BackgroundEnterotoxigenic Bacteroides fragilis (ETBF) is a toxin-producing bacteria thought to possibly promote colorectal carcinogenesis by modulating the mucosal immune response and inducing epithelial cell changes. Here, we aim to examine the association of colonic mucosal colonization with ETBF and the presence of a range of lesions on the colonic neoplastic spectrum.MethodsMucosal tissue from up to four different colonic sites was obtained from a consecutive series of 150 patients referred for colonoscopy. The presence and relative abundance of the B. fragilis toxin gene (bft) in each tissue sample was determined using quantitative PCR, and associations with clinicopathological characteristics were analysed.FindingsWe found a high concordance of ETBF between different colonic sites (86%). Univariate analysis showed statistically significant associations between ETBF positivity and the presence of low-grade dysplasia (LGD), tubular adenomas (TA), and serrated polyps (P-values of 0.007, 0.027, and 0.007, respectively). A higher relative abundance of ETBF was significantly associated with LGD and TA (P-values of < 0.0001 and 0.025, respectively). Increased ETBF positivity and abundance was also associated with left-sided biopsies, compared to those from the right side of the colon.ConclusionOur results showing association of ETBF positivity and increased abundance with early-stage carcinogenic lesions underlines its importance in the development of colorectal cancer, and we suggest that detection of ETBF may be a potential marker of early colorectal carcinogenesis.

We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5′ end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a T m of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.

Background and aims: Transforming growth factor b (TGF-b) is frequently involved in gastrointestinal carcinogenesis although its contribution to oesophageal adenocarcinoma (AC) and its precursor Barrett's oesophageal epithelium (BE) metaplasia are unclear. Methods: Expression of TGF-b signalling components was assessed by reverse transcription-polymerase chain reaction (PCR), western blot, and immunohistochemistry in oesophageal endoscopic biopsies and cell lines. Genomic alterations in SMAD4 were characterised by fluorescence in situ hybridisation, methylation specific PCR, and sequencing. Functional integrity of TGF-b signalling was assessed by characterisation of p21 and proliferation status. Smad4 negative BIC-1 cells were transiently transfected with smad4 and TGF-b responsiveness evaluated. Results: smad4 mRNA expression was progressively reduced in the metaplasia-dysplasia-adenocarcinoma sequence (p,0.01). A quarter of AC samples displayed an abnormal Smad4 protein isoform, with no corresponding changes in gene sequence or organisation. Methylation of smad4 has not been described previously but we found promoter methylation in 70% of primary AC samples. In 6/8 oesophageal cell lines, chromosomal rearrangements affected the smad4 locus. Lack of smad4 expression in BIC-1 cells occurred secondary to loss of one copy and extensive deletion of the second allele's promoter region. TGF-b dependent induction of p21 and downregulation of minichromosome maintenance protein 2 was lost in .80% of BE and AC. TGF-b failed to inhibit proliferation in 5/8 oesophageal cell lines. In BIC-1, the antiproliferative response was restored following transient transfection of smad4 cDNA. Conclusions: In BE carcinogenesis, downregulation of Smad4 occurs due to several different mechanisms, including methylation, deletion, and protein modification. Frequent alterations in TGF-b signalling lead to a functionally significant impairment of TGF-b mediated growth suppression.

The mouse H19 gene is expressed from the maternal chromosome exclusively. A 2-kb region at 2 to 4 kb upstream of H19 is paternally methylated throughout development, and these sequences are necessary for the imprinted expression of both H19 and the 5 -neighboring Igf2 gene. In particular, on the maternal chromosome this element appears to insulate the Igf2 gene from enhancers located downstream of H19. We analyzed the chromatin organization of this element by assaying its sensitivity to nucleases in nuclei. Six DNase I hypersensitive sites (HS sites) were detected on the unmethylated maternal chromosome exclusively, the two most prominent of which mapped 2.25 and 2.75 kb 5 to the H19 transcription initiation site. Five of the maternal HS sites were present in expressing and nonexpressing tissues and in embryonic stem (ES) cells. They seem, therefore, to reflect the maternal origin of the chromosome rather than the expression of H19. A sixth maternal HS site, at 3.45 kb upstream of H19, was detected in ES cells only. The nucleosomal organization of this element was analyzed in tissues and ES cells by micrococcal nuclease digestion. Specifically on the maternal chromosome, an unusual and strong banding pattern was obtained, suggestive of a nonnucleosomal organization. From our studies, it appears that the unusual chromatin organization with the presence of HS sites (maternal chromosome) and DNA methylation (paternal chromosome) in this element are mutually exclusive and reflect alternate epigenetic states. In addition, our data suggest that nonhistone proteins are associated with the maternal chromosome and that these might be involved in its boundary function.

This indicates that epigenetic changes affect the expression of the Smad4 protein in prostate cancer and points to methylation of the promoter as a novel marker of and contributor to the disease warranting further study.