Gene expression profiling in human esophageal cancers using cDNA microarray

Human esophageal cancer cell lines and human esophageal cancer tissues were profiled on cDNA microarrays. In esophageal cancer cell lines, KYAE and OE-33 (adenocarcinomas) were distinguished from KYSE series (squamous cell carcinomas). Although SK-GT-4 and TE7 were derived from adenocarcinomas, they had a comparatively similar expression profile to the KYSE series. A set of genes whose expression commonly either increased or decreased in cancer cell lines was identified. Genes that were characteristically expressed in KYAE and OE-33 were also identified. The gene expression profiles of cancer tissues (CTs) were remarkably different from those of the cancer cell lines (CCLs). Notable differences between CCLs and CTs were observed in matrix metalloproteinases, plasminogen activator, collagens, paxillin, and thrombospondin 2, etc., whose expression was not increased in CCLs but increased in CTs. Twenty-three genes were extracted to categorize patients according to their prognoses, and clustering analyses, using these genes, were performed successfully.     

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ～90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.     

Mining microarray expression data by literature profiling

Background  

The rapidly expanding fields of genomics and proteomics have prompted the development of computational methods for managing, analyzing and visualizing expression data derived from microarray screening. Nevertheless, the lack of efficient techniques for assessing the biological implications of gene-expression data remains an important obstacle in exploiting this information.     

Lectin histochemistry for in situ profiling of rat colon sialoglycoconjugates

The growing interest in glycoconjugates expressed and released by the epithelium of the intestinal mucosa is tightly related to the multiple functional roles attributed to sialic acid and its derivatives. In the present work, biotin and HRP conjugated lectins were used to detect the sialylation pattern and to identify specific structural features of sialoderivatives in the rat colon. In particular, the occurrence and distribution of sialic acids linked alpha2,6 to D-Gal/D-GalNAc and alpha2,3 to D-Gal were directly demonstrated with SNA and MAL II binding, respectively. In addition, in order to by-pass the specificity problems of SNA and MAL II as histochemical reagents, as well as to look for additional and complementary information about acetylation degree and sites, we combined sialidase digestion, potassium hydroxide deacetylation, and differential periodate oxidation with PNA and DBA binding. The data showed the distribution and structure of sialic acid-beta-D-Gal(1-3)-D-GalNAc and sialic acid-D-GalNac sequences, which proved to be widely distributed as cellular components or secretory products in surface goblet cells and crypt cells of the colonic epithelium. A high degree of O-acetylation, with acetyl groups mainly at 9 and 4 positions, was found, showing an increasing gradient from the proximal to distal portion of the colon. These results, which largely reproduce the sialylation pattern in other species, contribute new insights in defining the tissue specific expression of sialoderivatives in the colonic mucosa, and testify to their high heterogeneity which the wide range of sialic acid functional correlates in the intestinal tract depend on.     

Antibody profiling in plague patients by protein microarray

A protein microarray containing 144 known or putative virulence-related proteins of Yersinia pestis was used to evaluate the antibody responses of plague patients. Forty-two proteins were found to be expressed in vivo and antibodies against 14 of them were detected in all patients analyzed, providing potential candidates for novel protective antigens and novel serodiagnostic markers in Y. pestis. Moreover, the lack of antibody to LcrV in the five patients in Focus F might be a challenge to our understanding of the pathogenesis of Y. pestis.     

Lectin histochemical HPA-binding pattern of human breast and colon cancers is associated with metastases formation in severe combined immunodeficient mice

Metastasis formation is a major clinical problem in cancer treatment, and no significant progress in the treatment of metastatic spread has been made. This apparent lack of progress is partly caused by the absence of clinically relevant animal models of meta stases. The binding of the lectin Helix pomatia agglutinin (HPA) has been associated with a poor prognosis in breast and colon cancer patients. HPA-positive and -negative human breast and colon cancer cell lines were transplanted into severe combined immunodeficient (SCID) mice. HPA-positive breast cancer cell lines (MCF-7 and T47D) metastasized in SCID mice, whereas the HPA-negative ones (BT20, HS578T and HBL100) did not. The HPA-positive colon cancer cell line HT29 metastasized, while the HPA-negative ones (COLO320DM, SW480 and SW620) did not. However, in two of eight SCID mice inoculated with the HPA-negative colon cancer cell line, CACO2 metastatic deposits were found. Despite this exception, HPA binding is a good indicator of the metastasis of human breast and colon cancer cells in SCID mice: 23 out of 26 HPA-positive cancers metastasized, as opposed to only two out of 38 HPA-negative cancers. This experimental model is well suited for investigating the functional role of carbohydrate residues recognized by HPA in breast and colon cancer metastasis. This revised version was published online in November 2006 with corrections to the Cover Date.     

A quality-controlled microarray method for gene expression profiling

Gene expression profiling on microarrays is widely used to measure the expression of large numbers of genes in a single experiment. Because of the high cost of this method, feasible numbers of replicates are limited, thus impairing the power of statistical analysis. As a step toward reducing technically induced variation, we developed a procedure of sample preparation and analysis that minimizes the number of sample manipulation steps, introduces quality control before array hybridization, and allows recovery of the prepared mRNA for independent validation of results. Sample preparation is based on mRNA separation using oligo(dT) magnetic beads, which are subsequently used for first-strand cDNA synthesis on the beads. cDNA covalently bound to the magnetic beads is used as template for second-strand cDNA synthesis, leaving the intact mRNA in solution for further analysis. The quality of the synthesized cDNA can be assessed by quantitative polymerase chain reaction using 3'- and 5'-specific primer pairs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase. Second-strand cDNA is chemically labeled with fluorescent dyes to avoid dye bias in enzymatic labeling reactions. After hybridization of two differently labeled samples to microarray slides, arrays are scanned and images analyzed automatically with high reproducibility. Quantile-normalized data from five biological replica display a coefficient of variation 45% for 90% of profiled genes, allowing detection of twofold changes with false positive and false negative rates of 10% each. We demonstrate successful application of the procedure for expression profiling in plant leaf tissue. However, the method could be easily adapted for samples from animal including human or from microbial origin.     

An oligonucleotide microarray for mouse imprinted genes profiling

Genomic imprinting is an epigenetic phenomenon unique to mammals that causes some genes to be expressed according to their parental origin. It results in developmental asymmetry in the function of the parental genomes. We describe here a method for the profiling of imprinted genes based on the development of a mouse imprinting microchip containing oligonucleotides corresponding to 493 genes, including most of the known imprinted genes (IG = 63), genes involved in epigenetic processes (EPI = 15), in metabolism (= 147), in obesity (= 10) and in neurotransmission (= 256) and housekeeping reference genes (= 2). This custom oligonucleotide microarray has been constructed to make data analysis and handling more manageable than pangenomic microarrays. As a proof of concept we present the differential expression of these 493 genes in different tissues (liver, placenta, embryo) of C57BL6/J mice fed different diets. Appropriate experimental strategies and statistical tools were defined at each step of the data analysis process with regard to the different sources of constraints. Data were confirmed by expression analyses based on quantitative real-time PCR. These oligochips should make it possible to increase our understanding of the involvement of imprinted genes in the timing of expression programs, tissue by tissue, stage by stage, in response to nutrients, lifestyles and other as yet unknown critical environmental factors in a variety of physiopathological situations, and in animals of different strains, ages and sexes. The use of oligonucleotides makes it possible to expand this microchip to include the increasing number of imprinted genes discovered.     

Lectin histochemical HPA-binding pattern of human breast and colon cancers is associated with metastases formation in severe combined immunodeficient mice

Metastasis formation is a major clinical problem in cancer treatment, and no significant progress in the treatment of metastatic spread has been made. This apparent lack of progress is partly caused by the absence of clinically relevant animal models of meta stases. The binding of the lectin Helix pomatia agglutinin (HPA) has been associated with a poor prognosis in breast and colon cancer patients. HPA-positive and -negative human breast and colon cancer cell lines were transplanted into severe combined immunodeficient (SCID) mice. HPA-positive breast cancer cell lines (MCF-7 and T47D) metastasized in SCID mice, whereas the HPA-negative ones (BT20, HS578T and HBL100) did not. The HPA-positive colon cancer cell line HT29 metastasized, while the HPA-negative ones (COLO320DM, SW480 and SW620) did not. However, in two of eight SCID mice inoculated with the HPA-negative colon cancer cell line, CACO2 metastatic deposits were found. Despite this exception, HPA binding is a good indicator of the metastasis of human breast and colon cancer cells in SCID mice: 23 out of 26 HPA-positive cancers metastasized, as opposed to only two out of 38 HPA-negative cancers. This experimental model is well suited for investigating the functional role of carbohydrate residues recognized by HPA in breast and colon cancer metastasis. This revised version was published online in November 2006 with corrections to the Cover Date.     

Functional profiling of microarray experiments using text-mining derived bioentities

MOTIVATION: The increasing use of microarray technologies brought about a parallel demand in methods for the functional interpretation of the results. Beyond the conventional functional annotations for genes, such as gene ontology, pathways, etc. other sources of information are still to be exploited. Text-mining methods allow extracting informative terms (bioentities) with different functional, chemical, clinical, etc. meanings, that can be associated to genes. We show how to use these associations within an appropriate statistical framework and how to apply them through easy-to-use, web-based environments to the functional interpretation of microarray experiments. Functional enrichment and gene set enrichment tests using bioentities are presented.     

Expression profiling by whole-genome microarray hybridization reveals differential gene expression in breast cancer cell lines after lycopene exposure

The correlation between diet and variation in gene-expression is an important field which could be considered to approach cancer pathways comprehension. We examined the effects of lycopene on breast cancer cell lines using pangenomic arrays. Lycopene is derived predominantly from tomatoes and tomato products and there is some epidemiologic evidence for a preventive role in breast cancer. Previously, we investigated lycopene in breast cancer using a dedicated breast cancer microarray. To confirm these results and explore pathways other than those implicated in breast cancer, for this study we used pangenomic arrays containing 25,000 oligonucleotides. This in vitro study assayed two human mammary cancer cell lines (MCF-7 and MDA-MB-231), and a fibrocystic breast cell line (MCF-10a) treated or not with 10 μM lycopene for 48 h. A competitive hybridization was performed between Cy3-labeled lycopene treated RNA and Cy5-labeled untreated RNA to define differentially expressed genes. Using t-test analysis, a subset of 391 genes was found to be differentially modulated by lycopene between estrogen-positive cells (MCF-7) and estrogen-negative cells (MDA-MB-231, MCF-10a). Hierarchical clustering revealed 726 discriminatory genes between breast cancer cell lines (MCF-7, MDA-MB-231) and the fibrocystic breast cell line (MCF-10a). Modified gene expression was observed in various molecular pathways, such as apoptosis, cell communication, MAPK and cell cycle as well as xenobiotic metabolism, fatty acid biosynthesis and gap junctional intercellular communication.     

Expression profiling by whole-genome microarray hybridization reveals differential gene expression in breast cancer cell lines after lycopene exposure

The correlation between diet and variation in gene-expression is an important field which could be considered to approach cancer pathways comprehension. We examined the effects of lycopene on breast cancer cell lines using pangenomic arrays. Lycopene is derived predominantly from tomatoes and tomato products and there is some epidemiologic evidence for a preventive role in breast cancer. Previously, we investigated lycopene in breast cancer using a dedicated breast cancer microarray. To confirm these results and explore pathways other than those implicated in breast cancer, for this study we used pangenomic arrays containing 25,000 oligonucleotides. This in vitro study assayed two human mammary cancer cell lines (MCF-7 and MDA-MB-231), and a fibrocystic breast cell line (MCF-10a) treated or not with 10 microM lycopene for 48 h. A competitive hybridization was performed between Cy3-labeled lycopene treated RNA and Cy5-labeled untreated RNA to define differentially expressed genes. Using t-test analysis, a subset of 391 genes was found to be differentially modulated by lycopene between estrogen-positive cells (MCF-7) and estrogen-negative cells (MDA-MB-231, MCF-10a). Hierarchical clustering revealed 726 discriminatory genes between breast cancer cell lines (MCF-7, MDA-MB-231) and the fibrocystic breast cell line (MCF-10a). Modified gene expression was observed in various molecular pathways, such as apoptosis, cell communication, MAPK and cell cycle as well as xenobiotic metabolism, fatty acid biosynthesis and gap junctional intercellular communication.