoter methylation of E-cadherin . hMLH1 . and p16 genes in cells of various organs at autopsy, and compared the results those of nonneoplastic epithelia of a stomach. Methylation of these was not seen in nonneoplastic cells of from people who were 22 and younger (0%, 0 of 6). In contrast, and p16 were methylated in nonneoplastic epithelia of persons who were 45 or older. The numbers were 86% (12 of 14) and 29% (4 of respectively. E-cadherin methylation preferentially in the intestines, whereas p16 was almost restricted to the stomach. For obtained from patients stomach cancer, methylation was observed in both neoplastic and nonneoplastic gastric epithelia: 47% (44 of 94) and 67% (63 of 94) for . 32% (30 of 94) and 24% (23 of 94) for hMLH1 . and 22% (21 of 94) and 44% (41 of 94) for p16 . respectively. hMLH1 was not seen in nonneoplastic gastric from autopsy samples but significantly in samples from tissues of individuals with cancer. Therefore, detection of methylation in nonneoplastic gastric may be useful for screening patients who may be at of developing gastric cancer.

DNA methylation is a regulatory mechanism with loss of function of the gene. 1,2 In various types of tumors and cells that are CpG islands on promoters of genes are important in tumor generation are to be methylated. 3,4 Estrogen receptor in human colonic mucosa is to be hypermethylated with age, 5 as are the of IGF2 . MYOD1 . N33 . PAX6 . and in the normal colon. 6,7 Although the of age-related methylation is unknown, it is that age-related methylation only a subset of genes intensities that vary tissues. 7,8 Several factors may to age-related methylation, such as carcinogens, endogenously generated oxygen species, and host differences. 8 In gastric cancers, the of function of E-cadherin . hMLH1 . and p16 is linked to hypermethylation of CpG islands their promoters. 9-12 of these genes in nonneoplastic epithelia was recently reported. We reasoned that this during aging may be an early signal for the beginning of cancer

in the gastric epithelia. To test hypothesis, we have compared the methylation status of E-cadherin . . and p16 genes in nonneoplastic cells of obtained at autopsies from and old cadavers, with neoplastic and gastric epithelia of patients stomach cancer.

Materials and Methods

Samples

mucosal tissue samples obtained from the upper, and lower portions of the stomach, as as the esophagus, duodenum, jejunum, colon, rectum, liver, lung, and kidney at autopsies. The consisted of 11 males and 9 females, in age from 0.7 to 87 years (53 years in excluding a stillborn infant). gastric cancer samples and matching nonneoplastic gastric were obtained at surgery 94 patients (56 males and 38 females). The ranged in age from 32 to 89 years (64 in average). All samples were and stored at #x02212;80#x000b0;C until The tumors consisted of 62 early and 32 made up of 42 differentiated and 52 undifferentiated by examination. Genomic DNA was extracted standard procedures.

Bisulfite Modification and Methylation-Specific Chain Reaction (MSP)

of DNA samples with bisulfite all unmethylated cytosines to uracils, leaving methylated cytosines Briefly, 2 #x003bc;g of genomic DNA was by treatment with NaOH and by sodium bisulfite. The samples then purified using DNA purification resin (Promega, WI), treated with recovered in ethanol, and resuspended in 30 of distilled water. Amplification was in a 20-#x003bc;l reaction volume 2 #x003bc;l of GeneAmp PCR Gold (PE Applied Biosystems, Foster CA), 1.0 mmol/L of MgCl 2 . 1 of each primer, 0.2 mmol/L of and 1 U of Taq polymerase (AmpliTaq Gold DNA PE Applied Biosystems). Hot start chain reaction (PCR) was in a thermal cycler (GeneAmp PE Applied Biosystems) for 35 cycles, of which consisted of denaturation at for 15 seconds, annealing at 55#x000b0;C for 15 and extension at 72#x000b0;C for 30 seconds, by a final 7-minute extension at A positive control and negative (distilled water without were included for each The PCR products were separated on a 6% polyacrylamide gel. Primer for MSP were: sense, 5#x02032;-GGT GAA TTT TTA GTT AAT TAG CGG and antisense, 5#x02032;-CAT AAC TAA CCG AAA ACG CCG-3#x02032; bp) for methylated DNA of E-cadherin . sense, AGG TGA ATT TTT AGT TAA TTA GTG GTA-3#x02032;; and antisense, 5#x02032;-ACC CAT AAC TAA CCA AAA ACA (211 bp) for unmethylated DNA of E-cadherin no. L34545 ), 10 and sense, 5#x02032;-ACG TAG ACG TTT TAT TAG GGT and antisense, 5#x02032;-CCT CAT CGT AAC TAC CCG CG-3#x02032; bp) for methylated DNA of hMLH1 (accession no. ), 11 and sense, 5#x02032;-GGG TCG GAG GGG

GTT TTT TC-3#x02032; (nucleotides 1075 to and antisense, 5#x02032;-CAA CCG CCG AAC GCA CTC GA-3#x02032; 1152 to 1171) (97 bp) for methylated DNA of p16 no. X94154 ). All DNA samples were for bisulfite modification using the set for unmethylated E-cadherin.

Preparation of Positive Control

Analysis

Statistical comparisons performed using Fisher#x02019;s test. A P #x0003c; 0.05 was significant.

Results

Methylation in Samples

Interesting Articles

Tagged as:

Here you can write a commentary on the recording "Promoter Methylation Status of E-Cadherin, hMLH1, and p16 Genes in Nonneoplastic…".

Sign in, to write a review.

Twitter-news
Our partners
Follow us
Contact us
Our contacts

dima911@gmail.com

Born in the USSR

423360519

About this site

For all questions about advertising, please contact listed on the site.


Toyota cars catalog with specifications, pictures, ratings, reviews and discusssions about cars Toyota.