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Results for “Expression profiling”

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PubMed2005

Gene expression profiling of primary tumor cell populations using laser capture microdissection, RNA transcript amplification, and GeneChip microarrays.

Gene expression profiling from microdissected cell populations is a powerful approach to explore molecular processes involved in development and solid tumor biology. In this chapter, we detail robust and validated methods for tissue preparation and isolation of high-quality RNA from microdissected cell populations. A protocol is also provided for linear transcript amplification using as little as 10 ng of total RNA to produce labeled cRNA targets for hybridization to GeneChip high-density oligonucleotide microarrays. Particular emphasis is placed on troubleshooting each technical step in the protocol and measures of quality assurance for both RNA isolation and resulting microarray data.

Animals↗

PubMed2005

DNA microarray-based gene expression profiles of cytomegalovirus infection and acute rejection in liver transplants.

An association between cytomegalovirus (CMV) infection and alloresponse has been suggested. CMV increases inflammation and adhesion molecule expression in graft, and induces cytokines and growth factors, linked with transplant vasculopathy and chronic rejection. We have investigated the gene expression of various inflammatory factors in the CMV-associated immune response and compared this with the immune response of acute rejection in liver transplants by using DNA microarray technology. Gene expression was studied at mRNA level in biopsies from liver transplant patients experiencing CMV infection or acute rejection. RNA extracted from liver grafts after reperfusion was used as control material. Among the strongly upregulated genes in the specimens obtained from liver transplants during CMV infection were IFN-gamma, caspases 1 and 3, granzymes A and B, TGF-beta receptors II and III, IL-10 receptor alpha, VCAM-1, TNF receptor, IL-4, TNF-alpha, IL-10, IL-2 receptor beta, IL-1beta, PDGF-receptor beta, vascular adhesion protein-1, TGF-beta2, and ICAM-1. In biopsies with acute liver allograft rejection, the most significantly upregulated genes were MHC class II, IFN-gamma, caspases 1 and 3, IL-2R beta and gamma, granzymes A and B, VLA-4, L-selectin, E-selectin, VCAM-1, and IL-1beta. Upregulated genes common for CMV and alloresponse were granzyme A and B, E-selection, IFN-gamma, VCAM-1, VLA-4, TNF, caspases 1, 3, and 8, and PDGF. Microarray analysis defined different entities in the immune responses of CMV infection and acute rejection. The differences and similarities of the gene expression profiles related to those in CMV infection and rejection may help to understand the intragraft immunologic events.

Biopsy↗

PubMed2003

Analysis of gene expression profiles of gastric normal and cancer tissues by SAGE.

In an attempt to understand the molecular bases of gastric cancer progression, we have analyzed the differentially expressed genes in gastric cancer by SAGE. Four SAGE cDNA tag libraries were constructed from two sets of gastric cancer and normal tissues and 241,127 tags were obtained. By comparing the tags from cancer and normal tissues, 414 differentially expressed tags, representing 383 genes, were identified in cancer tissues (p </= 0.01). Of the 414 tags, 50 tags were previously unidentified and potentially novel genes. Although each gastric cancer tissue revealed more than 200 differentially expressed genes compared to the respective normal tissue, the number of genes with consistent regulation patterns in both cancer tissues was 51: 12 up-regulated and 39 down-regulated genes. The genes that showed consistent regulation patterns included well-known genes such as Trefoil factor 3, RegIV, gastric intrinsic factor, and lactotransferrin as well as a few novel candidates. Interestingly, the expression of several genes, such as osteoglycin, prostate stem cell antigen, and histone deacetylase 3, was variable in the two normal tissues but similar in the cancer tissues. The expression profiles of these genes in normal tissues, possibly due to genetic background, could greatly affect individual sensitivity to cancer development and/or progression. The genes identified in this study will provide useful target genes for diagnosis and molecular treatment of gastric cancer.

Adenocarcinoma↗

PubMed2003

Gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells.

OBJECTIVE: To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells. METHODS: Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with alpha-(32)P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by Atlas Image analysis software Version 1.01a. RESULTS: Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells. CONCLUSION: The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells.

Bone Marrow Cells↗

PubMed2002

Analysis of age-dependant alteration in the brain gene expression profile following induction of hydrocephalus in rats.

Hydrocephalus is associated with gradual progressive impairment and destruction of cerebral axons and neurons. To provide a comprehensive analysis of gene expression changes in brain due to experimental hydrocephalus we used a DNA microarray screening technique. Hydrocephalus was induced in 3-week-old and 8- to 10-week-old rats by injection of kaolin into cisterna magna. Following induction of hydrocephalus, samples of frontoparietal cerebrum were studied 3 and 36 weeks later in young rats and 1.5 weeks later in adult rats. At the transcriptional level, young rats with subacute hydrocephalus showed overexpression of genes involved in synaptic transmission in parallel to genes associated with protective and compensatory mechanisms. Those with chronic hydrocephalus exhibited some similar changes among synapse-related genes but suppression of other neuronal genes. Expression of myelin-related genes was increased in both groups of rats with early onset hydrocephalus but suppressed in adult rats with acute hydrocephalus. Changes in genes related to extracellular matrix molecules suggest that there might be remodeling in this compartment. Adult rats showed elevated expression of inflammatory genes, likely related to kaolin-induced inflammation, but they failed to show changes in genes involved in compensatory or protective mechanisms. These results indicate that there is an age- and duration-dependent difference in the gene expression profiles of kaolin-induced hydrocephalus and they present avenues for future research.

Aging↗

PubMed2003

Changes in gene expression profile by HCV core protein in cultured liver cells: analysis by DNA array assay.

In this study, we established hepatitis C virus (HCV) core-expressing mouse liver cells and investigated changes occurring in the gene expression profile accompanied by expression of the viral core protein using DNA array analysis. Both non-transformed and transformed mouse liver cells constitutively expressing the viral core protein were established by DNA transfection, and subjected to DNA array analysis. For the genes judged positive by DNA array, Northern blot analysis was done for corroboration. DNA array analysis revealed one up-regulated gene and three down-regulated genes by expression of the viral core protein in non-transformed liver cells. For the transformed liver cells, four enhanced and five suppressed genes were observed. The Northern blot corroboration analysis clarified two genes, osteopontin precursor and activating transcription factor 4, as being down-regulated by the viral core protein in both non-transformed and transformed liver cells, and three genes, cathepsin D, matrix metalloproteinase 14 and anti-proliferative B-cell translocation gene 2, as being up-regulated by the viral core protein in only transformed liver cells. In conclusion, a total of five genes were identified as viral core protein-responsive ones in the DNA array analysis. Alterations in the expression levels of these genes may be relevant to the viral core-mediated pathogenesis.

Journal Article↗

PubMed2003

Discovery of imprinted transcripts in the mouse transcriptome using large-scale expression profiling.

Candidate imprinted transcriptional units in the mouse genome were identified systematically from 27,663 FANTOM2 full-length mouse cDNA clones by expression profiling. Large-scale cDNA microarrays were used to detect differential expression dependent upon chromosomal parent of origin by comparing the mRNA levels in the total tissue of 9.5 dpc parthenogenote and androgenote mouse embryos. Of the FANTOM2 transcripts, 2114 were identified as candidates on the basis of the array data. Of these, 39 mapped to known imprinted regions of the mouse genome, 56 were considered as nonprotein-coding RNAs, and 159 were natural antisense transcripts. The imprinted expression of two transcripts located in the mouse chromosomal region syntenic to the human Prader-Willi syndrome region was confirmed experimentally. We further mapped all candidate imprinted transcripts to the mouse and human genome and were shown in correlation with the imprinting disease loci. These data provide a major resource for understanding the role of imprinting in mammalian inherited traits.

Animals↗

PubMed2002

Expression profiling of lineage differentiation in pluripotential human embryonal carcinoma cells.

Pluripotential human embryonal carcinoma (EC) cell linesundergo differentiation programs resembling those occurring in embryonal stem cells during development. Expression profiling was performed during the terminal differentiation of the EC cell line, NTera2/Clone D1 by all-trans-retinoic acid. Time-response analysis via clustering of >12,000 human transcripts revealed distinct stages in the transition from an EC cell to neuronal progenitor cells expressing patterning markers compatible with posterior hindbrain fates followed by the appearance of immature postmitotic neurons with an evolving synaptic apparatus. Global analysis of gene expression allows monitoring cell fate and differentiation of EC cells in vitro and may provide insight into human embryonal stem cell development.

Antineoplastic Agents↗

PubMed2006

[Gene expression profiles on three kinds of genotype hepatitis C virus core protein in Huh-7 cell line with microarray analysis].

OBJECTIVE: To analyze three kinds of genotype hepatitis C virus (HCV) core protein expressed in human hepatoma (Huh-7) cell line and to recognize HCV core proteins biological function and its pathogenic mechanism. METHODS: The Huh-7 cell expressed three kinds of core proteins were established respectively. Affymetrix human gene chip was used for identifying the gene expression dependently on Affymetrix's protocol. All genes changed by 3 or 1.5 folds between the transfected cells and a control cells were further analyzed, and annotated by using NetAffx analysis through Affymetrix website and were categorized based on their biological processes. RESULTS: The HCV-1b core protein caused 16 genes up/down-regulated expression, of which the immune response genes of PF4V1 and SPP1 were up-regulated 3.4 or 4.4 folds respectively. The HCV-2a core protein had caused the immune response gene CXCL5 and apoptosis gene BTF a down-regulated expression of 3.4 and 3.1 folds respectively, but caused the apoptosis genes of HRK and LZTS1 an up-regulated expression of 3.2 and 3.4 folds respectively. As compared with HCV 1b or 2a core protein, HCV-4b core protein caused 111 genes expression changing and it had more obvious effects on gene expression. If we applied 1.5 fold change for a comparison gene expression, a few of the same gene expression profiles might be caused by these two core proteins. CONCLUSION: The three kinds of HCV core protein should have its own expression character and be mainly shown in immune responses, signal transduction, apoptosis, etc. It should be helpful for our recognizing the HCV core protein biological function and its pathogenic mechanism.

Carcinoma, Hepatocellular↗

PubMed2000

Gene-expression profile of the ageing brain in mice.

Ageing of the brain leads to impairments in cognitive and motor skills, and is the major risk factor for several common neurological disorders such as Alzheimer disease (AD) and Parkinson disease (PD). Recent studies suggest that normal brain ageing is associated with subtle morphological and functional alterations in specific neuronal circuits, as opposed to large-scale neuronal loss. In fact, ageing of the central nervous system in diverse mammalian species shares many features, such as atrophy of pyramidal neurons, synaptic atrophy, decrease of striatal dopamine receptors, accumulation of fluorescent pigments, cytoskeletal abnormalities, and reactive astrocytes and microglia. To provide the first global analysis of brain ageing at the molecular level, we used oligonucleotide arrays representing 6,347 genes to determine the gene-expression profile of the ageing neocortex and cerebellum in mice. Ageing resulted in a gene-expression profile indicative of an inflammatory response, oxidative stress and reduced neurotrophic support in both brain regions. At the transcriptional level, brain ageing in mice displays parallels with human neurodegenerative disorders. Caloric restriction, which retards the ageing process in mammals, selectively attenuated the age-associated induction of genes encoding inflammatory and stress responses.

Aging↗

PubMed2001

Gene expression profiles of proliferating vs. G1/G0 arrested human leukemia cells suggest a mechanism for glucocorticoid-induced apoptosis.

Glucocorticoids (GC) have pronounced effects on metabolism, differentiation, proliferation, and cell survival (1). In certain lymphocytes and lymphocyte-related malignancies, GC inhibit proliferation and induce apoptotic cell death, which has led to their extensive use in the therapy of malignant lymphoproliferative disorders (2). Most of these effects result from regulation of gene expression via the GC receptor (GR), a ligand-activated transcription factor (3). Although hundreds of genes are regulated by GC (1), how certain biological GC effects relate to individual gene regulation remains enigmatic. To address this question with respect to GC-induced cell cycle arrest and apoptosis, we applied DNA chip technology (4, 5) to determine gene expression profiles in proliferating and G1/G0-arrested (by conditional expression of the CDK inhibitor p16/INK4a) acute lymphoblastic T cells undergoing GC-induced apoptosis. Of 7074 genes tested, 163 were found to be regulated by dexamethasone in the first 8 h in proliferating cells and 66 genes in G1/G0-arrested cells. An almost nonoverlapping set of genes (i.e., only eight genes) was coordinately regulated in proliferating and arrested cells. Analysis of the regulated genes supports the concept that GC-induced apoptosis results from positive GR autoregulation entailing persistent down-regulation of metabolic pathways critical for survival

Apoptosis↗

PubMed2006

Analysis of gene expression profiles of normal human nasal mucosa and nasal polyp tissues by SAGE.

BACKGROUND: A systemic determination of gene expression profiles in nasal polyp compared with normal nasal mucosa would contribute considerably to the investigation of the disease marker in various rhinopathy and general knowledge on the formation of human nasal polyp. OBJECTIVE: The aim of this study was to identify the transcriptome of the normal human nasal mucosa and nasal polyp by the serial analysis of gene expression. METHODS: mRNA was extracted from normal inferior turbinate mucosa and nasal polyp. Short sequences (tags), each one usually corresponding to a distinct transcript, was isolated and concatemerized into long DNA molecules, which were cloned and sequenced. RESULTS: We detected 65,305 tags for normal nasal mucosa and 55,829 tags for nasal polyp, representing 20,629 and 17,636 potential transcripts species, respectively. Of the unique tags encountered more than once, 92% (normal nasal mucosa) and 90% (nasal polyp) matched known genes or expressed sequence tags, whereas the remainder did not match any GenBank sequences. Therefore, 504 and 539 novel transcripts were identified in normal nasal mucosa and nasal polyp, respectively. Although the expression levels of most transcripts in both libraries were similar, 114 transcripts were differentially expressed at a statistically significant level (P < .05)-that is, 65 and 49 transcripts among them were expressed at a higher level in normal nasal mucosa and nasal polyp, respectively. CONCLUSION: This information should be very useful for basic knowledge as well as for future studies on pathophysiological conditions of human nasal mucosa, providing some clues to evaluate the altered factors in a variety of rhinopathies. CLINICAL IMPLICATIONS: The results of this study might contribute to general knowledge on the formation of human nasal polyp.

Adult↗

PubMed2000

Identification of novel hypoxia dependent and independent target genes of the von Hippel-Lindau (VHL) tumour suppressor by mRNA differential expression profiling.

The von Hippel-Lindau tumour suppressor gene (VHL) targets hypoxia inducible factor (HIF)-alpha subunits for ubiquitin dependent proteolysis. To better understand the role of this and other putative pathways of gene regulation in VHL function we subjected mRNA from VHL defective renal carcinoma cells and transfectants re-expressing a wild type VHL allele to differential expression profiling, and analysed VHL target genes for oxygen regulated expression. Among a group of newly identified VHL target genes the majority but not all were regulated by oxygen, indicating that whilst dysregulation of the HIF system makes a dominant contribution to alterations in transcription, VHL has other influences on patterns of gene expression. Genes newly defined as targets of the VHL/hypoxia pathway (conditionally downregulated by VHL in normoxic cells) include aminopeptidase A, collagen type V, alpha 1, cyclin G2, DEC1/Stra13, endothelin 1, low density lipoprotein receptor-related protein 1, MIC2/CD99, and transglutaminase 2. These genes have a variety of functions relevant to tumour biology. However, not all are connected with the promotion of tumour growth, some being pro-apoptotic or growth inhibitory. We postulate that co-ordinate regulation as part of the HIF pathway may explain this paradox, and that evolution of anti-apoptotic pathways may be required for tumour growth under VHL-dysregulation. Our results indicate that it will be necessary to consider the effects of abnormal activity in integral regulatory pathways, as well as the effects of individual genes to understand the role of abnormal patterns of gene expression in cancer.

Carcinoma, Renal Cell↗

PubMed2004

Dissecting progressive stages of 5-fluorouracil resistance in vitro using RNA expression profiling.

Resistance to anticancer drugs such as the widely used antimetabolite 5-fluorouracil (FU) is one of the most important obstacles to cancer chemotherapy. Using GeneChip arrays, we compared the expression profile of different stages of FU resistance in colon cancer cells after in vitro selection of low-, intermediate- and high-resistance phenotypes. Drug resistance was associated with significant changes in expression of 330 genes, mainly during early or intermediate stage. Functional annotation revealed a majority of genes involved in signal transduction, cell adhesion and cytoskeleton with subsequent alterations in apoptotic response, cell cycle control, drug transport, fluoropyrimidine metabolism and DNA repair. A set of 33 genes distinguished all resistant subclones from sensitive progenitor cells. In the early stage, downregulation of collagens and keratins, together with upregulation of profilin 2 and ICAM-2, suggested cytoskeletal changes and cell adhesion remodeling. Interestingly, 6 members of the S100 calcium-binding protein family were suppressed. Acquisition of the intermediate-resistance phenotype included upregulation of the well-known drug resistance gene ABCC6 (ATP-binding cassette subfamily C member 6). The very small number of genes affected during transition to high resistance included the primary FU target thymidylate synthase. Although limited to an in vitro model, our data suggest that resistance to FU cannot be explained by known mechanisms alone and substantially involves a wide molecular repertoire. This study emphasizes the understanding of resistance as a time-depending process: the cell is particularly challenged at the beginning of this process, while acquisition of the high-resistance phenotype seems to be less demanding.

Antimetabolites, Antineoplastic↗

PubMed2005

[Evolution of gene expression profile in 3 cases of acute myeloid leukemia].

OBJECTIVE: To investigate the mechanism of refractoriness of acute myeloid leukemia (AML) by studying the changes of gene mRNA expression from primary diagnosis to relapsed disease in AML. METHODS: Differences in gene expression profile of bone marrow mononuclear cells were compared between primary diagnosis and relapsed/refractory disease in 3 patients with M(2a) subtype of AML using Agilent human 1B 60mer oligonucleotide microarray. RESULTS: Common alterations were found in 10 genes among the 20173 genes tested at relapsed/refractory disease as compared with that at primary diagnosis in 3 patients. Of these 10 genes, 7 were up-regulated while 3 down-regulated at relapse in all the 3 patients. CONCLUSION: Development of relapsed/refractory disease in AML-M(2a) might be associated with the mRNA expression changes in the 10 genes tested including DAPK1. The alteration of these genes may be indications for the early diagnosis of refractoriness of AML, and these genes might provide new therapeutic targets for the treatment of refractory AML.

Adult↗

PubMed2004

Microarrays and the gene expression profile of a single cell.

With completion of the human genome sequence, it is now possible to study the expression of the entire human gene complement of approximately 30,000-35,000 genes. To accomplish this goal, microarrays have become the leading methodology for the analysis of global gene expression. Improvements in technology have increased the sensitivity of microarrays to the point where it is feasible to study gene expression in a small number of cells and even at the single cell level. A summary of developments in the area of expression profiling in single cells will be described, and the rationale for these types of studies will be presented. In addition, from a biologist's point of view, some bioinformatic challenges of expression analysis of single cells will be discussed.

Cell Physiological Phenomena↗

PubMedDate not supplied

RNA amplification, fidelity and reproducibility of expression profiling.

We quantitatively address the effect of T7 RNA amplification on expression profiling data, and answer the following questions: (1) What fraction of genes sampled is amplified non-linearly? (2) What is the effect of RNA amplification on comparative expression measurements? (3) If there is amplification bias, is the bias dependent on the degree of amplification or the amount of starting material and (4) Does amplification increase the overall variability of the results? We show that while there is significant amplification bias, the bias is consistent and generally has little effect on array comparisons between amplified samples.

HeLa Cells↗

PubMed2005

Gene expression profiling differentiates germ cell tumors from other cancers and defines subtype-specific signatures.

Germ cell tumors (GCTs) of the testis are the predominant cancer among young men. We analyzed gene expression profiles of 50 GCTs of various subtypes, and we compared them with 443 other common malignant tumors of epithelial, mesenchymal, and lymphoid origins. Significant differences in gene expression were found among major histological subtypes of GCTs, and between them and other malignancies. We identified 511 genes, belonging to several critical functional groups such as cell cycle progression, cell proliferation, and apoptosis, to be significantly differentially expressed in GCTs compared with other tumor types. Sixty-five genes were sufficient for the construction of a GCT class predictor of high predictive accuracy (100% training set, 96% test set), which might be useful in the diagnosis of tumors of unknown primary origin. Previously described diagnostic and prognostic markers were found to be expressed by the appropriate GCT subtype (AFP, POU5F1, POV1, CCND2, and KIT). Several additional differentially expressed genes were identified in teratomas (EGR1 and MMP7), yolk sac tumors (PTPN13 and FN1), and seminomas (NR6A1, DPPA4, and IRX1). Dynamic computation of interaction networks and mapping to existing pathways knowledge databases revealed a potential role of EGR1 in p21-induced cell cycle arrest and intrinsic chemotherapy resistance of mature teratomas.

Chromosomes, Human↗