Alterations in DNA methylation frequently occur in hepatocellular cancer (HCC). We have previously demonstrated that hypermethylation in candidate genes can be detected in plasma DNA prior to HCC diagnosis. To identify with a genome-wide approach additional genes hypermethylated in HCC that could be used for more accurate analysis of plasma DNA for early diagnosis, we analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Illumina methylation arrays that screen 26,486 autosomal CpG sites. After Bonferroni adjustment, a total of 2,324 CpG sites significantly differed in methylation level, with 684 CpG sites significantly hypermethylated and 1,640 hypomethylated in tumor compared to non-tumor tissues. Array data were validated with pyrosequencing in a subset of 5 of these genes; correlation coefficients ranged from 0.92 to 0.97. Analysis of plasma DNA from 38 cases demonstrated that 37% to 63% of cases had detectable hypermethylated DNA (≥5% methylation) for these 5 genes individually. At least one of these genes was hypermethylated in 87% of cases, suggesting that measurement of DNA methylation in plasma samples is feasible. The panel of methylated genes indentified in the current study will be further tested in large cohort of prospectively collected samples to determine their utility as early biomarkers of hepatocellular carcinoma.

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Mutations in Drosophila alpha spectrin cause larval lethality and defects in cell shape and adhesion (J. Lee et al., 1993, J. Cell Biol. 123, 1797-1809). Here we examined the effects of two lethal alpha spectrin alleles (alpha-specrg41 and alpha-specrg35) on development and function of the larval midgut. Homozygous null alpha-specrg41-mutant larvae exhibited a striking defect in middle midgut acidification. In contrast, many homozygous alpha-specrg35 mutants were capable of acidification, indicating partial function of the truncated alpha-specrg35 product. Acidification was also blocked by a mutation in the labial gene, which is required for differentiation of cuprophilic cells in the midgut, suggesting that these cells secrete acid. We found that two isoforms of spectrin (alphabeta and alphabetaH) are segregated within the basolateral and apical domains of cuprophilic cells, respectively. The most conspicuous defect in cuprophilic cells from labial and alpha spectrin mutants was in morphogenesis of the invaginated apical domain, although basolateral defects may also contribute to the acidification phenotype. Acid secretion in vertebrate systems is thought to involve the polarized activities of apical proton pumps and basolateral anion exchangers, both of which interact with spectrin. We propose that the alpha-specrg41 mutation in Drosophila interferes with the polarized activities of homologous molecules that drive acid secretion in cuprophilic cells.

The importance of imprinted genes in regulating feto-placental development has been long established. However, a comprehensive assessment of the role of placental imprinted gene expression on fetal growth has yet to be conducted. In this study, we examined the association between the placental expression of 108 established and putative imprinted genes and birth weight in 677 term pregnancies, oversampled for small for gestational age (SGA) and large for gestational age (LGA) infants. Using adjusted multinomial regression analyses, a 2-fold increase in the expression of 9 imprinted genes was positively associated with

Keywords: DNA methylation, diet, lifestyle factors, cancer As aberrant DNA methylation is associated with cancer and other human diseases, there are growing interests in determining how environmental exposures may affect patterns of DNA methylation. Previous studies found older monozygotic twins were more diverse in DNA methylation patterns than were younger monozygotic twins.17 There is also evidence that DNA methylation patterns in individuals change over time, 18 supporting aging and/or external environment factors affecting DNA methylation. However, which environmental exposures, among many that individuals are exposed to over the life course, affect patterns of DNA methylation remains elusive.Chronic inflammation constitutes an important mechanism for cancer. 19 The specific pathways by which inflammation causes cancer are not fully understood. Interleukin (IL-6) is a proinflammatory cytokine released by inflammatory cells and can activate intracellular transcription factors leading to tumorigenesis. There are some suggestions that IL-6 may stimulate tumor proliferation by altering patterns of DNA methylation, 20-22 and

Osteopontin (OPN) is a glycosylated, secreted phosphoprotein that functions both as a cell attachment and chemotactic factor. Elevated expression of OPN confers enhanced metastatic ability on transformed cells, suggesting that OPN may contribute to the malignant progression of tumors. Migration of mammary carcinoma cells is stimulated by OPN via interactions with integrins and CD44 cell surface receptors. We hypothesized that OPN modulates specific CD44 isoform expression to facilitate breast cancer cell migration. The 21NT tumorigenic human breast cancer cell line was examined for regulation of CD44 expression at both the mRNA and protein levels in response to an engineered increase in OPN expression under CMV promoter control. Significant up-regulation of CD44s isoform mRNA expression was observed, but no change in CD44v6, v8, v9 or v10 mRNA levels. While there were elevated levels of CD44s, v6 and v9 protein at the cell surface, at the level of total cellular protein only CD44s and v6 were markedly increased. This suggests that OPN can regulate CD44 expression at both transcriptional and post-transcriptional (both amount and localization of protein) levels. To validate the functional consequence of OPN regulation of CD44 expression, we demonstrate that OPN-mediated cell migration was reduced by exposure to a anti-pan CD44 antibody, and to anti-CD44v6 and anti-CD44v9 function-blocking antibodies. Our data provide evidence that in 21NT cells OPN enhances CD44s mRNA expression, increases cell surface expression of CD44 variant forms without a change in mRNA levels, and stimulates cell migration.

BackgroundEpidemiologic studies of cardiovascular disease risk factors and appropriate biomarkers in populations exposed to a wide range of arsenic levels are a public health research priority.ObjectiveWe investigated the relationship between inorganic arsenic exposure from drinking water and plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular adhesion molecule-1 (sVCAM-1), both markers of endothelial dysfunction and vascular inflammation, in an arsenic-exposed population in Araihazar, Bangladesh.MethodsThe study participants included 115 individuals with arsenic-related skin lesions participating in a 2 × 2 randomized, placebo-controlled, double-blind trial of vitamin E and selenium supplementation. Arsenic exposure status and plasma levels of sICAM-1 and sVCAM-1 were assessed at baseline and after 6 months of follow-up.ResultsBaseline well arsenic, a long-term measure of arsenic exposure, was positively associated with baseline levels of both sICAM-1 and sVCAM-1 and with changes in the two markers over time. At baseline, for every 1-μg/L increase in well arsenic there was an increase of 0.10 ng/mL [95% confidence interval (CI), 0.00–0.20] and 0.33 ng/mL (95% CI, 0.15–0.51) in plasma sICAM-1 and sVCAM-1, respectively. Every 1-μg/L increase in well arsenic was associated with a rise of 0.11 ng/mL (95% CI, 0.01–0.22) and 0.17 ng/mL (95% CI, 0.00–0.35) in sICAM-1 and sVCAM-1 from baseline to follow-up, respectively, in spite of recent changes in urinary arsenic as well as vitamin E and selenium supplementation during the study period.ConclusionsThe findings indicate an effect of chronic arsenic exposure from drinking water on vascular inflammation that persists over time and also suggest a potential mechanism underlying the association between arsenic exposure and cardiovascular disease.

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.