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PubMed2001

Gene expression profile analysis by DNA microarrays: promise and pitfalls.

DNA microarrays represent a technological intersection between biology and computers that enables gene expression analysis in human tissues on a genome-wide scale. This application can be expected to prove extremely valuable for the study of the genetic basis of complex diseases. Despite the enormous promise of this revolutionary technology, there are several issues and possible pitfalls that may undermine the authority of the microarray platform. We discuss some of the conceptual, practical, statistical, and logistical issues surrounding the use of microarrays for gene expression profiling. These issues include the imprecise definition of normal in expression comparisons; the cellular and subcellular heterogeneity of the tissues being studied; the difficulty in establishing the statistically valid comparability of arrays; the logistical logjam in analysis, presentation, and archiving of the vast quantities of data generated; and the need for confirmational studies that address the functional relevance of findings. Although several complicated issues must be resolved, the potential payoff remains large.

Gene Expression Profiling↗

PubMed2001

Prostanoid receptor gene expression profile in human trabecular meshwork: a quantitative real-time PCR approach.

PURPOSE: To assess the expression pattern of prostanoid receptor-encoding genes in trabecular meshwork (TM) of human donor eyes. METHODS: Disposed human donor eyes (n = 10) were obtained from the Cornea Bank, Amsterdam. The TM was dissected from the scleral tissue and homogenized in lysis buffer, and total RNA was isolated. The RNA was converted into cDNA and used as a template for noncompetitive quantitative real-time polymerase chain reaction (PCR) using green fluorescent dye to quantify the accumulation of double-stranded PCR product. Specific primers for four housekeeping genes and DP, EP(1), EP(2), EP(3,) EP(4), FP, IP, and TP receptor-encoding transcripts were developed and tested for their efficiency. RESULTS: The characterized expression profile was highly reproducible in all samples, with the EP(2) receptor-encoding transcript in the highest abundance, followed by FP, TP, IP, and EP(4) at levels that were approximately 10 to 15 times lower than that of the EP(2) subtype. DP and EP(3) were at the lowest levels, which were, on average, 45 times and 228 times lower than EP(2), respectively. CONCLUSIONS: These data show that all prostanoid receptors are expressed at different levels in human TM tissue. Because the gene expression of the EP(2) receptor is, on average, 15 times more abundant than that of the EP(4) receptor, it may be expected that the increase in flow and cAMP levels in response to the activation of the EP receptors by application of prostaglandin E(1) (PGE(1)), is primarily mediated by the EP(2) receptor. These data should be considered when designing prostanoid receptor mimetics intended to enhance the aqueous humor outflow through the TM and Schlemm's canal.

Actins↗

PubMed2001

MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia.

Acute lymphoblastic leukemias carrying a chromosomal translocation involving the mixed-lineage leukemia gene (MLL, ALL1, HRX) have a particularly poor prognosis. Here we show that they have a characteristic, highly distinct gene expression profile that is consistent with an early hematopoietic progenitor expressing select multilineage markers and individual HOX genes. Clustering algorithms reveal that lymphoblastic leukemias with MLL translocations can clearly be separated from conventional acute lymphoblastic and acute myelogenous leukemias. We propose that they constitute a distinct disease, denoted here as MLL, and show that the differences in gene expression are robust enough to classify leukemias correctly as MLL, acute lymphoblastic leukemia or acute myelogenous leukemia. Establishing that MLL is a unique entity is critical, as it mandates the examination of selectively expressed genes for urgently needed molecular targets.

Acute Disease↗

PubMed1999

Expression profile of the copper homeostasis gene, rAtox1, in the rat brain.

In humans the regulation of cellular copper homeostasis is essential for proper organ development and function. A novel cytosolic protein, named Atox 1, was recently identified in yeast that functions in shuttling intracellular mononuclear copper [Cu(I)] to copper-requiring proteins. Atox 1 and its human homolog, hAtox1, are members of an emerging family of proteins termed copper chaperones that are involved in the maintenance of copper homeostasis. Northern blot analysis demonstrates that Atox 1 is widely expressed at varying levels in a variety of rat tissues including brain. Using in situ hybridization histochemistry, we characterized the expression profile for the rat homolog of Atox1 (rAtox1) in the normal adult rat brain. There is widespread expression within the brain that appears to be primarily neuronal. The highest levels of Atox1 message consists of distinct neuronal subtypes that are also characterized by their high levels of metals like copper, iron, and zinc, which include the pyramidal neurons of the cerebral cortex and hippocampus in addition to the neurons of the locus coeruleus. The high levels of a metal chaperone like Atox1 in subsets of neurons that also sequester metals suggests that Atox1 may be important in maintaining the functionality of metal requiring enzymes. A detailed analysis of the restricted expression profile for a novel copper chaperone, rAtox1, is described in the adult rat CNS. Further analysis shows that Atoxl expression is associated with neuronal populations that sequester copper.

Animals↗

PubMed1998

Gene expression profiling of alveolar rhabdomyosarcoma with cDNA microarrays.

Several forms of human sarcoma, lymphoma, and leukemia are characterized by somatically acquired chromosome translocations that result in fusion genes that encode chimeric transcription factors with oncogenic properties. We have used cDNA microarrays containing 1238 cDNAs to investigate the gene expression profile of a group of seven alveolar rhabdomyosarcoma (ARMS) cell lines characterized by the presence of the PAX3-FKHR fusion gene. Using the method of multidimensional scaling to represent the relationships among the cell lines in two-dimensional Euclidean space, we determined that ARMS cells show a consistent pattern of gene expression, which allows the cells to be clustered together. By searching across the seven ARMS cell lines, we found that 37 of 1238 genes were most consistently expressed in ARMS relative to a reference cell line. Only three of these genes have been previously reported to be expressed in ARMS. Among these 37 were genes related to both primary (PAX3-FKHR) and secondary (CDK4) genetic alterations in ARMS. These results in ARMS demonstrate the potential of cDNA microarray technology to elucidate tumor-specific gene expression profiles in human cancers.

Chromosomes, Human, Pair 13↗

PubMed2001

Expression profile of 30,000 genes in rat hippocampus using SAGE.

Using the serial analysis of gene expression (SAGE) method, we generated a gene expression profile of the rat hippocampus. A total of 76,790 SAGE tags was analyzed, allowing identification of 28,748 different tag species, each representing a unique mRNA transcript. The tags were divided into different abundancy classes, ranging from tags that were detected over 500 times to tags encountered only once in the 76,790 tags analyzed. The mRNA species detected more than 50 times represented 0.3% of the total number of unique tags while accounting for 22% of the total hippocampal mRNA mass. The majority of tags were encountered 5 times or less. The genes expressed at the highest levels were of mitochondrial origin, consistent with a high requirement for energy in neuronal tissue. At a lower level of expression, several neuron-specific transcripts were encountered, encoding various neurotransmitter receptors, transporters, and enzymes involved in neurotransmitter synthesis and turnover, ion channels and pumps, and synaptic components. Comparison of relative expression levels demonstrated that glutamate receptors are the most frequent neurotransmitter receptors expressed in the hippocampus, consistent with the important role of glutamatergic neurotransmission in the hippocampus, while GABA receptors were present at approximately 10-fold lower levels. Several kinases were present including CaMKII, which was expressed at high levels, consistent with its being the most abundant protein in the spines of hippocampal pyramidal cells. This is the first extensive inventory of gene expression in the hippocampus and serves as a reference for future studies aimed at elucidating hippocampal transcriptional responses under various conditions.

Animals↗

PubMed2000

Gene expression profiling in an in vitro model of angiogenesis.

In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.

Cells, Cultured↗

PubMed2000

Expression profiling of neural cells reveals specific patterns of ethanol-responsive gene expression.

Adaptive changes in gene expression are thought to contribute to dependence, addiction and other behavioral responses to chronic ethanol abuse. DNA array studies provide a nonbiased detection of networks of gene expression changes, allowing insight into functional consequences and mechanisms of such molecular responses. We used oligonucleotide arrays to study nearly 6000 genes in human SH-SY5Y neuroblastoma cells exposed to chronic ethanol. A set of 42 genes had consistently increased or decreased mRNA abundance after 3 days of ethanol treatment. Groups of genes related to norepinephrine production, glutathione metabolism, and protection against apoptosis were identified. Genes involved in catecholamine metabolism are of special interest because of the role of this pathway in mediating ethanol withdrawal symptoms (physical dependence). Ethanol treatment elevated dopamine beta-hydroxylase (DBH, EC 1.14.17.1) mRNA and protein levels and increased releasable norepinephrine in SH-SY5Y cultures. Acute ethanol also increased DBH mRNA levels in mouse adrenal gland, suggesting in vivo functional consequences for ethanol regulation of DBH. In SH-SY5Y cells, ethanol also decreased mRNA and secreted protein levels for monocyte chemotactic protein 1, an effect that could contribute to the protective role of moderate ethanol consumption in atherosclerotic vascular disease. Finally, we identified a subset of genes similarly regulated by both ethanol and dibutyryl-cAMP treatment in SH-SY5Y cells. This suggests that ethanol and cAMP signaling share mechanistic features in regulating a subset of ethanol-responsive genes. Our findings offer new insights regarding possible molecular mechanisms underlying behavioral responses or medical consequences of ethanol consumption and alcoholism.

Animals↗

PubMed1999

In vivo gene expression profile analysis of human breast cancer progression.

The development and use of molecular-based therapy for breast cancer and other human malignancies will require a detailed molecular genetic analysis of patient tissues. The recent development of laser capture microdissection and high density cDNA arrays now provides a unique opportunity to generate gene expression profiles of cells from various stages of tumor progression as it occurs in the actual neoplastic tissue milieu. We report the combined use of laser capture microdissection and high-throughput cDNA microarrays to monitor in vivo gene expression levels in purified normal, invasive, and metastatic breast cell populations from a single patient. These in vivo gene expression profiles were verified by real-time quantitative PCR and immunohistochemistry. The combined use of laser capture microdissection and cDNA microarray analysis provides a powerful new approach to elucidate the in vivo molecular events surrounding the development and progression of breast cancer and is generally applicable to the study of malignancy.

Breast Neoplasms↗

PubMed2001

Expression profiles and intergenic structure of head-to-head oriented Brca1 and Nbr1 genes.

Mouse and human Brca1/BRCA1 and Nbr1/NBR2 genes lie head-to-head, being transcribed from the opposite strands. The normal function of Brca1 is associated with DNA damage-related responses and the maintenance of genome integrity. Mutations of this gene are major risk factors for developing breast and ovarian cancers. The function of Nbr1 is unknown. Here we analyzed expression profiles of Brca1 and a newly identified, evolutionary conserved isoform of Nbr1(1a) transcript. In adult testis, the Nbr1(1a) mRNA showed an 18-fold higher steady-state level than the ubiquitous Nbr1(1b) form and its expression was confined mainly to spermatids. The expression profile of Brca1 paralleled that of Nbr1(1b). The Nbr1(1a) mRNA was not significantly expressed in normal somatic tissues, but was found on a list of cDNA clones from a human myeloblast cell line. The newly defined intergenic region (289 bp) between Nbr1 and Brca1 in the mouse and rat corresponds well to that of human BRCA1-NBR2 region.

Animals↗

PubMed2002

Gene expression profiling following BMP-2 induction of mesenchymal chondrogenesis in vitro.

OBJECTIVE: This study aims to apply gene expression profiling technology to gain insight into the molecular regulation of mesenchymal chondrogenesis. METHODS: The experimental system consists of micromass cultures of C3H10T1/2 cells, a murine multipotential embryonic cell line, treated with the chondroinductive growth factor, bone morphogenetic factor-2 (BMP-2). In this system, chondrogenic differentiation characterized by both morphological changes and cartilage matrix gene expression has been shown to be completely dependent upon BMP-2 treatment and the high cell plating density of micromass cultures. To identify candidate genes that may have key functional roles in chondrogenesis, we have applied subtractive hybridization to isolate genes whose expression is significantly up- or down-regulated during chondrogenesis. RNA was isolated from micromass cultures treated with BMP-2 for 24 h and analysed for representational differences by means of a subtractive hybridization screening method. RESULTS: Sixteen different genes were identified whose expression was up-regulated between two- and 12-fold by B,P-2, and twelve different genes were identified whose expression was down-regulated between two- and seven-fold by BMP-2. CONCLUSIONS: The potential of this screening methodology to identify new BMP-2 regulated genes is suggested by the fact that a majority of the identified genes are indeed novel. Identification and characterization of these genes should provide insight as to how chondrogenesis is regulated and also should provide important new markers for the study of osteoarthritis.

Animals↗

PubMed2000

Individual variation in the expression profiles of nicotinic receptors in the olfactory bulb and trigeminal ganglion and identification of alpha2, alpha6, alpha9, and beta3 transcripts.

Nicotine evokes dose-dependent and often variable chemosensory responses in animals and humans. Earlier observations that nicotine binds to some nicotinic acetylcholine receptor (nAChR) subtypes in the olfactory bulb (OB) and trigeminal ganglion (TG) led us to investigate the complete nAChR expression profile in each tissue and to determine whether inter-individual differences exist in male and female rats. Total RNA was extracted from individual samples of dissected OB and TG and analyzed by a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay to determine the messenger RNA profiles of ten transcripts encoded by the alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, alpha9, beta2, beta3, and beta4 nAChR genes. We found that (a) in the OB, all animals expressed alpha2, alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunit mRNAs, whereas alpha6, beta3, and alpha9 transcripts were expressed in only 17, 28, and 33% of the animals, respectively, and (b) in the TG, all animals expressed alpha2, alpha3, alpha6, alpha7, beta2, and beta4 subunit mRNAs, whereas alpha9, beta3, alpha4, and alpha5 transcripts were expressed in 4, 38, 88, and 92% of the animals, respectively. These results also identified new subunits that are expressed in each tissue (alpha2, alpha6, alpha9, and beta3) and demonstrated that individual rats may have different tissue-specific expression profiles for alpha4, alpha5, alpha6, alpha9, and beta3 transcripts. Such variations are likely to be reflected in the composition of functional receptor subtypes in the rat OB and TG that have different activation and desensitization characteristics to acetylcholine and nicotine.

Acetylcholine↗

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↗

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↗

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↗

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↗

PubMed2001

Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by oligonucleotide arrays.

Using an oligonucleotide array containing sequences complementary to approximately 3200 full-length human cDNAs and 3400 expressed sequence tags (GeneChip, Affymetrix), mRNA expression patterns were probed in 18 colon adenocarcinomas and 4 adenomas. Paired normal tissue was available and analyzed for each of the tumors. Relatively few changes in transcript expression are associated with colon cancer. Nineteen transcripts (0.48% of those detected) had at least 4-10.5-fold higher mRNA expression in carcinoma compared with paired normal samples, whereas 47 transcripts (1.3% of those detected) had at least 4-38-fold or lower expression in the tumor tissue compared with the normal samples. Some of these differences were confirmed by reverse transcription-PCR. Many of these transcripts were already known to be abnormally expressed in neoplastic tissue in general, or colon cancer in particular, and several of these differences were also observed in premalignant adenoma samples. A two-way hierarchical clustering algorithm successfully distinguished adenoma from adenocarcinoma and normal tissue, generating a phylogenetic tree that appropriately represented the clinical relationship between the three tissue types included in the analysis. This supports the concept that genome-wide expression profiling may permit a molecular classification of solid tumors.

Adenocarcinoma↗