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At least 973 records · Page 54Linked to original sources
PubMed2004

Phenotypic and microarray gene expression analysis of tri-dimensional raft-modeled human head and neck squamous cell carcinoma.

OBJECTIVES: To describe the phenotypic and gene expression differences in monolayer and tri-dimensional cultures systems. METHODS: Normal oral epithelial cells (NOEC), primary head and neck squamous cell carcinoma (HNSCC), and HNSCC cell lines were used to create and study modeled tri-dimensional tissue. Using cDNA microarray analysis, monolayer and raft-modeled tri-dimensional HNSCC cell lines were compared. RESULTS: NOEC, HNSCC, and both together can be modeled with tri-dimensional differentiation and cytokeratin characteristics analogous to in vivo tissue. Modeling of primary HNSCC resulted in a morphology of invasive HNSCC with areas of direct collagen invasion and MMP2 expression. Gene array analysis suggests that the individual cell lines themselves are the primary gene expression predictor and not the presence of tri-dimensional tissue architecture. CONCLUSIONS: This tissue culture modeling system approximates the differentiation and tri-dimensional structure of in vivo tissues, and that raft modeled tri-dimensional HNSCC does not have a significantly different gene expression profile than the corresponding monolayer culture.

Carcinoma, Squamous Cell↗
PubMed1989

Fully automated measurements by light microscopy of tissue sections using a cellular array computer.

Software was developed for the acquisition, segmentation and analysis of microscopic OD-images on a VICOM digital image processor, extended with a VISIOMORPH morphoprocessor board. The delineation algorithms for peroxisomes, lysosomes, and nuclei in liver, kidney, and adrenal gland sections start by thresholding the difference between the original image and a low pass filtered version. The resulting binary mask is then processed by morphological operations in order to produce an object overlay. The efficiency of the programs is evaluated by comparing delineated objects at different OD-levels, created by varying the stain or by multiplying the original pixel values with constant factors. Manual delineation on some images is also used as a reference. More complex algorithms are used for the delineation of muscle fibres in ATP-ase-stained sections and immunocytochemically labelled cells in monolayer preparations. Muscle images from parallel sections with different stainings are matched with a coordinate transform, enabling the transfer of the object mask from a single delineated image to the unprocessed images and thus obtain all necessary information for fibre classification. After segmentation, the OD-images and their object overlays are fed into a data extraction program, measuring for each delineated object user-selected features. Data are sent to a VAX for statistical interpretation.

Adrenal Cortex↗
PubMed2004

cDNA array analysis of Japanese quail lines divergently selected for four-week body weight.

Decades of divergent selection for 4-wk BW has produced 3 lines of growth-selected Japanese quail. P line quail have been selected for >110 generations for 4-wk posthatch BW and are nearly 3-fold larger than the randomly bred control C line. The H line has been selected for high 4-wk BW for 52 generations and the L line has been selected for low 4-wk BW for 54 generations. To identify differentially expressed genes that may play a role in defining the differences in these lines, a DNA array containing 4,704 random anonymous cDNA clones from 8-d C line embryos was screened using isotopically-labeled cDNA from the different quail lines. Array analysis yielded 3 differentially expressed cDNA clones that were confirmed by Northern blot analysis. The 35-kD quail EB1 protein, previously unidentified, was shown to have elevated transcripts in the L line and decreased transcripts in the H and P lines, compared with the C line. It was also widely expressed in embryonic and adult tissues by BLASTN analysis of chicken expressed sequence tag (EST) libraries. Two other cDNA clones are novel sequences expressed at higher levels in the L line and at lower levels in the H and P lines, one of which was more selectively expressed in embryos and adult tissues by BLASTN analysis. These limited findings suggest that anonymous cDNA array analysis is a productive means to identify differentially expressed genes in growth-selected poultry.

Animals↗
PubMed2004

Spinal cord molecular profiling provides a better understanding of amyotrophic lateral sclerosis pathogenesis.

Research efforts in amyotrophic lateral sclerosis (ALS) have not yet provided a comprehensive explanation of the disease pathogenesis, which is emerging as a complex interaction between multiple factors. Gene expression studies traditionally based on single mRNA specie analysis have recently progressed to allow entire transcriptional profiles of affected tissues to be obtained through array-based methods. This experimental approach has significantly improved our understanding of the molecular changes occurring in ALS, although its limitations in the detection of low-abundance transcripts in tissues with a high level of complexity are becoming increasingly recognized. In this paper, experimental findings based on an expression study in post-mortem spinal cord from sporadic ALS individuals will be discussed in light of recently published data using array analysis in an animal model of the disease. Previous expression data obtained using conventional techniques are also compared. Through the analysis of the information arising from ALS post-mortem and animal model tissues studies, we have identified a pattern of molecular events in which factors implicated in the immune response, cytoprotection and growth-differentiation are differentially regulated in a time-dependent way from early to advanced stages of disease progression.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Cloning and characterization of a novel cardiac-specific kinase that interacts specifically with cardiac troponin I.

Cardiac-restricted genes play important roles in cardiovascular system. In an effort to identify such novel genes we identified a novel cardiac-specific kinase gene TNNI3K localized on 1p31.1 based on bioinformatics analyses. Sequence analysis suggested that TNNI3K is a distant family member of integrin-linked kinase. Northern blot and 76-tissue array analyses showed that TNNI3K is highly expressed in heart, but is undetectable in other tissues. Immunohistochemical analysis predominantly localized TNNI3K in the nucleus of cardiac myocytes. In vitro kinase assay showed that TNNI3K is a functional kinase. The yeast two-hybrid system showed that TNNI3K could directly interact with cardiac troponin I, results that were further confirmed by coimmunoprecipitation in vivo. Our data suggest that TNNI3K is a cardiac-specific kinase and play important roles in cardiac system.

Adult↗
PubMed2003

Genomic structure of human OKL38 gene and its differential expression in kidney carcinogenesis.

We previously demonstrated the growth inhibitory property of OKL38 and its possible roles in mammary carcinogenesis. To further understand the regulation and roles of OKL38 in tumorigenesis we proceeded to clone and characterize the human OKL38 gene and three of its variants with transcripts of 1.9, 2.2, and 2.4 kb. The human OKL38 gene spans approximately 18 kb and contains 8 exons and 7 introns with exon size ranging from 92 to 1270 bp. RT-PCR and sequence analysis suggest that different transcripts were arrived through differential promoter usage and alternate splicing. Multiple Tissue Expression array (MTE) and Multiple Tissue Northern blot (MTN) indicated that OKL38 was ubiquitously expressed in all tissues with high expression in liver, kidney, and testis. The cancer profiling array (CPA) of paired normal/tumor cDNA showed that OKL38 mRNA was down-regulated in 70% (14 of 20) of kidney tumors. Western analysis revealed that the OKL38 protein was undetectable in 78% (7 of 9 pairs) of kidney tumor tissues. Immunohistological analysis showed that 64% (14 of 22) of kidney tumors were either lost or underexpressed OKL38 protein compared with the adjacent normal tissue. A transfection study using OKL38-eGFP recombinant construct showed that overexpression of the 52 kDa OKL38 protein in A498 cells resulted in growth inhibition and cell death. This study demonstrates the complex genomic structure of the OKL38 gene and its growth inhibitory and cytotoxic properties. Our data suggest the potential use of OKL38 in diagnosis, prognosis, and/or treatment of kidney cancer.

Apoptosis Regulatory Proteins↗
PubMed2006

Molecular alterations of monophasic synovial sarcoma: loss of chromosome 3p does not alter RASSF1 and MLH1 transcriptional activity.

Differential diagnosis of monophasic synovial sarcoma requires the detection of specific biological markers. In this study we evaluated the presence of molecular alterations in 15 monophasic synovial sarcomas. Multiple changes affecting chromosome arms were detected by CGH-array in all microdissected cases available, and an association between gain or loss of specific regions harbouring cancer progression-associated genes and aneuploid status was found. The most frequent alteration was loss of 3p including 3p21.3-p23 region that, however, did not involve the promoter regions of the corresponding genes, RASSF1 and MLH1. Using Real-Time PCR, mRNA levels of both resulted moderately high compared to normal tissue; however, the weak to absent protein expression suggests RASSF1 and MLH1 post-transcription deregulation. Moreover, immunohistochemical analysis revealed that both mesenchymal and epithelial antigens were present in diploid tumours. These findings confirm the genetic complexity of monophasic synovial sarcoma and underline the need to integrate different analyses for a better knowledge of this tumour, essential to investigate new diagnostic and prognostic markers.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Screening for PTB domain binding partners and ligand specificity using proteome-derived NPXY peptide arrays.

Modular interaction domains that recognize peptide motifs in target proteins can impart selectivity in signaling pathways. Phosphotyrosine binding (PTB) domains are components of cytoplasmic docking proteins that bind cell surface receptors through NPXY motifs. We have employed a library of human proteome-derived NXXY sequences to explore PTB domain specificity and function. SPOTS peptide arrays were used to create a comprehensive matrix of receptor motifs that were probed with a set of 10 diverse PTB domains. This approach confirmed that individual PTB domains have selective and distinct recognition properties and provided a means to explore over 2,500 potential PTB domain-NXXY interactions. The results correlated well with previously known associations between full-length proteins and predicted novel interactions, as well as consensus binding data for specific PTB domains. Using the Ret, MuSK, and ErbB2 receptor tyrosine kinases, we show that interactions of these receptors with PTB domains predicted to bind by the NXXY arrays do occur in cells. Proteome-based peptide arrays can therefore identify networks of receptor interactions with scaffold proteins that may be physiologically relevant.

Adaptor Proteins, Signal Transducing↗
PubMed2002

[Preliminary analysis on the gene expression profiles of different types of gliomas with cDNA array].

BACKGROUND & OBJECTIVE: There is great signification to classify the molecular pathogenesis of gliomas. Gene chip is able to profile the gene expression of tumors, which may become a new method for studying molecular pathology of tumors. The cDNA was used in order to detect the differences in gene expression profiles of different types of gliomas. METHODS: cDNA probes tagged with 32P were converted from total RNA extracted from 4 samples of 3 different pathological types, including oligodendrogliomas, anaplastic astrocytomas and ependymomas, and one sample of normal human brain tissue as well, which were then hybridized to the Atlas array. After autoradiography, the gene expression profiles were analyzed with the special software and by the cluster analysis. RESULTS: As compared to the normal brain tissue, the number of differential expressed genes in these tumor samples ranging from 11 to 118 was found. These 4 samples could be divided into 3 kinds of types when 107 differential expressed genes were selected to be as the cluster analysis index. This result was in accordance with the neuropathological diagnosis. CONCLUSION: cDNA array is able to profile gene expression in different types of gliomas. Combined with the bioinformational method, we may further obtain useful information about molecular pathology of gliomas.

Gene Expression Profiling↗
PubMedDate not supplied

Profiling and verification of gene expression patterns in normal and malignant human prostate tissues by cDNA microarray analysis.

cDNA microarray technology allows the "profiling" of gene expression patterns for virtually any cellular material. In this study, we applied cDNA microarray technology to profile changes in gene expression associated with human prostate tumorigenesis. RNA prepared from normal and malignant prostate tissue was examined for the expression levels of 588 human genes. Four different methods for data normalization were utilized. Of these, normalization to ACTB expression proved to be the most rigorous technique with the least probability of producing spurious results. After normalization to ACTB expression, 15 of 588 (2.6%) genes examined by array analysis were differentially expressed by a factory of 2x or more in malignant compared to normal prostate tissues. The expression patterns for 8 of 15 genes have been reported previously in prostate tissues (TGFbeta3, TGFBR3, IGFII, IGFBP2, VEGF, FGF7, ERBB3, MYC), but those of seven genes are reported here for the first time (MLH1, CYP1B1, RFC4, EPHB3, MGST1, BTEB2, MLP). These genes describe at least four metabolic and signaling pathways likely disrupted in human prostate tumorigenesis. Reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analyses quantitated with reference to ACTB expression levels verified the trends in gene expression levels observed by array analysis for 14/15 and 8/8 genes, respectively. However, RT-PCR and Northern blot analyses accurately verified the "fold" differences in expression levels for only 6/15 (40%) and 7/8 (88%) of genes examined, respectively, demonstrating the need to better validate quantitative differences in gene expression revealed by array-based techniques.

Blotting, Northern↗
PubMed2004

Experiences from tissue microarray in soft tissue sarcomas.

The tissue microarray (TMA) technology was introduced in 1998 as a tissue preserving, high-throughput technique that allows studies of multiple markers in large sample sets. TMA slides can be analyzed using techniques such as immunohistochemistry and in situ hybridization and represents a powerful tool for the investigation of potential diagnostic and prognostic markers identified in DNA microarray studies. We review the TMA method, its reproducibility, advantages, limitations and future perspectives with specific focus on soft tissue sarcomas.

Biomarkers, Tumor↗
PubMed2003

Optimization of a beam shaping bolus for superficial microwave hyperthermia waveguide applicators using a finite element method.

Temperature inhomogeneity in hyperthermia treatments often limits the total thermal dose that can be delivered to the tumour region. To reduce such inhomogeneities, a prototype dynamically modifiable square array of saline-filled patches which attenuate microwave energy was developed for superficial treatments that use external microwave applicators. The array was situated inside the coupling water bolus that is often used with external applicators. The prototype has been previously tested clinically with promising results. A more complete theoretical analysis of the performance of this new bolus design and improvements to its design by modelling are presented here. The analysis was performed by performing five iterative simulations of the SAR pattern produced inside a tissue structure by a waveguide applicator with a water bolus containing the dynamic patch array attached. Between iterations the patch array configuration was modified in an attempt to improve the ability of the bolus to confine heating to an 'L'-shaped tumour region. These simulations were performed using the finite element method. The steady-state temperature profile was then computed using a finite element method based simulation of heat transfer that assumed a given applicator power level and water bolus temperature. Several iterations of these heat transfer simulations were performed with varying applicator power level and water bolus temperature to improve the confinement of heating to the target region. The analysis showed that the dynamic patch array should be capable of conforming heating to an 'L'-shaped target tumour region while limiting the heating to the surrounding normal tissue to an acceptable level.

Adipose Tissue↗
PubMed2004

Discrimination of melanocytic tumors by cDNA array hybridization of tissues prepared by laser pressure catapulting.

Gene expression profiling by cDNA array analysis in melanoma is hampered by the need for large amounts of RNA to prepare reliable probes for array hybridization. On the other hand, for ex vivo analysis of malignant cells from melanocytic tumors laser pressure catapulting is an essential prerequisite to obtain noncontaminated melanocytic preparations; however, laser pressure catapulting prepared material provides only nanogram amounts of RNA. In this study we present an approach to overcome these limitations by combining laser pressure catapulting and real-time polymerase chain reaction based SMART cDNA amplification technology. Reproducible and reliable hybridization patterns from about 500 laser pressure catapulting prepared cell equivalents from 22 cases of melanocytic tumors were generated using array analysis. Univariate analysis revealed significant differences of the expression pattern of melanocytic nevi, melanomas, and melanoma metastases. Multivariate analysis with four genes being the best univariate discriminative features (tyrosinase related protein 2, translation initiation factor 2 gamma, ubiquitine conjugating enzyme E2I and one expressed sequence tag) allowed clustering of nevi, melanomas, and melanoma metastases with an accuracy of 82%. Data validation was performed by additional quantitative reverse transcription-polymerase chain reaction (TaqMan-reverse transcription-polymerase chain reaction). Taken together, this study shows, that (1) array analysis is feasible on tumors with rather low cell numbers, and (2) differences in expression profiles allow discrimination between benign and malignant lesions. Expression patterns of marker genes defined in unequivocal histopathologic entities may improve the diagnostic and prognostic assessment of difficult melanocytic lesions, which is still the hardest problem in dermatopathology.

Biopsy↗
PubMed2004

Endothelin-1 induces expression of matrix-associated genes in lung fibroblasts through MEK/ERK.

The endothelins are a family of endothelium-derived peptides that possess a variety of biological activities, including potent vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and pulmonary fibrosis. Here, we use genome-wide expression array analysis to show that the addition of ET-1 (100 nm, 4 h) to normal lung fibroblasts directly induces expression of matrix and matrix-associated genes, including the profibrotic protein CCN2 (connective tissue growth factor, or CTGF). ET-1 induces the MEK/ERK MAP kinase pathway in fibroblasts. Blockade of the MEK/ERK kinase pathway with U0126 abrogates the ability of ET-1 to induce expression of matrix and matrix-associated mRNAs and the CCN2 protein. The CCN2 promoter possesses an ET-1 response element, which maps to the previously identified basal control element-1 (BCE-1) site. Our results suggest that ET-1 induces a program of matrix synthesis in lung fibroblasts and that ET-1 may play a key role in connective tissue deposition during wound repair and in pulmonary fibrosis.

Endothelin-1↗
PubMed2006

Loss of arginase I results in increased proliferation of neural stem cells.

Loss of arginase I (AI) results in a metabolic disorder characterized by growth retardation, increased mental impairment and spasticity, and potentially fatal hyperammonemia. This syndrome plus a growing body of evidence supports a role for arginase and arginine metabolites in normal neuronal development and function. Here we report our initial observations of the effects of AI loss on proliferation and differentiation of neural stem cells (NSCs) isolated from the germinal zones of embryonic and newborn AI knockout (KO) mice compared with heterozygous (HET) and wild-type (WT) control animals. By using both short and long-term proliferation assays (3 and 10 days, respectively), we found a 1.5-2-fold increase in the number of KO cells compared with WT. FACS analysis showed an increase in KO cells in the synthesis phase of the cell cycle vs. WT cells. After NSC differentiation, AI-deficient cells expressed beta-tubulin, SMI81 (SNAP25), glial fibrillary acidic protein, and CNPase, which are markers consistent with neurons, astrocytes, and oligodendrocytes. Many KO cells exhibited a more mature morphology and expressed mature neuronal markers that were decreased or not present in HET or WT cells. Limited, comparative expression array and quantitative RT-PCR analysis identified differences in the levels of several mRNAs encoding structural, signaling, and arginine metabolism proteins between KO and WT cells. The consequence of these changes may contribute to the differential phenotypes of KO vs. WT cells. It appears that AI may play an important and unanticipated role in growth and development of NSCs.

Animals↗
PubMed2005

Characterization of free-floating spheres from human trabecular meshwork (HTM) cell culture in vitro.

It has been observed in several tissues that direct isolation of cells in serum-free media and on nonadhesive substrates results in the formation of spherical clusters of cells known as free-floating spheres. Such free-floating spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Our goal was to isolate and characterize such free-floating spheres from HTM cell primary cultures. For this purpose, HTM cells were incubated in serum-free media and on a nonadhesive substrate. Individual free-floating spheres generated in these conditions were isolated in 96-well plates, and their proliferative capacity was evaluated by monitoring their size increase over time. The expression of the TM markers, MGP and CHI3L1, was examined using recombinant adenoviruses containing the respective promoters. Morphology of the free-floating spheres was analysed in semithin sections, and the gene expression profile was obtained using Human Genome U133 Plus 2.0 Affymetrix microarrays. HTM cells incubated in serum-free media and on nonadhesive substrate generated free-floating spheres that could be grown for more than 3 months. Addition of serum to the culture media promoted the attachment of the spheres to the substrate, migration of cells from the spheres, and differentiation into cells phenotypically similar to normal TM cells. Gene profiling analysis demonstrated strong similarities between the gene expression profiles of the spheres and HTM cell monolayers. Both infection with the recombinant adenoviruses and gene array analysis demonstrated the expression of CHI3L1 and MGP, indicating that free-floating spheres likely originate from HTM cells. Gene array analysis also showed expression of the marker for neural precursor cells nestin, as well as leukemia inhibitory factor, a gene involved in the maintenance of the undifferentiated state of progenitor cells. Analysis of semithin sections indicated that these TM free-floating spheres were highly dynamic structures demonstrating a distinct radial gradient of cell proliferation, survival, and apoptosis. Extensive up- and down-regulation of gene expression was associated with the processes of sphere attachment and cell migration after the addition of serum. These results suggest that HTM primary cultures might contain relatively undifferentiated or progenitor cells. The availability of TM progenitor cell cultures could constitute a useful tool to investigate cell therapy approaches targeting the TM in glaucoma.

Adipokines↗
PubMed2003

Overlapping microarray profiles of dentate gyrus gene expression during development- and epilepsy-associated neurogenesis and axon outgrowth.

Neurogenesis and axon outgrowth are features shared by normal nervous system development and certain forms of epileptogenesis. This observation has led to the hypothesis that some aspects of normal development and epileptogenesis have common molecular mechanisms. To test this hypothesis, we have used DNA microarray analysis to characterize gene expression in the dentate gyrus and identify genes exhibiting similar patterns of regulation during development and epileptogenesis. Of more than 8000 sequences surveyed, over 600 were regulated during development or epileptogenesis, and 37 of these were either upregulated or downregulated during both processes. In situ hybridization analysis of a subset of these "commonality genes" confirmed the patterns of regulation predicted by the microarray data in most cases and demonstrated various spatial and temporal patterns of commonality gene expression. Of the 25 named commonality genes in which some functional characteristics are known, 11 have been implicated in cell morphology and axon outgrowth or cellular proliferation and fate determination. This enrichment for candidate plasticity-related genes supports the concept that developmental mechanisms contribute to network alterations associated with epileptogenesis and offers a useful strategy for identifying molecules that may play a role in both of these processes.

Analysis of Variance↗