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PubMed2004

Gene expression in giant-cell tumors.

Malignant transformation is thought to be associated with changes in the expression of a number of genes, and this alteration in gene expression is considered critical to the development of the malignant phenotype. In this study, gene expression in 8 samples of giant-cell tumor (GCT) of bone, as well as in bone at the site of osteoarthritis and in a variety of normal tissues, was determined at Gene Logic Inc (Gaithersburg, Md) with the use of Affymetrix GeneChip U_133 arrays containing approximately 40,000 genes/expressed sequence tags (ESTs). Gene-expression analysis was performed with the use of the Gene Logic GeneExpress Software System. Differences in gene expression between GCTs and bone were observed. In addition, genes expressed uniquely in GCTs among these and 519 samples from 20 other tissue types were identified. Some of the genes that were found to be overexpressed in GCTs, such as tartrate-resistant acid phosphatase and the lysosomal H + -transporting ATPase, are also expressed by osteoclasts. Osteoprotegrin ligand (OPGL) was also selectively overexpressed in GCTs. The genes found to be overexpressed in GCTs appear to reflect the genetic profile of osteoclast-lineage cells and also the genetic profile of an osteoclastogenic environment. The genes identified in this study may play a role in the pathogenesis of GCTs, confirm the likely importance of OPGL in GCT pathogenesis, and may indicate other possible targets to which antitumor therapy could be directed.

Adolescent↗

PubMed2006

Cloning, characterization and tissue-specific expression of a cDNA encoding a novel EMBRYONIC FLOWER 2 gene (OsEMF2) in Oryza sativa.

EMBRYONIC FLOWER 2 (EMF2) gene plays a major role in maintain vegetative development and repress flower development. Here, we present the cloning, characterization and tissue-specific expression of a putative EMF2 (OsEMF2) gene in Oryza sativa. The full-length cDNA of OsEMF2 was 1899 bp and contained an 1872 bp open reading frame (ORF) encoding a 624 amino acid protein. Homologous analysis showed that OsEMF2 contain a single conserved C2H2-type zinc finger motif. Sequence alignment shows that there is a homology between the deduced amino acid sequence of OsEMF2 and EMF2 in Zea mays (55%). Moreover, pI of OsEMF2 are predicted. The tissue-specific expression pattern of OsEMF2 reveals that it is abundant in shoot apical meristem and inflorescence meristem, while its expression level is much lower in leaf, root, immature seed and callus.

Amino Acid Sequence↗

PubMed2001

Tissue microarray profiling of cancer specimens and cell lines: opportunities and limitations.

The implementations of high-throughput genetic technologies, such as oligonucleotide microarrays, generate myriad points of data. The identified potential candidate genes need to be further characterized and selected using a large number of well-characterized tumors and stringent criteria. Tissue microarrays allow for such high-throughput expression profiling of tumor samples, providing, in addition, information at the microanatomical level. Different techniques could be applied for identification of specific phenotypic (immunohistochemistry and in situ hybridization) or genotypic (fluorescence in situ hybridization) alterations, holding strong potential for translational research. Tissue microarrays consisting of 0.6-mm biopsies of paraffin-embedded tissues are well validated and have been used for various clinicopathological studies. This review discusses the technical considerations for construction of such arrays from paraffin-embedded tissues and cell lines and outlines their potential for clinical research applications. The use of paraffin-embedded tissues has some limitations with regard to analysis of RNA or certain proteins. To overcome such limitations, we have developed a cryoarray strategy allowing for the processing of multiple frozen tissue specimens and/or cell lines on a single tissue block. These approaches offer the opportunity to conduct pilot and validation studies of potential targets using clinical samples linked to clinicopathological databases.

Animals↗

PubMed2006

Identification of novel genes associated with astrocytoma progression using suppression subtractive hybridization and real-time reverse transcription-polymerase chain reaction.

To identify novel genes involved in glioma progression we performed suppression subtractive hybridization combined with cDNA array analysis on 4 patients with primary low-grade gliomas of World Health Organization (WHO) grade II that recurred as secondary glioblastomas (WHO grade IV). Eight genes showing differential expression between primary and recurrent tumors in 3 of the 4 patients were selected for further analysis using real-time reverse transcription-PCR on a series of 10 pairs of primary low-grade and recurrent high-grade gliomas as well as 42 astrocytic gliomas of different WHO grades. These analyses revealed that 5 genes, i.e., AMOG (ATP1B2, 17p13.1), APOD (3q26.2-qter), DMXL1 (5q23.1) DRR1 (TU3A, 3p14.2) and PSD3 (KIAA09428/HCA67/EFA6R, 8p22), were expressed at significantly lower levels in secondary glioblastomas as compared to diffuse astrocytomas of WHO grade II. In addition, AMOG, DRR1 and PSD3 transcript levels were significantly lower in primary glioblastomas than in diffuse astrocytomas. Treatment of glioma cell lines with 5-aza-2'-deoxycytidine and trichostatin A resulted in increased expression of AMOG and APOD transcripts. Sequencing of sodium bisulfite-modified DNA demonstrated AMOG promoter hypermethylation in the glioma cell lines and 1 primary anaplastic astrocytoma with low AMOG expression. Taken together, we identified interesting novel candidate genes that likely contribute to glioma progression and provide first evidence for a role of epigenetic silencing of AMOG in malignant glioma cells.

Adenosine Triphosphatases↗

PubMed2001

Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays.

We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

Animals↗

PubMed2004

Differential gene expression in neural stem cells and oligodendrocyte precursor cells: a cDNA microarray analysis.

The use of neural stem cells (NSCs) or their progeny oligodendrocyte precursor cells (OPCs) represents a promising repair strategy for many neurological disorders. However, the molecular events and biological features during the transition from NSCs to OPCs remain unclear. In the present study, we isolated NSCs from the embryonic rat forebrain and induced them into OPCs by using B104 conditioned medium (B104CM) in vitro. We then employed cDNA array technology to compare changes in gene expression between the two cell populations. Among 1,176 genes examined, 40 were differentially expressed, and some of them may be involved in OPC differentiation from NSCs. Our findings thus provide new insights into the molecular basis of differentiation of OPCs from NSCs.

Animals↗

PubMed2004

The human LAPTM4b transcript is upregulated in various types of solid tumours and seems to play a dual functional role during tumour progression.

LAPTM4b (lysosome associated protein transmembrane 4 beta) was recently identified as a gene overexpressed in human hepatocellular carcinoma and belongs to the mammalian LAPTM family. By analysing genome-wide expression profiles of microdissected solid tumour samples by the means of Affymetrix GenChip hybridisation, we found LAPTM4b to be upregulated in 88% (23/26) of lung and in 67% (18/27) of colon carcinoma patients. Northern blots revealed additionally an overexpression of LAPTM4b in the majority of carcinomas of the uterus (30/44), breast (27/53) and ovary (11/16). Other members of the LAPTM family were not overexpressed in the investigated tumour samples according to GeneChip hybridisation data. Northen blot and quantitative RT-PCR on different normal tissues, detected highest levels of LAPTM4b mRNA in uterus, heart and skeletal muscle. Due to sequence analysis of bilaterian LAPTM proteins we suggests the presence of four transmembrane helices per protein, which are probably packed together by hydrophobic forces that are excerted by several evolutionary conserved aromatic residues within the alpha-helices. We discuss an active role for LAPTM4b during disease progression of malignant cells and conclude that its putative dual functional involvement in tumour cell proliferation as well as in multidrug-resistance may represent LAPTM4b as a target suitable for development of novel therapeutic agents.

Amino Acid Sequence↗

PubMed2005

Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling.

A great need exists for the systematic generation of specific antibodies to explore the human proteome. Here, we show that antibodies specific to human proteins can be generated in a high-throughput manner involving stringent affinity purification using recombinant protein epitope signature tags (PrESTs) as immunogens and affinity-ligands. The specificity of the generated affinity reagents, here called mono-specific antibodies (msAb), were validated with a novel protein microarray assay. The success rate for 464 antibodies generated towards human proteins was more than 90% as judged by the protein array assay. The antibodies were used for parallel profiling of patient biopsies using tissue microarrays generated from 48 human tissues. Comparative analysis with well-characterized monoclonal antibodies showed identical or similar specificity and expression patterns. The results suggest that a comprehensive atlas containing extensive protein expression and subcellular localization data of the human proteome can be generated in an efficient manner with mono-specific antibodies.

Animals↗

PubMed2006

Application of microarray technology in primate behavioral neuroscience research.

Gene expression profiling of brain tissue samples applied to DNA microarrays promises to provide novel insights into the neurobiological bases of primate behavior. The strength of the microarray technology lies in the ability to simultaneously measure the expression levels of all genes in defined brain regions that are known to mediate behavior. The application of microarrays presents, however, various limitations and challenges for primate neuroscience research. Low RNA abundance, modest changes in gene expression, heterogeneous distribution of mRNA among cell subpopulations, and individual differences in behavior all mandate great care in the collection, processing, and analysis of brain tissue. A unique problem for nonhuman primate research is the limited availability of species-specific arrays. Arrays designed for humans are often used, but expression level differences are inevitably confounded by gene sequence differences in all cross-species array applications. Tools to deal with this problem are currently being developed. Here we review these methodological issues, and provide examples from our experiences using human arrays to examine brain tissue samples from squirrel monkeys. Until species-specific microarrays become more widely available, great caution must be taken in the assessment and interpretation of microarray data from nonhuman primates. Nevertheless, the application of human microarrays in nonhuman primate neuroscience research recovers useful information from thousands of genes, and represents an important new strategy for understanding the molecular complexity of behavior and mental health.

Animals↗

PubMed2003

Identification of differentially expressed genes in coronary atherosclerotic plaques from patients with stable or unstable angina by cDNA array analysis.

The composition of atherosclerotic plaques is a crucial factor in determining rupture, thrombosis and clinical events. In this study, we analyzed gene expression in coronary plaques from patients with stable or unstable angina using gene arrays. Total RNA was extracted from eight plaques collected by therapeutic directional coronary atherectomy. cDNA probes, generated by amplification, were hybridized to nylon arrays containing 482 genes. Here we report the results for the inflammation, adhesion and hemostasis subsets. Many genes not previously associated with atherosclerosis, such as the lymphocyte adhesion molecule MadCAM, were expressed in the plaques. anova analysis showed higher tissue factor (TF) expression in unstable angina samples. Five genes were expressed at lower levels in unstable angina samples: anticoagulant protein S, cyclooxygenase (COX)-1, interleukin (IL)-7 and chemokines monocyte chemotactic protein (MCP)-1 and -2. Gene arrays provide a new approach to study plaque composition and identify candidate markers of plaque instability.

Angina Pectoris↗

PubMed2003

Effects of oral adsorbent on gene expression profile in uremic rat kidney: cDNA array analysis.

BACKGROUND: An oral adsorbent, AST-120, is effective in removing such uremic toxins as indoxyl sulfate and delays the progression of chronic renal failure. To elucidate the molecular mechanisms underlying the renoprotective effects of AST-120, the complementary DNA (cDNA) array method was used to survey the alteration in gene expression profiles of uremic rat kidneys in response to AST-120. METHODS: Six weeks after five-sixth nephrectomy, 10 uremic rats were divided into two groups: those administered AST-120 and control uremic rats. Rats subjected to sham operation also were included as normal rats. After administration of AST-120 for 18 weeks, renal tissues were analyzed by cDNA array. RESULTS: Among the 343 genes selected as expressed, control uremic rats showed significantly increased levels of 139 genes and significantly decreased levels of 45 genes compared with normal rats. AST-120 treatment attenuated expression levels of 43 of the 139 upregulated renal genes and 21 of the 45 downregulated renal genes in uremic rat kidney. Those genes could be subdivided into several functional categories, including cytokine (transforming growth factor-beta1 [TGF-beta1], etc), intracellular signaling, transcription, translation, channel and transporter (organic anion transporter 1 [OAT1], etc), metabolism, and protease and its inhibitor. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that AST-120 significantly increased OAT1 renal expression and tended to decrease TGF-beta1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and endothelin-1 renal expression in uremic rats. There were statistically significant positive correlations between cDNA array-based and RT-PCR-based gene expression levels of TGF-beta1, TIMP-1, and endothelin-1. CONCLUSION: The cDNA array method determines changes in the gene expression profile in uremic rat kidney in response to AST-120 and provides new insights into elucidation of the molecular mechanism underlying the renoprotective effects of AST-120.

Administration, Oral↗

PubMed1998

Tissue microarrays for high-throughput molecular profiling of tumor specimens.

Many genes and signalling pathways controlling cell proliferation, death and differentiation, as well as genomic integrity, are involved in cancer development. New techniques, such as serial analysis of gene expression and cDNA microarrays, have enabled measurement of the expression of thousands of genes in a single experiment, revealing many new, potentially important cancer genes. These genome screening tools can comprehensively survey one tumor at a time; however, analysis of hundreds of specimens from patients in different stages of disease is needed to establish the diagnostic, prognostic and therapeutic importance of each of the emerging cancer gene candidates. Here we have developed an array-based high-throughput technique that facilitates gene expression and copy number surveys of very large numbers of tumors. As many as 1000 cylindrical tissue biopsies from individual tumors can be distributed in a single tumor tissue microarray. Sections of the microarray provide targets for parallel in situ detection of DNA, RNA and protein targets in each specimen on the array, and consecutive sections allow the rapid analysis of hundreds of molecular markers in the same set of specimens. Our detection of six gene amplifications as well as p53 and estrogen receptor expression in breast cancer demonstrates the power of this technique for defining new subgroups of tumors.

Animals↗

PubMed2006

Characterization of a method for profiling gene expression in cells recovered from intact human prostate tissue using RNA linear amplification.

Coupling array technology to laser capture microdissection (LCM) has the potential to yield gene expression profiles of specific cell populations within tissue. However, remaining problems with linear amplification preclude accurate expression profiling when using the low nanogram amounts of RNA recovered after LCM of human tissue. We describe a novel robust method to reliably amplify RNA after LCM, allowing direct probing of 12K gene arrays. The fidelity of amplification was demonstrated by comparing the ability of amplified RNA (aRNA) versus that of native RNA to identify differentially expressed genes between two different cell lines, demonstrating a 99.3% concordance between observations. Array findings were validated by quantitative polymerase chain reaction analysis of a randomly selected subset of 32 genes. Using LCM to recover normal (N=5 subjects) or cancer (N=3) cell populations from intact human prostate tissue, three differentially expressed genes were identified. Independent investigators have previously identified differential expression of two of these three genes, hepsin and beta-microseminoprotein, in prostate cancer. Taken together, the current study demonstrates that accurate gene expression profiling can readily be performed on specific cell populations present within complex tissue. It also demonstrates that this approach efficiently identifies biologically relevant genes.

Cell Line, Tumor↗

PubMed2003

Identification of cathepsin B as a mediator of neuronal death induced by Abeta-activated microglial cells using a functional genomics approach.

Alzheimer's disease is a progressive neurodegenerative disease characterized by senile plaques, neurofibrillary tangles, dystrophic neurites, and reactive glial cells. Activated microglia are found to be intimately associated with senile plaques and may play a central role in mediating chronic inflammatory conditions in Alzheimer's disease. Activation of cultured murine microglial BV2 cells by freshly sonicated Abeta42 results in the secretion of neurotoxic factors that kill primary cultured neurons. To understand molecular pathways underlying Abeta-induced microglial activation, we analyzed the expression levels of transcripts isolated from Abeta42-activated BV2 cells using high density filter arrays. The analysis of these arrays identified 554 genes that are transcriptionally up-regulated by Abeta42 in a statistically significant manner. Quantitative reverse transcription-PCR was used to confirm the regulation of a subset of genes, including cysteine proteases cathepsin B and cathepsin L, tissue inhibitor of matrix metalloproteinase 2, cytochrome c oxidase, and allograft inflammatory factor 1. Small interfering RNA-mediated silencing of the cathepsin B gene in Abeta-activated BV2 cells diminished the microglial activation-mediated neurotoxicity. Moreover, CA-074, a specific cathepsin B inhibitor, also abolished the neurotoxic effects caused by Abeta42-activated BV2 cells. Our results suggest an essential role for secreted cathepsin B in neuronal death mediated by Abeta-activated inflammatory response.

Alzheimer Disease↗

PubMed2006

A novel mammalian bubblegum-related acyl-CoA synthetase restricted to testes and possibly involved in spermatogenesis.

We have characterized a new, membrane-associated acyl-CoA synthetase (ACS), termed bubblegum-related protein (BGR), which upon functional analysis demonstrated ACS activity capable of activating long- and very long-chain fatty acids. By multiple tissue RNA array and Northern blot analyses, human BGR mRNA was exclusively detected in testes. Murine Bgr mRNA was specifically expressed in pubertal and adult testes and was further demonstrated to be enriched in germ cells and Sertoli cells while present at a lower level in Leydig cells both by in situ hybridization and cell type fractionation. The complex 5'-end of the BGR mRNA appears to underlie translational control leading to differential utilization of alternative translation start sites. Thus, the BGR gene expands the bubblegum ACS family with a testes-specific, developmentally regulated member that may play a role in spermatogenesis.

Adult↗

PubMed2005

Elucidation of IL-1/TGF-beta interactions in mouse chondrocyte cell line by genome-wide gene expression.

OBJECTIVE: To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases. METHODS: The H4 chondrocyte cell line was validated by comparing metalloproteinase expression profile with intact murine cartilage by reverse transcription polymerase chain reaction. Genome-wide gene expression in the H4 cells in response to IL-1 and TGF-beta, alone and in combination, was analyzed by using oligonucleotide arrays negotiating approximately 12,000 genes. RESULTS: The response of cartilage and the H4 cell line to IL-1 and TGF-beta was comparable. Oligonucleotide array analysis demonstrated a mutual but asymmetrical antagonism as the dominant mode of interaction between IL-1 and TGF-beta. Cluster analysis revealed a remarkable selectivity in the mode of action exerted by TGF-beta on IL-1 regulated genes: antagonistic on pro-inflammatory genes whereas additive on growth regulators such as vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF). While the former cluster underlined the protective effect of TGF-beta, the latter underscored the adverse effect of TGF-beta. We further identified potentially novel classes of target genes under control of TGF-beta such as ras family, histones, proteasome components, and ubiquitin family, highlighting the importance of such genes in TGF signaling besides the well-characterized SMAD pathway. CONCLUSIONS: We identified a cluster of genes as potential targets mediating the adverse effect of TGF-beta such as fibrosis. Transcriptional regulation of ras GTPase and ubiquitin/proteasome pathways is likely to be a novel mechanism mediating the effect of TGF-beta and its interaction with IL-1. These down-stream genes and pathways can be targets in future therapy.

Animals↗

PubMed2004

Differential domain evolution and complex RNA processing in a family of paralogous EPB41 (protein 4.1) genes facilitate expression of diverse tissue-specific isoforms.

The EPB41 (protein 4.1) genes epitomize the resourcefulness of the mammalian genome to encode a complex proteome from a small number of genes. By utilizing alternative transcriptional promoters and tissue-specific alternative pre-mRNA splicing, EPB41, EPB41L2, EPB41L3, and EPB41L1 encode a diverse array of structural adapter proteins. Comparative genomic and transcript analysis of these 140- to 240-kb genes indicates several unusual features: differential evolution of highly conserved exons encoding known functional domains interspersed with unique exons whose size and sequence variations contribute substantially to intergenic diversity; alternative first exons, most of which map far upstream of the coding regions; and complex tissue-specific alternative pre-mRNA splicing that facilitates synthesis of functionally different complements of 4.1 proteins in various cells. Understanding the splicing regulatory networks that control protein 4.1 expression will be critical to a full appreciation of the many roles of 4.1 proteins in normal cell biology and their proposed roles in human cancer.

Alternative Splicing↗

PubMed2006

Gene expression changes during step-wise differentiation of embryonic stem cells along the inner ear hair cell pathway.

CONCLUSION: Our study outlines an alternative approach for the selection and investigation of genes involved in inner ear function. OBJECTIVE: To gain understanding of the gene pathways involved in the development of the normal cochlea. MATERIALS AND METHODS: Microarray technology currently offers the most efficient approach to investigate gene expression and identify pathways involved in cell differentiation. Epidermal growth factor (EGF) induces cultures derived from the organ of Corti to proliferate and produce new hair cells. Since pluripotent embryonic stem (ES) cells have the capacity to generate all tissues, we induced murine ES cells to differentiate towards ectodermal and neuroectodermal cell types and from there investigated their commitment towards the hair cell lineage in the presence of EGF. Cells were collected at three points along the differentiation pathway and their expression profiles were determined using the Soares NMIE mouse inner ear cDNA library printed in microarray format. RESULTS: Three genes up-regulated after addition of EGF (serine (or cysteine) proteinase inhibitor, clade H, member 1 (Serpinh1), solute carrier family 2 (facilitated glucose transporter), member 10 (Slc2a10) and secreted acidic cysteine-rich glycoprotein (Sparc)) were selected for further analysis and characterization. Of the three genes, Serpinh1 and Slc2a10 have never been implicated in the hearing process.

Animals↗