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At least 271 records · Page 15Linked to original sources
PubMed2000

Functional discovery via a compendium of expression profiles.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Cell Wall↗
PubMedDate not supplied

Early gene expression profile of human skin to injury using high-density cDNA microarrays.

Disturbances in normal wound healing may be traced to perturbations in gene expression following injury. To decipher normal and abnormal genetic responses to cutaneous injury, baseline gene expression of uninjured skin and injured skin must be better defined. Our aim for this study was to determine the gene expression profile of human skin immediately following injury using cDNA microarrays. Samples of normal and injured skin were obtained from 5 healthy females undergoing breast reduction surgery. Specimens of the epidermis and dermis were obtained at 30 minutes and 1 hour after the initial injury. RNA was extracted, reverse transcribed into cDNA and hybridized onto high-density cDNA microarray membranes of 4,000 genes. At 30 minutes, injury resulted in a consistent increase (> 2x) in gene expression of 124 out of 4,000 genes (3%). These genes were primarily involved in transcription and signaling. None of the 4,000 genes were decreased (< 2x) at 30 minutes. At 1 hour only 46 out of the 4,000 genes were increased in expression (1.15%) but 264 out of 4,000 (6.6%) genes were decreased greater than 2 fold, indicating a silencing of many structural genes. We have identified several genes, namely, suppressor of cytokine signaling-1, rho HP1, and BB1, that are highly expressed after injury and may have an unappreciated role in regulating the initial inflammatory response. These data provide an initial high-throughput analysis of gene expression immediately following human skin injury and show the utility and future importance of high-throughput analysis in skin biology and wound repair.

Adult↗
PubMed2002

Expression profiling in transformed human B cells: influence of Btk mutations and comparison to B cell lymphomas using filter and oligonucleotide arrays.

We have used both Clontech Atlas Human Hematology/Immunology cDNA microarrays, containing 588 genes, and Affymetrix oligonucleotide U95Av2 human array complementary to more than 12,500 genes to get a global view of genes expressed in Epstein-Barr virus (EBV)-transformed B cells and genes regulated by Bruton's tyrosine kinase (Btk). We compared EBV-transformed wild-type (WT) B cells from a healthy individual, WT1 and an X-linked agammaglobulinemia (XLA) patient cell line, XLA1, using the Clontech filters arrays. Eleven genes were > or =1.9-fold induced in absence of functional Btk. Furthermore, we analyzed a second patient cell line, XLA2, and compared this to two WT cell lines using oligonucleotide arrays. A total of 391 genes were found to be differentially expressed, including kinases and transcriptions factors. Furthermore, one expressed sequence tag and eight complementary DNA clones with unknown function were down-regulated in XLA2, indicating their biological role. Higher-fold inductions, Fyn (39.5), Hck (15.5) and Cyp1B1 (5.8), were observed using oligonucleotide array and were confirmed using real-time PCR for Fyn (20.8), Hck (6.7) and Cyp1B1 (10). Two genes, B cell translocation gene1 (BTG1) and B cell-specific OCT binding factor-1 (OBF-1) were induced > or =1.9-fold in both XLA1 and XLA2 analyzed by Atlas filter arrays andAffymetrix chips, respectively. Data from both filter and oligonucleotide arrays were compared to the gene clusters of a previously published lymphoma expression profile by linking to the UniGene transcript database. Our findings demonstrate for the first time the use of microarray to study the influence of Btk mutations and the use of functional annotation and validation of expression data by comparison of microarray analyses.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2000

The gene expression profile of human umbilical vein endothelial cells stimulated by tumor necrosis factor alpha using DNA microarray analysis.

Stimulation of vascular endothelial cells by tumor necrosis factor alpha (TNFalpha) plays a critical role in the pathogenesis of inflammation and vascular diseases. Changes in the gene expression profile in cultured human umbilical vein endothelial cells (HUVEC) treated with TNFalpha was analyzed with high-density oligonucleotide arrays comprised of 35,000 genes. TNFalpha stimulation profoundly induced genes involved in signal transduction, leukocyte adhesion and chemoattraction. ICAM-1 mRNA (fold change 111.9) was most profoundly induced followed by TNFalpha receptor-associated factor 1 (TRAF1) (95.5), Bcl3 (71.8), IL8 (65.4), fractalkaine (62.4), E-selectin (48.0), lymphotoxin beta (41.3) and VCAM-1 (31.7). In addition to these previously known genes, 18 poorly characterized or novel genes known as ESTs profoundly induced by TNFalpha. Initial sequencing analysis identified three of these the genes for squalene epoxydase, chromodomain helicase DNA binding protein 4, and CLP respectively. Further analysis of these genes will provide important information about TNFalpha signaling and function in vascular endothelial cells.

Cell Line↗
PubMed2001

The consequences of chromosomal aneuploidy on gene expression profiles in a cell line model for prostate carcinogenesis.

Here we report the genetic characterization of immortalized prostate epithelial cells before and after conversion to tumorigenicity using molecular cytogenetics and microarray technology. We were particularly interested to analyze the consequences of acquired chromosomal aneuploidies with respect to modifications of gene expression profiles. Compared with nontumorigenic but immortalized prostate epithelium, prostate tumor cell lines showed high levels of chromosomal rearrangements that led to gains of 1p, 5, 11q, 12p, 16q, and 20q and losses of 1pter, 11p, 17, 20p, 21, 22, and Y. Of 5700 unique targets on a 6.5K cDNA microarray, approximately 3% were subject to modification in expression levels; these included GRO-1, -2, IAP-1,- 2, MMP-9, and cyclin D1, which showed increased expression, and TRAIL, BRCA1, and CTNNA, which showed decreased expression. Thirty % of expression changes occurred in regions the genomic copy number of which remained balanced. Of the remainder, 42% of down-regulated and 51% of up-regulated genes mapped to regions present in decreased or increased genomic copy numbers, respectively. A relative gain or loss of a chromosome or chromosomal arm usually resulted in a statistically significant increase or decrease, respectively, in the average expression level of all of the genes on the chromosome. However, of these genes, very few (e.g., 5 of 101 genes on chromosome 11q), and in some instances only two genes (MMP-9 and PROCR on chromosome 20q), were overexpressed by > or =1.7-fold when scored individually. Cluster analysis by gene function suggests that prostate tumorigenesis in these cell line models involves alterations in gene expression that may favor invasion, prevent apoptosis, and promote growth.

Aneuploidy↗
PubMed2001

Arrays for protein expression profiling: towards a viable alternative to two-dimensional gel electrophoresis?

Two-dimensional gel electrophoresis (2-DE) is used as a platform method for the measurement of protein expression patterns within cells, tissues or organisms. This approach can support expression profiling of several thousand proteins in multiple samples and as such it is currently unrivaled as a tool for the analysis of protein expression, which is a key component of the rapidly expanding field of proteomics. However, 2-DE has a number of significant limitations and as a consequence, alternative approaches for the measurement of expression of proteins within complex samples are actively being explored. Here we review some existing and emerging methods for protein expression analysis. In particular, we review a range of technologies that might be integrated to support the development of 'arrays' or 'chips' for rapid, high-throughput analysis of protein expression in a manner analogous to the current use of DNA arrays for mRNA expression analysis. We conclude that such separation-independent platforms may ultimately supersede two-dimensional (2-D) gel-based analyses for global protein expression analysis but that before this the technologies might provide important new platforms for diagnostic and prognostic monitoring of diseases.

Databases, Protein↗
PubMed2000

Expression profiling of human sulfotransferase and sulfatase gene superfamilies in epithelial tissues and cultured cells.

The bioavailability of drugs administered topically or orally depends on their metabolism by epithelial enzymes such as the cytosolic sulfotransferases (SULT). Reverse transcriptase-polymerase chain reaction (RT-PCR) methods were established to detect expression of 8 SULT genes and 4 arylsulfatase (ARS) genes in human tissues of epithelial origin and in cultures of normal and transformed (cancer) cells. The results indicate: (i) SULT 1A1, 1A3, ARSC, and ARSD genes are ubiquitously expressed; (ii) expression is frequently similar between cell lines and corresponding tissues; (iii) SULT gene expression in normal cultured cells is generally comparable to the expression in associated transformed (cancer) cell lines; (iv) SULT 1A1 promoter usage is mainly tissue specific; however, both promoters are frequently used in SULT 1A3 expression; and (v) the expression profile of SULT 1A1, 1A3, 1E1, and 2B1a/b suggests that one or more of these isoforms may be involved in the cutaneous sulfoconjugation of minoxidil and cholesterol.

Biological Availability↗
PubMed2000

Analysis of prolactin-modulated gene expression profiles during the Nb2 cell cycle using differential screening techniques.

BACKGROUND: Rat Nb2-11C lymphoma cells are dependent on prolactin for proliferation and are widely used to study prolactin signaling pathways. To investigate the role of this hormone in the transcriptional mechanisms that underlie prolactin-stimulated mitogenesis, five different techniques were used to isolate differentially expressed transcripts: mRNA differential display, representational difference analysis (RDA), subtractive suppressive hybridization (SSH), analysis of weakly expressed candidate genes, and differential screening of an organized library. RESULTS: About 70 transcripts were found to be modulated in Nb2 cells following prolactin treatment. Of these, approximately 20 represent unknown genes. All cDNAs were characterized by northern blot analysis and categorized on the basis of their expression profiles and the functions of the known genes. We compared our data with other cell-cycle-regulated transcripts and found several new potential signaling molecules that may be involved in Nb2 cell growth. In addition, abnormalities in the expression patterns of several transcripts were detected in Nb2 cells, including the constitutive expression of the immediate-early gene EGR-1. Finally, we compared the differential screening techniques in terms of sensitivity, efficiency and occurrence of false positives. CONCLUSIONS: Using these techniques to determine which genes are differentially expressed in Nb2 lymphoma cells, we have obtained valuable insight into the potential functions of some of these genes in the cell cycle. Although this information is preliminary, comparison with other eukaryotic models of cell-cycle progression enables identification of expression abnormalities and proteins potentially involved in signal transduction, which could indicate new directions for research.

Animals↗
PubMed2002

Gene-expression profile of collagen-induced arthritis.

To provide a global analysis of genes involved in the inflammatory process in joints of DBA/1J mice suffering from collagen induced arthritis (CIA) we used oligonucleotide microarrays representing approximately 11,000 genes to determine the gene expression profile of the inflamed paws at peak of disease, and compared them to normal tissue. Peak of disease was determined from clinical evaluation of disease and histopathology of joints. Of the 11,000 genes assayed, 223 showed differential expression of four fold or more (187 upregulated and 36 downregulated). Ninety-five of the genes observed had well-characterized full length sequences in databases, and 128 were unknown (Ests). Inflammation resulted in a profile of increased gene expression of matrix metalloproteinases, immune-related, extra-cellular matrix and cell adhesion molecules, as well as molecules involved in cell division and transcription; differential regulation of molecules involved in signal transduction, protein synthesis and metabolism. Of the 55 genes with known chromosomal locations nine mapped to previously identified QTL, contributing to susceptibility or severity of CIA, i.e. MHC class I, II, Basigin, FAP, Cathepsin K, CD 53, RAF1, glucagon, and retinal taurine transporter. The profile of gene expression supports current theoretical models of disease progression and might open new perspectives for both diagnosis and treatment of arthritis.

Animals↗
PubMed2002

Differential gene expression profiling of adult murine hematopoietic stem cells.

Hematopoietic stem cells (HSCs) have self-renewal capacity and multilineage developmental potentials. The molecular mechanisms that control the self-renewal of HSCs are still largely unknown. Here, a systematic approach using bioinformatics and array hybridization techniques to analyze gene expression profiles in HSCs is described. To enrich mRNAs predominantly expressed in uncommitted cell lineages, 54 000 cDNA clones generated from a highly enriched population of HSCs and a mixed population of stem and early multipotent progenitor (MPP) cells were arrayed on nylon membranes (macroarray or high-density array), and subtracted with cDNA probes derived from mature lineage cells including spleen, thymus, and bone marrow. Five thousand cDNA clones with very low hybridization signals were selected for sequencing and further analysis using microarrays on glass slides. Two populations of cells, HSCs and MPP cells, were compared for differential gene expression using microarray analysis. HSCs have the ability to self-renew, while MPP cells have lost the capacity for self-renewal. A large number of genes that were differentially expressed by enriched populations of HSCs and MPP cells were identified. These included transcription factors, signaling molecules, and previously unknown genes.

Animals↗
PubMed2000

Gene expression profiles in normal and Otx2-/- early gastrulating mouse embryos.

The mouse Otx2 gene is a homeobox transcription factor required as early as gastrulation for the proper development of the head. We compared gene expression profiles in wild-type and Otx2(-/-) 6.5 days postcoitum embryos by using a serial analysis of gene expression assay adapted to microdissected structures. Among a broader list, the study of six genes found to be differentially expressed allows defining a role for Otx2 in the orchestration of cell movements leading to the adequate organization of the embryo before gastrulation.

Animals↗
PubMed2001

Expression domains in the skin of genes affected by the nude mutation and identified by gene expression profiling.

Although several genes affected by the nude mutation inactivating the transcription factor Whn have recently been identified, a comprehensive molecular analysis of the nude phenotype is still missing. Gene expression profiling of wildtype and nude mice back skin reveals several so far unknown differences in mRNA levels and demonstrates that microarray hybridization is ideal to identify even quantitative changes in expression. Some genes are upregulated in the absence of Whn. Most of the differentially expressed genes are downregulated in nude skin. Our results identify metallothionein IV among these genes. This is the first report on metallothionein IV expression in the murine hair follicle; its expression domain almost completely overlaps that of Whn.

Animals↗
PubMed2002

Molecular classification of human diffuse gliomas by multidimensional scaling analysis of gene expression profiles parallels morphology-based classification, correlates with survival, and reveals clinically-relevant novel glioma subsets.

There are several currently employed classification systems for diffuse gliomas that sort tumors based on histological features. Contemporary molecular techniques, however, offer the promise of improved tumor classification and resultant patient stratification for treatment and prognosis. In particular, gene expression profiling has shown exceptional promise for providing an alternative and more objective molecular approach to glioma classification. In this study, we used cDNA array technology to profile the gene expression of 30 primary human glioma tissue samples comprising 4 different glioma subtypes as defined by current World Health Organization (WHO 2000) criteria: glioblastoma (GM, WHO grade IV), anaplastic astrocytoma (AA, WHO grade III), anaplastic oligodendroglioma (AO, WHO grade III), and oligodendroglioma (OL, WHO grade II). Gene expression data alone were used to group the tumors using multidimensional scaling, which is an unsupervised statistical method. Results show that impressive separation of the 4 glioma subtypes can be achieved solely on the basis of molecular data. In addition, a subcluster of 3 glioblastomas was identified as distinct from other GMs and from the oligodendroglial tumors. These 3 patients have shown extended survival compared to other GMs in the study. Survival analysis of the full data set revealed a good correlation with the molecular classification. Results of this proof-of-principle study demonstrate that molecular profiling alone can recapitulate conventional histologic classification and grading with high fidelity. In addition, results show that the molecular approach to tumor classification can generate clinically meaningful patient stratification, and, more importantly, is an efficient class-discovery tool for human gliomas, permitting the identification of previously unrecognized, clinically relevant tumor subsets.

Algorithms↗
PubMed2001

Quantitative gene expression profiles of human liver-derived cell lines exposed to moderate hypoxia.

AIMS: To obtain better understanding of molecular events following critical oxygen shortage in liver tissue, we performed a large-scale comparison of gene expression profiles in four human liver cell lines, Chang, Hep3B, HuH7, and HepG2. METHODS: We used Atlas cDNA expression arrays from Clontech for initial screening, and quantitative real-time RT-PCR to assess the statistical significance of observed changes. RESULTS: RT-PCR analysis confirmed significantly changed levels of 24 transcripts after 24 hours incubation in 1% O(2). Among the genes most robustly up-regulated were plasminogen activator inhibitor-1 (PAI-1), insulin-like growth factor binding protein-3, and glyceraldehyde-3-phosphate dehydrogenase. Only PAI-1 was concordantly up-regulated in all four cell lines. Conversely, most down-regulated were the stress response genes, including several heat shock proteins, yet only the expression of flap endonuclease-1 was significantly decreased in all cell lines. When comparing individual cell lines, the HepG2 cells displayed a pattern of general down-regulation (83%), followed by Hep3B with 58% of genes down-regulated. In the Chang and HuH7 cells, however, the expression of most genes, 50% and 67%, respectively, remained unchanged. CONCLUSION: These studies provide information that is of importance for improved insight into the responses of not only liver tumor cells but also normal liver tissue in the conditions where physiological oxygenation is severely impaired.

Carcinoma, Hepatocellular↗
PubMed2001

Gene-expression profiling of human osteoblasts following treatment with the ionic products of Bioglass 45S5 dissolution.

The effect of the ionic products of Bioglass 45S5 dissolution on the gene-expression profile of human osteoblasts was investigated by cDNA microarray analysis of 1,176 genes. Treatment with the ionic products of Bioglass 45S5 dissolution increased the levels of 60 transcripts twofold or more and reduced the levels of five transcripts to one-half or less than in control. Markedly up-regulated genes included RCL, a c-myc responsive growth related gene, cell cycle regulators such as G1/S specific cyclin D1, and apoptosis regulators including calpain and defender against cell death (DAD1). Other significantly up-regulated genes included the cell surface receptors CD44 and integrin beta1, and various extracellular matrix regulators including metalloproteinases-2 and -4 and their inhibitors TIMP-1 and TIMP-2. The identification of differentially expressed genes by cDNA microarray analysis has offered new insights into the mode of action of bioactive glasses and has proven to be an effective tool in evaluating their osteoproductive properties.

Biocompatible Materials↗
PubMed2001

Distinct gene expression profiling in chronic lymphocytic leukemia with 11q23 deletion.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with regard to its clinical course. The limitations of the methods currently available for prognostic assessment in CLL do not allow accurate prediction of the risk of disease progression in individual patients. The recently developed cDNA array technique provides a unique opportunity to study gene expression in various malignancies. To identify new molecular markers for prognostication of CLL patients, we analyzed cDNA arrays by using hierarchical clustering and standard statistic t-test on 34 CLL patients. We found significant expression differences in 78 genes compared to the reference tonsillar B lymphocytes. A cluster of genes, LCP1, PARP, BLR1, DEK, NPM, MCL1, SLP76, STAM, HIVEP1, EVI2B, CD25, HTLF, HIVEP2, BCL2, MNDA, PBX3, EB12, TCF1, CGRP, CD14, ILB, GZMK, GPR17 and CD79B, was associated (P < 0.05) with the unfavorable 11q deletion and also with the unfavorable Binet stages B and C. We present here gene expression profiling that is associated with CLL patients with the 11q23 deletion. Many of the genes in the cluster have not previously been shown to be related to the initiation or progression of CLL. These novel findings provide fundamental information for further attempts to understand the interaction of the clustered genes in the leukomogenesis of CLL in order to better design treatments aimed at specific molecular target(s).

Aged↗
PubMed2001

Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease.

Little is known about the genetic regulation of medulloblastoma dissemination, but metastatic medulloblastoma is highly associated with poor outcome. We obtained expression profiles of 23 primary medulloblastomas clinically designated as either metastatic (M+) or non-metastatic (M0) and identified 85 genes whose expression differed significantly between classes. Using a class prediction algorithm based on these genes and a leave-one-out approach, we assigned sample class to these tumors (M+ or M0) with 72% accuracy and to four additional independent tumors with 100% accuracy. We also assigned the metastatic medulloblastoma cell line Daoy to the metastatic class. Notably, platelet-derived growth factor receptor alpha (PDGFRA) and members of the downstream RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway are upregulated in M+ tumors. Immunohistochemical validation on an independent set of tumors shows significant overexpression of PDGFRA in M+ tumors compared to M0 tumors. Using in vitro assays, we show that platelet-derived growth factor alpha (PDGFA) enhances medulloblastoma migration and increases downstream MAP2K1 (MEK1), MAP2K2 (MEK2), MAPK1 (p42 MAPK) and MAPK3 (p44 MAPK) phosphorylation in a dose-dependent manner. Neutralizing antibodies to PDGFRA blocks MAP2K1, MAP2K2 and MAPK1/3 phosphorylation, whereas U0126, a highly specific inhibitor of MAP2K1 and MAP2K2, also blocks MAPK1/3. Both inhibit migration and prevent PDGFA-stimulated migration. These results provide the first insight into the genetic regulation of medulloblastoma metastasis and are the first to suggest a role for PDGFRA and the RAS/MAPK signaling pathway in medulloblastoma metastasis. Inhibitors of PDGFRA and RAS proteins should therefore be considered for investigation as possible novel therapeutic strategies against medulloblastoma.

Butadienes↗
PubMed1999

Prostate cancer expression profiling by cDNA sequencing analysis.

Prostate cancer is a frequently diagnosed solid tumor that is originated mostly from prostate epithelium. One of the key issues in prostate cancer research is to develop molecular markers that can effectively detect and distinguish the progression and malignancy of prostate tumors. Automated, single-pass cDNA sequencing was utilized to rapidly identify expressed genes in a number of cDNA libraries constructed from various normal and tumor prostatic tissues. These included cell lines as well as short-term epithelial culture. A total of 6604 expressed sequence tags (ESTs) were generated and searched against on-line nucleotide and protein databases. A relational database centric software system was constructed to process, store, and analyze EST data rapidly. cDNA contigs were also obtained by assembly of multiple EST sequences. Protein structural signatures were annotated using motif analysis tools including BLOCKS and an in-house-designed neural network. Cross-library comparisons revealed their unique gene expression profiles. Several differentially expressed cDNA clones were identified, and their expression patterns were confirmed by RNA dot blot and RT-PCR analyses.

Cell Line↗