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Effects of carbonic anhydrase VIII deficiency on cerebellar gene expression profiles in the wdl mouse.

Recently, the waddles (wdl) mouse was identified as a carbonic anhydrase VIII (Car8) mutant. The mutation is associated with marked deficiency of Car8, an inositol triphosphate receptor 1-binding protein expressed at high levels in cerebellar Purkinje cells. To help unravel the molecular aberrations contributing to motor dysfunction in wdl mice, cerebellar gene expression profiles were examined in the mutants and their wild-type littermates. Genes involved in signaling, cell division, zinc ion-binding, synapse integrity and plasticity were downregulated in wdl mice. Several of the upregulated genes encode proteins that function in the Golgi apparatus which suggests that Car8 deficiency has important effects on synaptic vesicle formation and transport.

Animals↗

Gene expression profiling between embryonic and larval stages of the silkworm, Bombyx mori.

To elucidate the molecular mechanisms associated with metamorphic phenomenon relating to Bombyx mori, an important organism in the sericulture industry, we identified genes that are expressed in the different developmental stages, specifically the embryonic (ES) and larval (LS) stages of B. mori. Of 8230 high-quality ESTs from two full-length enriched cDNA libraries, 3442 of the ES ESTs were coalesced into 1325 clusters, while 4788 were coalesced into 927 clusters. The functional classification of these ESTs based on Gene Ontology showed that the types of genes that are associated with oxidoreductase activity, enzyme inhibition, and larval development were highly observed in LS, whereas the types of genes that are involved in nucleotide binding, enzyme activity, and protein transport activity were highly observed in ES. In addition, when the gene expression profile between ES and LS was examined by counting the EST frequencies in each library, 69 genes were identified as being either up- or down-regulated in the larval stage compared to the embryonic stage (P>0.99) and this was confirmed by semi-quantitative RT-PCR. The results show that genes involved in proteolysis and peptidolysis, and lipid and carbohydrate metabolism were dramatically up-regulated in LS, while those related to protein metabolism, DNA/RNA, and coenzymes were highly down-expressed. In particular, a GO analysis of these genes revealed that genes that are involved in hydrolase activity were observed to be highly expressed in amount as well as diversity in LS, while those involved in nucleic acid binding were highly expressed in ES. These data may contribute to elucidating genetic events that distinguish the developmental stage and to our understanding of the metamorphosis of B. mori.

Animals↗

Expression profiling reveals distinct sets of genes altered during induction and regression of cardiac hypertrophy.

Although cardiac hypertrophy has been the subject of intensive investigation, regression of hypertrophy has been significantly less studied, precluding large-scale analysis of the relationship between these processes. In the present study, using pharmacological models of cardiac hypertrophy in mice, expression profiling was performed with fragments of more than 4,000 genes to characterize and contrast expression changes during induction and regression of hypertrophy. Administration of angiotensin II and isoproterenol by osmotic minipump produced increases in heart weight (15 and 45%, respectively) that returned to preinduction size after drug withdrawal. From multiple expression analyses of left ventricular RNA isolated at daily time-points during cardiac hypertrophy and regression, we identified sets of genes whose expression was altered at specific stages of this process. While confirming the participation of 25 genes or pathways previously shown to be altered by hypertrophy, a larger set of 30 genes was identified whose expression had not previously been associated with cardiac hypertrophy or regression. Of the 55 genes that showed reproducible changes during the time course of induction and regression, 32 genes were altered only during induction, and 8 were altered only during regression. This study identified both known and novel genes whose expression is affected at different stages of cardiac hypertrophy and regression and demonstrates that cardiac remodeling during regression utilizes a set of genes that are distinct from those used during induction of hypertrophy.

Angiotensin II↗

Characterization of medroxyprogesterone and DMBA-induced multilineage mammary tumors by gene expression profiling.

Mouse mammary tumors arising during medroxyprogesterone-DMBA-mediated mammary carcinogenesis comprised three distinct phenotypes: adenocarcinoma, squamous cell carcinoma, and myoepithelial carcinoma. The molecular signature for each of the three tumor subsets was characterized by gene microarray analysis, and three distinct sets of gene expression profiles were obtained that were corroborated in part by quantitative RT-PCR and immunohistochemistry. These results suggest that this carcinogenesis and gene expression model will be useful for rapidly assessing the histopathological differences arising in mammary carcinogenesis and the effects of tumor promoting or chemoprevention agents.

9,10-Dimethyl-1,2-benzanthracene↗

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

Aged↗

Gene-expression profiles in hereditary breast cancer.

DNA microarray technology is revolutionizing the way fundamental biologic questions are addressed in the postgenomic era. In this study by Hedenfalk et al., the authors attempted to identify discrete gene expression profiles for patients with known hereditary breast cancers caused by mutations in BRCA1 and BRCA2, both of which increase the lifetime risk of developing breast cancer. The genome-wide perspective identified discrete sets of genes that discriminated between BRCA, BRCA2, and sporadic breast tumors. This commentary discusses some limitations of studying a small number of cases. The authors also address the need for validation on independent tumor sets, and the potential benefit of multivariable-type analyses to consider other potential confounding factors such as tumor grade, receptor status, tumor stage, and treatment information.

BRCA1 Protein↗

Gene expression profiles in different stages of mouse spermatogenic cells during spermatogenesis.

During spermatogenesis, diploid stem cells differentiate, undergo meiosis and spermiogenesis, and transform into haploid spermatozoa. Various factors have been demonstrated to regulate this marvelous process of differentiation, but the expression of only a few genes specifically involved in spermatogenesis has been studied. In the present study, different types of spermatogenic cells were isolated from Balb/c mice testes of different ages using the velocity sedimentation method, and we determined the expression profiles of 1176 known mouse genes in six different types of mouse spermatogenic cells (primitive type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, round spermatids, and elongating spermatids) using Atlas cDNA arrays. Of the 1176 genes on the Atlas Mouse 1.2 cDNA Expression Arrays, we detected 181 genes in primitive type A spermatogonia, 256 in type B spermatogonia, 221 in preleptotene spermatocytes, 160 in pachytene spermatocytes, 141 in round spermatids, and 126 in elongating spermatids. A number of genes were detected as differential expression (up-regulation or down-regulation). Fourteen of the differentially expressed genes have been further confirmed by reverse transcription-polymerase chain reaction for their expression characterizations in different types of spermatogenic cells. These results provide more information for further studies into spermatogenesis-related genes and may lead to the identification of genes with potential relevance to spermatogenesis.

Animals↗

EDA targets revealed by skin gene expression profiles of wild-type, Tabby and Tabby EDA-A1 transgenic mice.

Mutations in the EDA gene cause anhidrotic ectodermal dysplasia (EDA), with lesions in skin appendage formation. To begin to analyze EDA pathways, we have used expression profiling on 15,000-gene mouse cDNA microarrays, comparing adult mouse skin from wild-type, EDA-defective (Tabby) mice, and Tabby mice supplemented with the EDA-A1 isoform, which is sufficient to rescue multiple Tabby phenotypes. Given the sensitivity of the current microarray system, 8500 genes (60%) were estimated to be expressed, including transcription factors and growth-regulatory genes that had not previously been identified in skin; but only 24 (0.16%), one-third of them novel, showed significant differences between wild type and Tabby. An additional eight genes not included in the 15,000 gene set were shown to have expression differences by real-time RT-PCR. Sixteen of 32 affected genes were restored significantly toward wild-type levels in EDA-A1 transgenic Tabby mice. Significant up-regulation in Tabby skin was observed for several dermal matrix genes, including Col1a1, Col1a2, Col3a1 and SPARC: In contrast, down-regulation occurred for the NEMO/NF-kappa B pathway, already implicated in skin appendage formation, and even more markedly for a second pathway, JNK/c-jun/c-fos and their target genes, that has not previously been clearly associated with skin development. These data are consistent with the regulation of the NF-kappa B pathway by EDA, and support its involvement in the regulation of the JNK pathway as well.

Animals↗

Gene expression profiling in polycythaemia vera: overexpression of transcription factor NF-E2.

Summary The molecular aetiology of polycythaemia vera (PV) remains unknown and the differential diagnosis between PV and secondary erythrocytosis (SE) can be challenging. Gene expression profiling can identify candidates involved in the pathophysiology of PV and generate a molecular signature to aid in diagnosis. We thus performed cDNA microarray analysis on 40 PV and 12 SE patients. Two independent data sets were obtained: using a two-step training/validation design, a set of 64 genes (class predictors) was determined, which correctly discriminated PV from SE patients. Separately 253 genes were identified to be upregulated and 391 downregulated more than 1.5-fold in PV compared with healthy controls (P < 0.01). Of the genes overexpressed in PV, 27 contained Sp1 sites: we therefore propose that altered activity of Sp1-like transcription factors may contribute to the molecular aetiology of PV. One Sp1 target, the transcription factor NF-E2 [nuclear factor (erythroid-derived 2)], is overexpressed 2- to 40-fold in PV patients. In PV bone marrow, NF-E2 is overexpressed in megakaryocytes, erythroid and granulocytic precursors. It has been shown that overexpression of NF-E2 leads to the development of erythropoietin-independent erythroid colonies and that ectopic NF-E2 expression can reprogram monocytic cells towards erythroid and megakaryocytic differentiation. Transcription factor concentration may thus control lineage commitment. We therefore propose that elevated concentrations of NF-E2 in PV patients lead to an overproduction of erythroid and, in some patients, megakaryocytic cells/platelets. In this model, the level of NF-E2 overexpression determines both the severity of erythrocytosis and the concurrent presence or absence of thrombocytosis.

Blotting, Northern↗

Impact of nonsense-mediated mRNA decay on the global expression profile of budding yeast.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd(+)) and mutant (Nmd(-)) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% +/- 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd(-) strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are available.

Codon, Initiator↗

Expression profiling and prediction of distant metastases in head and neck squamous cell carcinoma.

BACKGROUND: For breast and prostate cancer, a gene expression signature of the tumour is associated with the development of distant metastases. Regarding head and neck squamous cell carcinoma (HNSCC), the only known risk factor is the presence of > or =3 tumour-positive lymph nodes. AIM: To evaluate whether a HNSCC gene expression signature can discriminate between the patients with and without distant metastases. METHODS: Patients with HNSCC with and without distant metastases had >3 tumour-positive lymph nodes, and did not differ with respect to other risk factors. Statistical analysis was carried out using Student's t test, as well as statistical analysis of microarrays (SAM), to assess the false discovery rate for each gene. These analyses were supplemented with a newly developed method that computed deviations from gaussian-order statistics (DEGOS). To validate the platform, normal mucosa of the head and neck was included as control. RESULTS: 2963 genes were differently expressed between HNSCC and normal mucosa (t test; p<0.01). More rigorous statistical analysis with SAM confirmed the differential expression of most genes. The comparison of genes in HNSCC with and without metastases showed 150 differently expressed genes (t test; p<0.01), none of which, however, could be confirmed using SAM or DEGOS. CONCLUSIONS: No evidence for a metastasis signature is found, and gene expression profiling of HNSCC has seemingly no value in determining the risk of developing distant metastases. The absence of such a signature can be understood when it is realised that, for HNSCC in contrast with breast cancer, the lymph nodes are a necessary in-between station for haematogenous spread.

Adult↗

cDNA expression profiling of chondrosarcomas: Ollier disease resembles solitary tumours and alteration in genes coding for components of energy metabolism occurs with increasing grade.

Conventional central chondrosarcomas are malignant cartilaginous tumours, occasionally arising secondary to either solitary or multiple (Ollier disease) enchondromas. Recurrences may have progressed in grade. The aims of the present study were to identify putative differences in gene expression between solitary and Ollier disease-related tumours, and to elucidate signalling pathways involved in tumour progression by genome-wide cDNA expression analysis. Arrays enriched for cartilage-specific cDNAs and genes involved in general tumourigenesis were used to analyse enchondromas (n = 3, two with Ollier disease), chondrosarcomas of different grades (n = 19, three with Ollier disease), normal resting-zone cartilage (n = 2), and chondrosarcoma cells in culture (n = 7). The arrays were analysed by unsupervised hierarchical clustering, significant analysis of microarray, and T-tests. Confirmation of data was performed by immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Ollier disease cases and solitary tumours revealed similar expression profiles, suggesting that the same signalling pathways are involved in tumourigenesis. Interestingly, JunB protein expression was significantly higher in grade I chondrosarcomas than in enchondromas (p = 0.009), which could be of diagnostic relevance. Upon chondrosarcoma progression, matrix-associated genes are down-regulated, reflecting the histology of high-grade tumours. An increase in glycolysis-associated, and a decrease in oxidative phosphorylation-related, genes was found in high-grade tumours. These findings suggest an adaptation in energy supply upon progression towards higher grade.

Adolescent↗

Gene expression profile differences in high and low metastatic human ovarian cancer cell lines by gene chip.

OBJECTIVES: To study the difference between gene expressions of high (H0-8910PM) and low (HO-8910) metastatic human ovarian carcinoma cell lines and screen novel associated genes by cDNA microarray. METHODS: cDNA retro-transcribed from equal quantities of mRNA derived from high and low metastatic tumor cells or normal ovarian tissues were labeled with Cy5 and Cy3 fluorescein as probes. The mixed probe was hybridized with two pieces of BioDoor 4096 double dot human whole gene chip and scanned with a ScanArray 3000 laser scanner. The acquired image was analyzed by ImaGene 3.0 software. RESULTS: A total of 355 genes with expression levels more than 3 times larger were found by comparing the HO-8910 cell with normal ovarian epithelial cells. A total of 323 genes with expression levels more than 3 times larger in HO-8910PM cells compared to normal ovarian epithelium cells were also detected. A total of 165 genes whose expression levels were more than two times those of HO-8910PM cells compared to their mother cell line (HO-8910) were detected. Twenty-one genes with expression levels > 3 times were found from comparison of these two tumor cell lines. CONCLUSIONS: cDNA microarray techniques are effective in screening differential gene expression between two human ovarian cancer cell lines (H0-8910PM; HO-8910) and normal ovarian epithelial cells. These genes may be related to the genesis and development of ovarian carcinoma. Analysis of the human ovarian cancer gene expression profile with cDNA microarray may help in gene diagnosis, treatment and prevention.

Female↗

Gene expression profiles in the acutely dissected human aorta.

OBJECTIVES: heritable connective tissue abnormalities and arterial hypertension may predispose to aortic dissection. This study evaluates gene expression profiles in the acutely dissected human aorta. DESIGN, MATERIALS AND METHODS: Atlas Human Broad Arrays I, II, and III (Clontech) were used to compare gene expression in acutely dissected (6 patients) and normal ascending aortas (6 multiorgan donors). The tissues were also compared macroscopically. RESULTS: of 3537 genes analysed, 1250 (35%) were expressed in aortic tissue. For statistical analysis we focused on 627 genes, which had an intensity>0.95 of the mean patients or controls. Dissected and adjacent macroscopically intact aorta displayed similar gene expression patterns. On the contrary, 66 genes were expressed significantly different in dissected aorta, compared with undiseased control aorta of multiorgan donors. Genes, predominantly upregulated in dissection, are involved in inflammation, in extracellular matrix proteolysis, in proliferation, translation and transcription. Predominantly downregulated genes code for extracellular matrix proteins, adhesion proteins and cytoskeleton proteins. CONCLUSION: our results demonstrate for the first time the complexity of the dissecting process on a molecular level. The ultimate dissection seems to be the dramatic endpoint of a long-lasting process of degradation and insufficient remodelling of the aortic wall. Altered patterns of gene expression suggest a pre-existing structural failure of the aortic wall, resulting in dissection.

Acute Disease↗

Characterization of the breast cancer associated ATM 7271T>G (V2424G) mutation by gene expression profiling.

Mutations in ATM are responsible for the autosomal recessive disorder ataxia telangiectasia. Heterozygous mutations in ATM have been associated with an elevated risk of breast cancer. We previously reported one breast cancer family in which ATM 7271T>G (V2424G) segregated with disease, and apparently acted in a dominant negative manner. We now report the screening of 782 multiple-case breast cancer families that identified two additional index cases with ATM 7271T>G. Phylogenetic sequence analysis showed that V2424 is a highly conserved residue, and that the 2424G variant is likely to interfere with function. To elucidate the consequences of this mutation, we expression profiled wild-type, heterozygous, and homozygous lymphoblastoid cell lines (LCLs) from Scottish and Australian families using an oligonucleotide microarray. Cluster analysis revealed 77 genes that were differentially expressed in homozygous and heterozygous V2424G cells (compared to wild-type) and 11 genes differentially expressed in the homozygous cells. We also evaluated the profiles of LCLs after exposure to ionizing radiation (IR) and identified 77 genes that were differentially expressed in wild-type cells, but not in homozygous or heterozygous V2424G cells. We validated the expression differences by RT-PCR in additional heterozygous V2424G LCLs from another breast cancer family. We found no consistent cytotoxicity or abrogation of ATM kinase activity after IR in seven heterozygous V2424G LCLs, compared to wild-type LCLs, but did find an increase in the number of chromosomal aberrations. These data suggest that the V2424G missense mutation acts largely as a dominant negative in terms of the associated expression profiles.

Amino Acid Sequence↗

[Gene expression profiles and effects of transforming growth factor-beta1 intervention in Peyronie's disease].

OBJECTIVE: To demonstrate molecular insight into the pathology of Peyronie's disease (PD). A preliminary profile of differential gene expression between the PD plaque and control tunica albuginea was obtained with DNA microarrays. Also, to investigate the effect of intervention in PD cells, transforming growth factor-beta1 (TGF-beta1) was recruited to treat PD cell lines. METHODS: Three PD plaques and control tunica albugineas were constructed and studied. cDNA probes were prepared from RNA isolated from those cells and hybridized with the Clontech Atlas 3.6 Array. Relative changes of greater than 2.0 defined up-regulation and down-regulation, respectively. The expression of selected individual gene MCP-1 and the effect of TGF-beta1 on MCP-1 were analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: Some up-regulated genes in the PD plaque detected by the Clontech assay were screened, one of them was monocyte chemotactic protein. One involved the pathogenesis of PD as a downstream gene and responded to the TGF-beta1 treatment but not CTGF. The results were also confirmed by TR-PCR in all the types of cell. CONCLUSIONS: The cell lines from plaque tissue and normal tunica from men with PD were successfully established. The findings indicate a potential role for MCP-1 over expression in the pathogenesis of PD as a downstream gene regulated by some genes and could be a new therapeutic target in PD. The information may allow a better understanding of the basic mechanisms involved in the etiology and pathogenesis of PD. Furthermore, it may permit some strategies of therapeutic interventions combine routine methods with Chinese herbal medicine.

Cell Line↗

Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803.

The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.

Adaptation, Physiological↗

Expression profiling of microdissected matched prostate cancer samples reveals CD166/MEMD and CD24 as new prognostic markers for patient survival.

In order to screen for differentially expressed genes that might be useful in diagnosis or therapy of prostate cancer we have used a custom made Affymetrix GeneChip containing 3950 cDNA fragments. Expression profiles were obtained from 42 matched pairs of mRNAs isolated from microdissected malignant and benign prostate tissues. Applying three different bioinformatic approaches to define differential gene expression, we found 277 differentially expressed genes, of which 98 were identified by all three methods. Fourteen per cent of these genes were not found in other expression studies, which were based on bulk tissue. Resultant candidate genes were further validated by quantitative RT-PCR, mRNA in situ hybridization and immunohistochemistry. AGR2 was over-expressed in 89% of prostate carcinomas, but did not have prognostic significance. Immunohistologically detected over-expression of MEMD and CD24 was identified in 86% and 38.5% of prostate carcinomas respectively, and both were predictive of PSA relapse. Combined marker analysis using MEMD and CD24 expression proved to be an independent prognostic factor (RR = 4.7, p = 0.006) in a Cox regression model, and was also superior to conventional markers. This combination of molecular markers thus appears to allow improved prediction of patient prognosis, but should be validated in larger studies.

Activated-Leukocyte Cell Adhesion Molecule↗