The expression of type 1 ribbriae (pili) of Escherichia coli is turned on and off at the transcriptional level at a high frequency (10-3 per cell per generation) in a process termed phase variation. Using Southern blot and DNA sequence analysis, we have detected a genomic rearrangement in the switch region immediately upstream of the fimbrial structural gene. This rearrangement involves an invertible 314-basepair segment of DNA whose alternating orientation apparently results in the on-and-off activation of a promoter that determines the state of fimbrial expression.Type 1 fimbriae (pili) are major surface appendages that mediate binding of Escherichia coli to eukaryotic cells by a ligand-receptor mechanism that is sensitive to the presence of mannose. The fimbrial protein adhesin is thought to be a virulence factor during the initial colonization stage. During the invasive stage it would seem to be to the pathogen's advantage not to express adhesins that could mediate binding to phagocytic cells. A genetic regulatory system that would result in an on-and-off expression of an adhesin would itself be a virulence factor (1).The phase variation between fimbriate (Fim+) and nonfimbriate (Fim-) E. coli cells occurs at the transcriptional level (2). In addition, it has been shown that a cis-dominant DNA switch is turned on and off by means of a trans-active factor (3) different from that reported by Orndorff and Falkow (4). Here we report that the molecular basis of this switch is the inversion of a relatively small segment of DNA that results in the alternating activation of the fimbrial promoter in correlation with phase variation. This system resembles in general that which controls flagellar phase variation in Salmonella (5) but differs both in its recombinase specificity and in its small size, so that the element does not encode its own recombinase. MATERIALS AND METHODS Bacterial Strains and Media. E. coli K-12 strain CSH50[ara A(lac-pro) rpsL thi] was the parental strain used. The genetic construction of strain VL412 and bacteriophage X412 has been presented in detail (3). 5-Bromo-4-chloro-3-indolyl ,B-D-galactopyranoside (X-Gal) RESULTSThe Lac-E. coli K-12 strain CSH50, used in the genetic constructions outlined below, undergoes fimbrial phase variation. Eisenstein (2) described the construction of afim-lac operon fusion in this strain and used its oscillating Lac';±Lac-phenotype to show that phase variation is under transcriptional control (2). The fim', fim-lac merodiploid strain VL412 (Fig. 1) was constructed through integration of a specialized X phage, carrying the fim-lac operon fusion, by homologous recombination with the fimD gene (3). This merodiploid strain exhibits independent, reversible, and noncoordinated phase variation of both the Lac and Fim phenotypes, which indicates the presence of two cis-acting switches, each adjacent to its respective operon (3). Ultraviolet induction of VL412 yields the phage X412, which alternates between a very dark blue (Dk) and light blue (Lt) plaque phe...

A signi®cant portion of gastric cancers exhibit defective DNA mismatch repair, manifested as microsatellite instability (MSI). High-frequency MSI (MSI-H) is associated with hypermethylation of the human mut-L homologue 1 (hMLH1) mismatch repair gene promoter and diminished hMLH1 expression in advanced gastric cancers. However, the relationship between MSI and hMLH1 hypermethylation has not been studied in early gastric neoplasms. We therefore investigated hMLH1 hypermethylation, hMLH1 expression and MSI in a group of early gastric cancers and gastric adenomas. Sixty-four early gastric neoplasms were evaluated, comprising 28 adenomas, 18 mucosal carcinomas, and 18 carcinomas with super®cial submucosal invasion but clear margins. MSI was evaluated using multiplex uorescent PCR to amplify loci D2S123, D5S346, D17S250, BAT 25 and BAT 26. Methylation-speci®c PCR was performed to determine the methylation status of hMLH1. In two hypermethylated MSI-H cancers, hMLH1 protein expression was also evaluated by immunohistochemistry. Six of sixty-four early gastric lesions were MSI-H, comprising 1 adenoma, 4 mucosal carcinomas, and 1 carcinoma with super®cial submucosal invasion. Two lesions (one adenoma and one mucosal carcinoma) demonstrated low-frequency MSI (MSI-L). The remaining 56 neoplasms were MSI-stable (MSI-S). Six of six MSI-H, one of two MSI-L, and none of thirty MSI-S lesions showed hMLH1 hypermethylation (P50.001). Diminished hMLH1 protein expression was demonstrated by immunohistochemistry in two of two MSI-H hypermethylated lesions. hMLH1 promoter hypermethylation is signi®cantly associated with MSI and diminished hMLH1 expression in early gastric neoplasms. MSI and hypermethylation-associated inactivation of hMLH1 are more prevalent in early gastric cancers than in gastric adenomas. Thus, hypermethylation-associated inactivation of the hMLH1 gene can occur early in gastric carcinogenesis. Oncogene (2001) 20, 329 ± 335.

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Cholangiocarcinomas (CCA) are aggressive cancers, with a high mortality and poor survival rate. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of the late diagnosis secondary to relatively poor accuracy diagnostic means. MicroRNAs (miRs) are involved in every cancer examined, but they have not been evaluated in primary CCA. In this study, miR arrays were performed on 5 primary CCAs and 5 normal bile duct specimens (NBD). Several miRs were dysregulated, and miR-21 was overexpressed, in CCAs. miR-21 differential expression in these 10 specimens was verified with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). To validate these findings, qRT-PCR for miR-21 was then performed on 18 additional primary CCAs and 12 normal liver specimens. MiR-21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the Receiver Operating Characteristic (ROC) curve of 0.995. Inhibitors of miR-21 increased protein levels of programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinases 3 (TIMP3). Notably, messenger RNA (mRNA) levels of TIMP3 were significantly lower in CCAs than in normals. Conclusions MiR-21 is overexpressed in human CCAs. Furthermore, miR-21 may be oncogenic, at least in part, by inhibiting PDCD4 and TIMP3. Finally, these data suggest that TIMP3 is a candidate tumor suppressor gene in the biliary tree.

Background & Aims-Barrett's esophagus (BE) is a highly premalignant disease that predisposes to the development of esophageal adenocarcinoma (EAC); however, the involvement of microRNAs (miRs) in BE-EAC carcinogenic progression is not known.

Patients with Barrett's esophagus (BE) are at increased risk of developing esophageal adenocarcinoma (EAC). Clinical neoplastic progression risk factors, such as age and the length of the esophageal BE segment, have been identified. However, improved molecular biomarkers predicting increased progression risk are needed for improved risk assessment and stratification. Using realtime quantitative methylation-specific PCR, we screened 10 genes (HPP1, RUNX3, RIZ1, CRBP1, 3-OST-2, APC, TIMP3, p16, MGMT, p14) for promoter hypermethylation in 77 EAC, 93 BE, and 64 normal esophagus (NE) specimens. A subset of genes manifesting significant differences in methylation frequencies between BE and EAC was then analysed in 20 dysplastic specimens. All 10 genes except p14 were frequently methylated in EACs, with RUNX3, HPP1, CRBP1, RIZ1, and OST-2 representing novel methylation targets in EAC and/or BE. p16, RUNX3, and HPP1 displayed increasing methylation frequencies in BE vs EAC. Furthermore, these increases in methylation occurred early, at the interface between BE and low-grade dysplasia (LGD). To demonstrate the silencing effect of hypermethylation, we selected the EAC cells BIC1, in which the HPP1 promoter is natively methylated, and subjected them to 5-aza-2 0 -deoxycytidine (Aza-C) treatment. Real-time RT-PCR indicated increased HPP1 mRNA levels after 3 days of Aza-C treatment, as well as decreased levels of methylated HPP1 DNA. Hypermethylation of a subset of six genes (APC, TIMP3, CRBP1, p16, RUNX3, and HPP1) was then tested in a retrospective longitudinal study of 99 BE and nine LGD specimens obtained from 53 BE patients undergoing surveillance endoscopy. Only highgrade dysplasia (HGD) or EAC were defined as progression end points. Two patient groups were compared: eight progressors (P) and 45 nonprogressors (NP), using Cox proportional hazards models to determine the relative progression risks of age, BE segment length, and methylation events. Multivariate analyses revealed that only hypermethylation of p16 (odds ratio (OR) 1.74, 95% confidence interval (CI) 1.33-2.20), RUNX3 (OR 1.80, 95% CI 1.08-2.81), and HPP1 (OR 1.77, 95% CI 1.06-2.81) were independently associated with an increased risk of progression, whereas age, BE segment length, and hypermethylation of TIMP3, APC, or CRBP1 were not independent risk factors. In combined analyses, risk was detectable up to, but not earlier than, 2 years preceding neoplastic progression. Hypermethylation of p16, RUNX3, and HPP1 in BE or LGD may represent independent risk factors for the progression of BE to HGD or EAC. These findings have implications regarding risk stratification, early EAC detection, and the appropriate endoscopic surveillance interval for patients with BE.

The E-cadherin promoter frequently undergoes hypermethylation in human gastric cancers, particularly those of the undifferentiated-scattered histologic subtype. E-cadherin promoter hypermethylation is associated with decreased expression and may occur early in gastric carcinogenesis.