Raman micro-spectroscopy for accurate identification of primary human bronchial epithelial cells.

Live cell Raman micro-spectroscopy is emerging as a promising bioanalytical technique for label-free discrimination of a range of different cell types (e.g. cancer cells and fibroblasts) and behaviors (e.g. apoptosis). The aim of this study was to determine whether confocal Raman micro-spectroscopy shows sufficient sensitivity and specificity for identification of primary human bronchial epithelial cells (HBECs) to be used for live cell biological studies in vitro. We first compared cell preparation substrates and media, considering their influence on lung cell proliferation and Raman spectra, as well as methods for data acquisition, using different wavelengths (488 nm, 785 nm) and scan protocols (line, area). Evaluating these parameters using human lung cancer (A549) and fibroblast (MRC5) cell lines confirmed that line-scan data acquisition at 785 nm using complete cell media on a quartz substrate gave optimal performance. We then applied our protocol to acquisition of data from primary human bronchial epithelial cells (HBEC) derived from three independent sources, revealing an average sensitivity for different cell types of 96.3% and specificity of 95.2%. These results suggest that Raman micro-spectroscopy is suitable for delineating primary HBEC cell cultures, which in future could be used for identifying different lung cell types within co-cultures and studying the process of early carcinogenesis in lung cell culture

Extent of differential allelic expression of candidate breast cancer genes is similar in blood and breast.

INTRODUCTION: Normal gene expression variation is thought to play a central role in inter-individual variation and susceptibility to disease. Regulatory polymorphisms in cis-acting elements result in the unequal expression of alleles. Differential allelic expression (DAE) in heterozygote individuals could be used to develop a new approach to discover regulatory breast cancer susceptibility loci. As access to large numbers of fresh breast tissue to perform such studies is difficult, a suitable surrogate test tissue must be identified for future studies. METHODS: We measured differential allelic expression of 12 candidate genes possibly related to breast cancer susceptibility (BRCA1, BRCA2, C1qA, CCND3, EMSY, GPX1, GPX4, MLH3, MTHFR, NBS1, TP53 and TRXR2) in breast tissue (n = 40) and fresh blood (n = 170) of healthy individuals and EBV-transformed lymphoblastoid cells (n = 19). Differential allelic expression ratios were determined by Taqman assay. Ratio distributions were compared using t-test and Wilcoxon rank sum test, for mean ratios and variances respectively. RESULTS: We show that differential allelic expression is common among these 12 candidate genes and is comparable between breast and blood (fresh and transformed lymphoblasts) in a significant proportion of them. We found that eight out of nine genes with DAE in breast and fresh blood were comparable, as were 10 out of 11 genes between breast and transformed lymphoblasts. CONCLUSIONS: Our findings support the use of differential allelic expression in blood as a surrogate for breast tissue in future studies on predisposition to breast cancer.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal.

BACKGROUND: The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/WV mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells. METHOD: The gene expression profile of the gastric fundus of wild type and W/WV mice was assayed by murine microarray analysis displaying a total of 8734 elements. Queried genes from the microarray analysis were confirmed by semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: Twenty-one genes were differentially expressed in wild type and W/WV mice. Eleven transcripts had 2.0-2.5 fold higher mRNA expression in W/WV gastric fundus when compared to wild type tissues. Ten transcripts had 2.1-3.9 fold lower expression in W/WV mutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function. CONCLUSIONS: These data provides evidence that several important genes have significantly changed in the murine fundus of W/WV mutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission

Differential gene expression profile in the small intestines of mice lacking pacemaker interstitial cells of Cajal.

BACKGROUND: We previously identified eight known and novel genes differentially expressed in the small intestines of wild type and W/WV mice, which have greatly reduced populations of the interstitial cells of Cajal, that are responsible for the generation of electrical slow waves, by using a differential gene display method. METHODS: By using the same method we isolated additional candidate genes that were specifically down- or up-regulated in W/WV mice. Novel transcripts were designated as DDWMEST. RESULTS: We isolated seven candidates that were specifically down- or up-regulated in W/WV mice. Two novel transcripts, DDWMEST 1 and -91 were increased in both fed and fasted W/WV mice. Expression of another five genes was suppressed in W/WV mice: ARG2 (Arginase II), ONZIN (encoding leukemia inhibitory factor regulated protein), and three novel transcripts: DDWMEST62, -84, and -100. Together with the previous report, we identified fifteen differentially expressed genes in total in the small intestines of W/WV mice. Eight of these genes were reduced in the jejunums of W/WV mice compared to age matched wild type mice, whereas the other seven genes showed an increase in expression. Differential expression was the same in fasted and fed animals, suggesting that the differences were independent of the dietetic state of the animal. CONCLUSIONS: Several known and novel genes are differentially expressed in the small intestines of W/WV mice. Differential gene comparison might contribute to our understanding of motility disorders associated with the loss of the interstitial cells of Cajal.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Common variation in EMSY and risk of breast and ovarian cancer: a case-control study using HapMap tagging SNPs.

BACKGROUND: EMSY could be involved in low-level susceptibility to breast and ovarian cancer. Gene amplification is seen in a proportion of breast and ovarian tumours and correlates with poor prognosis in breast cancer patients. Furthermore, the EMSY protein silences a transcription activation domain in BRCA2 exon 3. METHODS: We used a genetic association study design to determine if common genetic variation (frequency > or = 5%) in EMSY was associated with breast or ovarian cancer risk in the British population. Haplotype tagging single-nucleotide polymorphisms (htSNPs) were selected from the HapMap database and genotyped using Taqman in two large study sets of white British women (n [breast set] = 2343 cases and 2284 controls, n [ovarian set] = 864 cases and 864 controls). HapMap data might be insufficient to tag genetic variation in EMSY comprehensively. We therefore screened the gene promoter and coding sequences with denaturing high performance liquid chromatography in order to identify additional SNPs that are most likely to be functional. RESULTS: HapMap data on 22 SNPs show that 4 htSNPs tag 4 common haplotypes: rs2282611 (5'up t > g), rs4245443 (IVS7 g > a), rs2513511 (IVS16 a > g), rs2155220 (3'down c > t). We observed no association between any of the genotypes or associated haplotypes and breast or ovarian cancer risk. Seventeen out of the 18 remaining HapMap polymorphisms (94%) were well tagged by the 4 selected htSNPs (r2s > 0.8). Genotype frequencies for two further SNPs identified by screening and located near exon-intron boundaries, rs2508740 (IVS9 a > g) and rs11600501 (IVS10 c > t), were also similar in cases and controls. In order to simulate unidentified SNPs, we performed the leave-one-out cross-validation procedure on the HapMap data; over 95% of the common genetic variation was well represented by tagging polymorphisms. We are therefore likely to have tagged any common, functional variants present in our population. CONCLUSION: We found no association between common genetic variation in EMSY and risk of breast or ovarian cancer in two large study sets of white British women

Common ERBB2 polymorphisms and risk of breast cancer in a white British population: a case-control study.

INTRODUCTION: About two-thirds of the excess familial risk associated with breast cancer is still unaccounted for and may be explained by multiple weakly predisposing alleles. A gene thought to be involved in low-level predisposition to the disease is ERBB2 (HER2). This gene is involved in cell division, differentiation, and apoptosis and is frequently amplified in breast tumours. Its amplification correlates with poor prognosis. Moreover, the coding polymorphism I655V has previously been associated with an increased risk of breast cancer. METHODS: We aimed to determine if common polymorphisms (frequency >or= 5%) in ERBB2 were associated with breast cancer risk in a white British population. Five single-nucleotide polymorphisms (SNPs) were selected for study: SNP 1 near the promoter, SNP 2 in intron 1, SNP 3 in intron 4, SNP 4 in exon 17 (I655V), and SNP 5 in exon 27 (A1170P). We tested their association with breast cancer in a large case-control study (n = 2192 cases and 2257 controls). RESULTS: There were no differences in genotype frequencies between cases and controls for any of the SNPs examined. To investigate the possibility that a common polymorphism not included in our study might be involved in breast cancer predisposition, we also constructed multilocus haplotypes. Our set of SNPs generated all existing (n = 6) common haplotypes and no differences were seen in haplotype frequencies between cases and controls (P = 0.44). CONCLUSIONS: In our population, common ERBB2 polymorphisms are not involved in predisposition to breast cancer

Common variants in the ATM, BRCA1, BRCA2, CHEK2 and TP53 cancer susceptibility genes are unlikely to increase breast cancer risk.

INTRODUCTION: Certain rare, familial mutations in the ATM, BRCA1, BRCA2, CHEK2 or TP53 genes increase susceptibility to breast cancer but it has not, until now, been clear whether common polymorphic variants in the same genes also increase risk. METHODS: We have attempted a comprehensive, single nucleotide polymorphism (SNP)- and haplotype-tagging association study on each of these five genes in up to 4,474 breast cancer cases from the British, East Anglian SEARCH study and 4,560 controls from the EPIC-Norfolk study, using a two-stage study design. Nine tag SNPs were genotyped in ATM, together with five in BRCA1, sixteen in BRCA2, ten in CHEK2 and five in TP53, with the aim of tagging all other known, common variants. SNPs generating the common amino acid substitutions were specifically forced into the tagging set for each gene. RESULTS: No significant breast cancer associations were detected with any individual or combination of tag SNPs. CONCLUSION: It is unlikely that there are any other common variants in these genes conferring measurably increased risks of breast cancer in our study population.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are