Identification and characterization of stemlike cells in human esophageal adenocarcinoma and normal epithelial cell lines

ObjectiveRecent studies have suggested that human solid tumors may contain subpopulations of cancer stem cells with the capacity for self-renewal and the potential to initiate and maintain tumor growth. The aim of this study was to use human esophageal cell lines to identify and characterize putative esophageal cancer stem cell populations.MethodsTo enrich stemlike cells, Het-1A (derived from immortalized normal esophageal epithelium), OE33, and JH-EsoAd1 (each derived from primary esophageal adenocarcinomas) were cultured using serum-free media to form spheres. A comprehensive analysis of parent and spheroid cells was performed by flow cytometry, Western blot analysis, immunohistochemistry and polymerase chain reaction array to study cancer stem cell-related genes, colony formation assays to assess clonogenicity, xenotransplantation to assess tumorigenicity, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays to assess chemosensitivity to 5-fluorouracil and Cisplatin.ResultsFor all cell lines, clonogenicity, tumorigenicity, and chemoresistance to 5-fluorouracil and Cisplatin were significantly higher than for spheroid cells compared with parent cells. Spheroids exhibited an increased frequency of cells expressing integrin α6bri/CD71dim, and Achaete-scute complex homolog 2 messenger RNA and protein were also significantly overexpressed in spheroid cells compared with parent cells.ConclusionsThe higher clonogenicity, tumorigenicity, and drug resistance exhibited by spheroids derived from Het-1A, OE33, and JH-EsoAd1 reflects an enrichment of stemlike cell populations within each esophageal cell line. Esophageal cells enriched for integrin α6bri/CD71dim and/or overexpressing Achaete-scute complex homolog 2 would appear to represent at least a subpopulation of stemlike cells in Het-1A, OE33, and JH-EsoAd1

Waiting for surgery from the patient perspective

The aim of this study was to perform a systematic review of the impact of waiting for elective surgery from the patient perspective, with a focus on maximum tolerance, quality of life, and the nature of the waiting experience. Searches were conducted using Medline, PubMed, CINAHL, EMBASE, and HealthSTAR. Twenty-seven original research articles were identified which included each of these three themes. The current literature suggested that first, patients tend to state longer wait times as unacceptable when they experienced severe symptoms or functional impairment. Second, the relationship between length of wait and health-related quality of life depended on the nature and severity of proposed surgical intervention at the time of booking. Third, the waiting experience was consistently described as stressful and anxiety provoking. While many patients expressed anger and frustration at communication within the system, the experience of waiting was not uniformly negative. Some patients experienced waiting as an opportunity to live full lives despite pain and disability. The relatively unexamined relationship between waiting, illness and patient experience of time represents an area for future research

Quercetin elevates p27Kip1 and arrests both primary and HPV16 E6/E7 transformed human keratinocytes in G1

Our previous work with primary bovine fibroblasts demonstrated that quercetin, a potent mutagen found in high levels in bracken fern (Pteridium aquilinum), arrested cells in G1 and G2/M, in correlation with p53 activation. The expression of bovine papillomavirus type 4 (BPV-4) E7 overcame this arrest and lead to the development of tumorigenic cells lines (Beniston et al., 2001). Given the possible link between papillomavirus infection, bracken fern in the diet and cancer of the upper gastrointestinal (GI) tract in humans, we investigated whether a similar situation would occur in human cells transformed by human papillomavirus type 16 (HPV-16) oncoproteins. Quercetin arrested primary human foreskin keratinocytes in G1. Arrest was linked to an elevation of the cyclin-dependent kinase inhibitor (cdki) p27Kip1. Expression of the HPV16 E6 and E7 oncoproteins in transformed cells failed to abrogate cell cycle arrest. G1 arrest in the transformed cells was also linked to an increase of p27Kip1 with a concomitant reduction of cyclin E-associated kinase activity. This elevation of p27Kip1 was due not only to increased protein half-life, but also to increased mRNA transcription

EFFECTS OF BARLEY FLOUR ADDITION AND BAKING TEMPERATURE ON Β-GLUCANS CONTENT AND BISCUITS PROPERTIES

The aim of this study was to investigate opportunities to improve the nutritional value of biscuits. Therefore, the content of β-glucans, physical, chemical and sensory properties of biscuits were determined in relation to a share of added barley flour and a baking temperature. Five different blends of barley and wheat were used for biscuit production: barley/wheat flours in combinations: 0/100; 25/75; 50/50; 75/25 and 100/0 according to the procedure described in AACC method 10-52. The temperatures used for baking were 150 and 205°C for 15 and 11 min, respectively. The obtained results showed the higher β-glucans content in samples when share of barley flour in biscuit formula was higher. The same trend was found on both baking temperatures. Besides, the share of barley flour in samples significantly influenced physical, chemical and sensory parameters. In addition, different baking temperatures affected significant differences between samples according to all parameters investigated, except N (%) and ash (%). After 6 months, β-glucan content was significantly lower in samples with high share of barley flour (75 and 100%), at both baking temperatures

Alternative splicing and differential subcellular localization of the rat FGF antisense gene product

<p>Abstract</p> <p>Background</p> <p>GFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS) gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species including rat. In the present study we focused on elucidating the expression and subcellular distribution of alternatively spliced rat GFG isoforms.</p> <p>Results</p> <p>RT-PCR and immunohistochemistry revealed tissue-specific GFG mRNA isoform expression and subcellular distribution of GFG immunoreactivity in cytoplasm and nuclei of a wide range of normal rat tissues. FGF-2 and GFG immunoreactivity were co-localized in some, but not all, tissues examined. Computational analysis identified a mitochondrial targeting sequence (MTS) in the N-terminus of three previously described rGFG isoforms. Confocal laser scanning microscopy and subcellular fractionation analysis revealed that all rGFG isoforms bearing the MTS were specifically targeted to mitochondria whereas isoforms and deletion mutants lacking the MTS were localized in the cytoplasm and nucleus. Mutation and deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization.</p> <p>Conclusion</p> <p>Previous findings strongly support a role for the FGF antisense RNA as a regulator of FGF2 expression. The present study demonstrates that the antisense RNA itself is translated, and that protein isoforms resulting form alternative RNA splicing are sorted to different subcellular compartments. FGF-2 and its antisense protein are co-expressed in many tissues and in some cases in the same cells. The strong conservation of sequence and genomic organization across animal species suggests important functional significance to the physical association of these transcript pairs.</p

MiRNA-Related SNPs and Risk of Esophageal Adenocarcinoma and Barrett's Esophagus: Post Genome-Wide Association Analysis in the BEACON Consortium.

Incidence of esophageal adenocarcinoma (EA) has increased substantially in recent decades. Multiple risk factors have been identified for EA and its precursor, Barrett's esophagus (BE), such as reflux, European ancestry, male sex, obesity, and tobacco smoking, and several germline genetic variants were recently associated with disease risk. Using data from the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) genome-wide association study (GWAS) of 2,515 EA cases, 3,295 BE cases, and 3,207 controls, we examined single nucleotide polymorphisms (SNPs) that potentially affect the biogenesis or biological activity of microRNAs (miRNAs), small non-coding RNAs implicated in post-transcriptional gene regulation, and deregulated in many cancers, including EA. Polymorphisms in three classes of genes were examined for association with risk of EA or BE: miRNA biogenesis genes (157 SNPs, 21 genes); miRNA gene loci (234 SNPs, 210 genes); and miRNA-targeted mRNAs (177 SNPs, 158 genes). Nominal associations (P0.50), and we did not find evidence for interactions between variants analyzed and two risk factors for EA/BE (smoking and obesity). This analysis provides the most extensive assessment to date of miRNA-related SNPs in relation to risk of EA and BE. While common genetic variants within components of the miRNA biogenesis core pathway appear unlikely to modulate susceptibility to EA or BE, further studies may be warranted to examine potential associations between unassessed variants in miRNA genes and targets with disease risk.This work was supported by the National Institutes of Health [R01CA136725 to T.L.V. and D.C.W, T32CA009168 to T.L.V, and K05CA124911 to T.L.V.]. Additional funding sources for individual studies included in the BEACON GWAS, and for BEACON investigators, have been acknowledged previously (16).This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.012861

Polymorphisms in Genes of Relevance for Oestrogen and Oxytocin Pathways and Risk of Barrett's Oesophagus and Oesophageal Adenocarcinoma: A Pooled Analysis from the BEACON Consortium.

BACKGROUND: The strong male predominance in oesophageal adenocarcinoma (OAC) and Barrett's oesophagus (BO) continues to puzzle. Hormonal influence, e.g. oestrogen or oxytocin, might contribute. METHODS: This genetic-epidemiological study pooled 14 studies from three continents, Australia, Europe, and North America. Polymorphisms in 3 key genes coding for the oestrogen pathway (receptor alpha (ESR1), receptor beta (ESR2), and aromatase (CYP19A1)), and 3 key genes of the oxytocin pathway (the oxytocin receptor (OXTR), oxytocin protein (OXT), and cyclic ADP ribose hydrolase glycoprotein (CD38)), were analysed using a gene-based approach, versatile gene-based test association study (VEGAS). RESULTS: Among 1508 OAC patients, 2383 BO patients, and 2170 controls, genetic variants within ESR1 were associated with BO in males (p = 0.0058) and an increased risk of OAC and BO combined in males (p = 0.0023). Genetic variants within OXTR were associated with an increased risk of BO in both sexes combined (p = 0.0035) and in males (p = 0.0012). We followed up these suggestive findings in a further smaller data set, but found no replication. There were no significant associations between the other 4 genes studied and risk of OAC, BO, separately on in combination, in males and females combined or in males only. CONCLUSION: Genetic variants in the oestrogen receptor alpha and the oxytocin receptor may be associated with an increased risk of BO or OAC, but replication in other large samples are needed

Risk of Esophageal Adenocarcinoma Decreases With Height, Based on Consortium Analysis and Confirmed by Mendelian Randomization

Background & Aims Risks for some cancers increase with height. We investigated the relationship between height and risk of esophageal adenocarcinoma (EAC) and its precursor, Barrett's esophagus (BE). Methods We analyzed epidemiologic and genome-wide genomic data from individuals of European ancestry in the Barrett's and Esophageal Adenocarcinoma Consortium, from 999 cases of EAC, 2061 cases of BE, and 2168 population controls. Multivariable logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (95% CI) for associations between height and risks of EAC and BE. We performed a Mendelian randomization analysis to estimate an unconfounded effect of height on EAC and BE using a genetic risk score derived from 243 genetic variants associated with height as an instrumental variable. Results Height was associated inversely with EAC (per 10-cm increase in height: OR, 0.70; 95% CI, 0.62–0.79 for men and OR, 0.57; 95% CI 0.40–0.80 for women) and BE (per 10-cm increase in height: OR, 0.69; 95% CI, 0.62–0.77 for men and OR, 0.61; 95% CI, 0.48–0.77 for women). The risk estimates were consistent across strata of age, education level, smoking, gastroesophageal reflux symptoms, body mass index, and weight. Mendelian randomization analysis yielded results quantitatively similar to those from the conventional epidemiologic analysis. Conclusions Height is associated inversely with risks of EAC and BE. Results from the Mendelian randomization study showed that the inverse association observed did not result from confounding factors. Mechanistic studies of the effect of height on EAC and BE are warranted; height could have utility in clinical risk stratification

A genome-wide association study identifies new susceptibility loci for esophageal adenocarcinoma and Barrett's esophagus.

Esophageal adenocarcinoma is a cancer with rising incidence and poor survival. Most such cancers arise in a specialized intestinal metaplastic epithelium, which is diagnostic of Barrett's esophagus. In a genome-wide association study, we compared esophageal adenocarcinoma cases (n = 2,390) and individuals with precancerous Barrett's esophagus (n = 3,175) with 10,120 controls in 2 phases. For the combined case group, we identified three new associations. The first is at 19p13 (rs10419226: P = 3.6 × 10(-10)) in CRTC1 (encoding CREB-regulated transcription coactivator), whose aberrant activation has been associated with oncogenic activity. A second is at 9q22 (rs11789015: P = 1.0 × 10(-9)) in BARX1, which encodes a transcription factor important in esophageal specification. A third is at 3p14 (rs2687201: P = 5.5 × 10(-9)) near the transcription factor FOXP1, which regulates esophageal development. We also refine a previously reported association with Barrett's esophagus near the putative tumor suppressor gene FOXF1 at 16q24 and extend our findings to now include esophageal adenocarcinoma

Obesity and Risk of Esophageal Adenocarcinoma and Barrett’s Esophagus: A Mendelian Randomization Study

Data from observational studies suggest that body mass index (BMI) is causally related to esophageal adenocarcinoma (EAC) and its precursor, Barrett’s esophagus (BE). However, the relationships may be affected by bias and confounding