Microarray Suite Research Articles

Genome-wide CNV analysis uncovers novel pathogenic regions in cohort of five multiplex families with neurodevelopmental disorders

Structural reorganization of chromosomes by genomic duplications and/or deletions are known as copy number variations (CNVs). Pathogenic and disease susceptible CNVs alter gene dosage and its phenotypic expression that often leads to human genetic diseases including Neurological disorders. CNVs affecting same common genes in multiple neurodevelopmental disorders can better explain the shared clinical and genetic aetiology across brain diseases. Our study presents the novel copy number variations in a cohort of five multiplex consanguineous families with intellectual disability, microcephaly, ASD, epilepsy, and neurological syndromic features. Cytoscan HD microarray suite has revealed genome wide deletions, duplications and LOH regions which are co-segregating in the family members for the rare neurodevelopmental syndromic phenotypes. This study identifies 1q21.1 microduplication, 16p11.2 microduplication, Xp11.22 microduplication, 4p12 microdeletion and Xq21.1 microdeletion that significantly contribute to primary disease onset and its progression for the first time in Pakistani families. Our study has potential impact on the understanding of pathogenic genetic predisposition for appearance of complex and heterogeneous neurodevelopmental disorders with otherwise unexplained syndromic features. Identification of altered gene dosage across the genome is helpful in improved diagnosis, better disease management in day-to-day life activities of patients with cognitive impairment and genetic counselling of families where consanguinity is a tradition. Our study will contribute to expand the knowledge of genotype-phenotype expression and future gateways in therapeutics and precision medicine research will be open in Pakistan.

Effect of Mongolian warm acupuncture on the gene expression profile of rats with insomnia.

The mechanism of Mongolian warm acupuncture (MWA) for the treatment of insomnia has not been previously reported. To investigate the effect of MWA on gene expression profile in the p-chlorophenylalanine (PCPA)-induced rat model of insomnia. A rat model of insomnia was established and the animals were divided into five groups: control, PCPA (untreated), PCPA+estazolam, PCPA+MA (manual acupuncture), and PCPA+MWA. The rats were euthanased at 7 days after treatment, and hypothalamic tissue was harvested to extract total RNA for the analysis of gene expression profile. Micro-array and Partek Genomics Suite analysis system were used to analyse differential expression of genes between groups. Furthermore, ingenuity pathways analysis was used to analyse the main regulators. After treatment, in rats with improved sleep, micro-array data from the follow-up phase compared with baseline showed that MWA down-regulated 11 genes compared with the control group and 16 genes compared with the PCPA group. Six genes were selected following the micro-array detection to perform quantitative polymerase chain reaction (qPCR) verification, and the results showed that the coincidence rate was up to 90%, which verified the reliability of the microarray results. Compared with the PCPA group, transcription levels of Egr 1, Btg2 and BDNF in the PCPA+MWA group were up-regulated (P<0.05). In combination, the findings of this study suggests that MWA is efficacious at improving sleep in an experimental rat model of insomnia.

Application of a Blood-based Dynamic Genome Signature: How MAS5/RMA/PLIER Normalization Batches Affect Measured Gene Expression Profiling Stability in Clinical Diagnosis

Objective: A number of studies compare the microarray normalization methods MAS5 (Microarray Suite Version 5), RMA (Robust Multi-array Average) and PLIER (Probe Logarithmic Error Intensity Estimate) with respect to the rate at which genes of interest are identified. Here we evaluate and compare the stability of the measured gene expression when identical or technical replicate arrays are analyzed in batches of differing sizes and composition. These variations in measured gene expression have implications for clinical applications, which have requirements that differ significantly from those of research applications.Methods: We evaluated the samples from data set E-MTAB-1532, available on ArrayExpress, a public repository of microarray data using the MAS5, RMA, and PLIER methods. We then evaluated a sample run as triplicate arrays and compared results among the different normalization methods.Results and conclusion: Our study found that for some applications MAS5 is superior to the other methods, although the MAQC (Micro Array Quality Control) project, which extensively evaluated the performance of the platforms, reached a different conclusion.

Comparative transcriptomics indicate changes in cell wall organization and stress response in seedlings during spaceflight.

Plants will play an important role in the future of space exploration as part of bioregenerative life support. Thus, it is important to understand the effects of microgravity and spaceflight on gene expression in plant development. We analyzed the transcriptome of Arabidopsis thaliana using the Biological Research in Canisters (BRIC) hardware during Space Shuttle mission STS-131. The bioinformatics methods used included RMA (robust multi-array average), MAS5 (Microarray Suite 5.0), and PLIER (probe logarithmic intensity error estimation). Glycome profiling was used to analyze cell wall composition in the samples. In addition, our results were compared to those of two other groups using the same hardware on the same mission (BRIC-16). In our BRIC-16 experiments, we noted expression changes in genes involved in hypoxia and heat shock responses, DNA repair, and cell wall structure between spaceflight samples compared to the ground controls. In addition, glycome profiling supported our expression analyses in that there was a difference in cell wall components between ground control and spaceflight-grown plants. Comparing our studies to those of the other BRIC-16 experiments demonstrated that, even with the same hardware and similar biological materials, differences in results in gene expression were found among these spaceflight experiments. A common theme from our BRIC-16 space experiments and those of the other two groups was the downregulation of water stress response genes in spaceflight. In addition, all three studies found differential regulation of genes associated with cell wall remodeling and stress responses between spaceflight-grown and ground control plants.

Additional annotation enhances potential for biologically-relevant analysis of the Illumina Infinium HumanMethylation450 BeadChip array

BackgroundMeasurement of genome-wide DNA methylation (DNAm) has become an important avenue for investigating potential physiologically-relevant epigenetic changes. Illumina Infinium (Illumina, San Diego, CA, USA) is a commercially available microarray suite used to measure DNAm at many sites throughout the genome. However, it has been suggested that a subset of array probes may give misleading results due to issues related to probe design. To facilitate biologically significant data interpretation, we set out to enhance probe annotation of the newest Infinium array, the HumanMethylation450 BeadChip (450 k), with >485,000 probes covering 99% of Reference Sequence (RefSeq) genes (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA). Annotation that was added or expanded on includes: 1) documented SNPs in the probe target, 2) probe binding specificity, 3) CpG classification of target sites and 4) gene feature classification of target sites.ResultsProbes with documented SNPs at the target CpG (4.3% of probes) were associated with increased within-tissue variation in DNAm. An example of a probe with a SNP at the target CpG demonstrated how sample genotype can confound the measurement of DNAm. Additionally, 8.6% of probes mapped to multiple locations in silico. Measurements from these non-specific probes likely represent a combination of DNAm from multiple genomic sites. The expanded biological annotation demonstrated that based on DNAm, grouping probes by an alternative high-density and intermediate-density CpG island classification provided a distinctive pattern of DNAm. Finally, variable enrichment for differentially methylated probes was noted across CpG classes and gene feature groups, dependant on the tissues that were compared.ConclusionDNAm arrays offer a high-throughput approach for which careful consideration of probe content should be utilized to better understand the biological processes affected. Probes containing SNPs and non-specific probes may affect the assessment of DNAm using the 450 k array. Additionally, probe classification by CpG enrichment classes and to a lesser extent gene feature groups resulted in distinct patterns of DNAm. Thus, we recommend that compromised probes be removed from analyses and that the genomic context of DNAm is considered in studies deciphering the biological meaning of Illumina 450 k array data.

Cyclin D1 Is a Selective Modifier of Androgen-dependent Signaling and Androgen Receptor Function

D-type cyclins regulate cellular outcomes in part through cyclin-dependent, kinase-independent mechanisms that modify transcription factor action, and recent in vivo studies showed that cyclin D1 associates with a large number of transcriptional regulators in cells of the retina and breast. Given the frequency of cyclin D1 alterations in cancer, it is imperative to delineate the molecular mechanisms by which cyclin D1 controls key transcription factor networks in human disease. Prostate cancer was used as a paradigm because this tumor type is reliant at all stages of the disease on androgen receptor (AR) signaling, and cyclin D1 has been shown to negatively modulate AR-dependent expression of prostate-specific antigen (KLK3/PSA). Strategies were employed to control cyclin D1 expression under conditions of hormone depletion, and the effect of cyclin D1 on subsequent androgen-dependent gene expression was determined using unbiased gene expression profiling. Modulating cyclin D1 conferred widespread effects on androgen signaling and revealed cyclin D1 to be a selective effector of hormone action. A subset of androgen-induced target genes, known to be directly regulated by AR, was strongly suppressed by cyclin D1. Analyses of AR occupancy at target gene regulatory loci of clinical relevance demonstrated that cyclin D1 limits AR residence after hormone stimulation. Together, these findings reveal a new function for cyclin D1 in controlling hormone-dependent transcriptional outcomes and demonstrate a pervasive role for cyclin D1 in regulating transcription factor dynamics.

Experimental observation on the alteration of metastatic potential and its gene function net of residual hepatocellular carcinoma following palliative liver resection

To investigate the effects of palliative liver resection on metastatic potential and its gene function net of residual hepatocellular carcinoma (HCC) in nude mice model. Orthotopic HCC model was established by implantation of human HCC cell line MHCC97H xenografts with a high metastatic potential. Thirty-six HCC-bearing nude mice were randomized into 3 groups at 14 days post-operation, including palliative resection group (HCC samples A, B), control group 1 (sham operation, HCC sample C1) and control group 2 (without intervention, HCC sample C2). Six mice in each group were sacrificed by cervical dislocation at 14 days after palliative resection. Oligo tumor metastasis microarray and GEArray expression analysis suite software were employed for gene analysis. The methods of support vector machine (SVM), gene significance analysis and gene correlation degree analysis were used to search the markers capable of differentiating the metastatic potential of HCC. Gene function net was constructed based on special gene clustering analysis and multi-dimensional scale. Real-time PCR was applied to detect the mRNA expression. The remaining mice were sacrificed at 35 days after palliative resection for the examination of pulmonary metastasis. SAS 8.2 software was used for statistical analysis. Gene analysis showed that for different gene expression among groups, the density of gene net in palliative group (B, 0.0670) were higher than those in control groups (A, 0.0145; C1, 0.0210; C2, 0.0146), the condensation degree of gene net in residual HCC (B, 0.1940) was higher than that in its own control group (A, 0.0098). Human gene metastasis suppressor 1 (MTSS1) that encodes a protein of missing in metastasis B (MIM-B) was situated in the central position of gene function net of residual HCC. The relative MIM-B mRNA expression examined by Real-time PCR was significantly up-regulated in residual HCC (B, 0.283 +/- 0.023) as comparison was made among groups (A, 0.142 +/- 0.018; C1, 0.177 +/- 0.054; C2, 0.156 +/- 0.017, all P < 0.05). The number of lung metastatic nodules in the palliative group (14.3 +/- 4.7) was more than that in the sham group (8.7 +/- 3.6) at 35 days post-resection (P < 0.01). Palliative liver resection enhances the pulmonary metastatic potential of residual HCC in nude mice model. MIM-B may be one of the key therapeutic targets for HCC patients.

Abstract 4655: Gene expression signatures of microsatellite-stable stage III colorectal adenocarcinomas based on the p53 status

Abstract Background: Microsatellite (MS) instability and p53 mutations have been implicated in development, progression and metastasis of colorectal cancer (CRCs). Thus, to identify aggressive tumor subsets, the gene expression profiles of Stage- III MS-stable (MSS), sporadic CRCs were analyzed based on their p53 gene status. Methods: DNA samples extracted from 136 Stage-III formalin-fixed paraffin embedded CRC tissues were analyzed for MSS and for p53 mutations. Further, mRNA samples extracted from 20 (10 each of p53-mutated and wild-type) corresponding snap-frozen Stage-III-MSS-CRCs were profiled for differential gene expression. Affymetrix Human Genome U133 Plus 2.0 arrays were used for gene expression profiling; and Affymetrix Microarray Suite (MAS 5.0), gene ontology annotation, and KEGG pathways were used for data analysis. Differentially expressed (DE) genes were confirmed by measuring mRNA levels by RT-PCR and by the quantitative nuclease protection assay. Results: A higher incidence of p53 mutations was found in MSS (65 of 113, 58%) than in MSI (6 of 23, 30%) phenotypes. Kaplan-Meier analyses demonstrated that patients with MSS-CRCs exhibiting p53 mutations had poor overall survival when compared to p53-wt-phenotypes (log rank, p=0.031). Gene expression analyses identified 116 DE genes (73 down-regulated and 43 up-regulated). All of these passed the stringent background filter (intensity p-value ≤0.001 and intensity ≥3 SD above mean background) and DE ratio (χ2 p-value ≤0.01 &amp; fold change ≥2.0). GBP1, PSMB9, TRIM29, BST2 and TFF1, that have prognostic value in CRCs, were up-regulated in p53-mutated-MSS-CRCs but down-regulated in p53-wt-MSS-CRCs. Further, genes that regulate cell-cycle arrest (CDKN1B) and tumor growth suppression (ACVR1B, SESN1, WFDC1 and IRF7) were down-regulated in p53-mutated-MSS-CRCs, but up-regulated in p53-wt-MSS-CRCs. The genes presented here are the top-ranked (top-5) genes in their respective groups. The mRNA expression levels of these DE genes in all 136 CRCs were confirmed on two different platforms. Conclusion: Our findings suggest that mutant- and wt-p53 Stage-III-MSS-CRCs have distinct gene expression profiles and appear to be useful in identifying aggressive Stage III subsets and for the assessment of clinical outcomes. These studies are supported by grants from the National Institute of Health/National Cancer Institute (U54-CA118948, RO1-CA98932, and R03CA139629). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4655.

Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain

We have observed extensive interindividual differences in DNA methylation of 8590 CpG sites of 6229 genes in 153 human adult cerebellum samples, enriched in CpG island "shores" and at further distances from CpG islands. To search for genetic factors that regulate this variation, we performed a genome-wide association study (GWAS) mapping of methylation quantitative trait loci (mQTLs) for the 8590 testable CpG sites. cis association refers to correlation of methylation with SNPs within 1 Mb of a CpG site. 736 CpG sites showed phenotype-wide significant cis association with 2878 SNPs (after permutation correction for all tested markers and methylation phenotypes). In trans analysis of methylation, which tests for distant regulation effects, associations of 12 CpG sites and 38 SNPs remained significant after phenotype-wide correction. To examine the functional effects of mQTLs, we analyzed 85 genes that were with genetically regulated methylation we observed and for which we had quality gene expression data. Ten genes showed SNP-methylation-expression three-way associations-the same SNP simultaneously showed significant association with both DNA methylation and gene expression, while DNA methylation was significantly correlated with gene expression. Thus, we demonstrated that DNA methylation is frequently a heritable continuous quantitatively variable trait in human brain. Unlike allele-specific methylation, genetic polymorphisms mark both cis- and trans-regulatory genetic sites at measurable distances from their CpG sites. Some of the genetically regulated DNA methylation is directly connected with genetically regulated gene expression variation.

Abstract A32: Identification of biomarkers in normal human breast cells regulated by the cancer preventive rexinoid LG100268

Abstract Background: Drugs targeting estrogen signaling or production have become useful in preventing estrogen receptor-positive breast cancers, but not estrogen receptor-negative (ER-negative) breast cancers. Our laboratory has demonstrated that rexinoids prevent ER-negative mammary tumors in transgenic mice by 90%. However, since these agents are not 100% effective, we are investigating the mechanism by which rexinoids prevent cancer to develop more efficacious prevention strategies. We hypothesized that by identifying molecules regulated by the rexinoid LG100268, future chemopreventive agents could be developed to target these molecules alone or in combination with rexinoids to totally prevent breast cancer development. To investigate this hypothesis we utilized an Affymetrix genome array to identify biomarkers regulated by LG100268. Methods: Cell counts were used to confirm rexinoid-induced growth suppression in normal human mammary epithelial cells (HMECs). TUNEL analysis was conducted to determine whether the growth suppression was a result of induction of apoptosis. We conducted Affymetrix analysis using 10ug total RNA from HMECs treated with 0.1% vehicle or 1uM LG100268 for 1 hour or 24 hours. cRNA was hybridized with the GeneChip® Expression Analysis Array (U133) from Affymetrix. Results were analyzed using Microarray Suite 5.0 (Affymetrix). Genes to be considered for further confirmation had a fold change of &amp;gt; 1.5 or &amp;lt; 0.8 (p &amp;lt; 0.05) for up- or down-regulated genes, respectively. Geneontology analysis identified candidate genes involved in proliferation, cell cycle and metabolism as genes to consider for further analysis. Quantitative RT-PCR (qRT-PCR) and standard western blot techniques were used to confirm regulation of selected up- and down-regulated genes at the mRNA and protein levels, respectively. Results: Cell count analysis confirmed that LGD1069 greatly suppressed growth of HMEC cells, while LG100268 slightly suppressed HMEC growth. TUNEL analysis indicated that neither rexinoid treatment induced a significant amount of apoptosis in HMEC cells, indicating that the induction of growth suppression was not a result of apoptosis. A full list of genes up- and down-regulated by LGD100268 will be presented. Of those genes selected for further study, qRT-PCR analysis confirmed that LG100268 up-regulated stanniocalcin 1 (STC1), carbonic anhydrase IX (CA9), angiopoietin-like 4 (ANGPTL4), transglutaminase 2 (TGM2), and acyl-CoA synthetase long-chain family member 3 (ACSL3). Confirmed down-regulated genes at the mRNA level included cyclin B1 (CCNB1). Additional validation studies are ongoing. These up- and down-regulated genes may be useful targets for chemopreventive drug development. Conclusions: These experiments identified a list of biomarkers that are regulated by LG100268. Such rexinoid-regulated molecules represent possible targets for breast cancer preventative therapy, alone or in combination with rexinoids. Support: NCI R01 CA78480 (PHB and IPU), and NIH/NCI T32 CA90221 (JMR). Citation Information: Cancer Prev Res 2010;3(1 Suppl):A32.

Comparative gene expression profiles between heterotic and non-heterotic hybrids of tetraploid Medicago sativa

BackgroundHeterosis, the superior performance of hybrids relative to parents, has clear agricultural value, but its genetic control is unknown. Our objective was to test the hypotheses that hybrids expressing heterosis for biomass yield would show more gene expression levels that were different from midparental values and outside the range of parental values than hybrids that do not exhibit heterosis.ResultsWe tested these hypotheses in three Medicago sativa (alfalfa) genotypes and their three hybrids, two of which expressed heterosis for biomass yield and a third that did not, using Affymetrix M. truncatula GeneChip arrays. Alfalfa hybridized to approximately 47% of the M. truncatula probe sets. Probe set signal intensities were analyzed using MicroArray Suite v.5.0 (MAS) and robust multi-array average (RMA) algorithms. Based on MAS analysis, the two heterotic hybrids performed similarly, with about 27% of genes showing differential expression among the parents and their hybrid compared to 12.5% for the non-heterotic hybrid. At a false discovery rate of 0.15, 4.7% of differentially expressed genes in hybrids (~300 genes) showed nonadditive expression compared to only 0.5% (16 genes) in the non-heterotic hybrid. Of the nonadditively expressed genes, approximately 50% showed expression levels that fell outside the parental range in heterotic hybrids, but only one of 16 showed a similar profile in the non-heterotic hybrid. Genes whose expression differed in the parents were three times more likely to show nonadditive expression than genes whose parental transcript levels were equal.ConclusionThe higher proportions of probe sets with expression level that differed from the parental midparent value and that were more extreme than either parental value in the heterotic hybrids compared to a non-heterotic hybrid were also found using RMA. We conclude that nonadditive expression of transcript levels may contribute to heterosis for biomass yield in alfalfa.

High-throughput Biology in the Postgenomic Era

High-throughput Biology in the Postgenomic Era

Scavenger Chemokine (CXC Motif) Receptor 7 (CXCR7) Is a Direct Target Gene of HIC1 (Hypermethylated in Cancer 1)

The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) that is epigenetically silenced in many human tumors and is essential for mammalian development encodes a sequence-specific transcriptional repressor. The few genes that have been reported to be directly regulated by HIC1 include ATOH1, FGFBP1, SIRT1, and E2F1. HIC1 is thus involved in the complex regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. We performed genome-wide expression profiling analyses to identify new HIC1 target genes, using HIC1-deficient U2OS human osteosarcoma cells infected with adenoviruses expressing either HIC1 or GFP as a negative control. These studies identified several putative direct target genes, including CXCR7, a G-protein-coupled receptor recently identified as a scavenger receptor for the chemokine SDF-1/CXCL12. CXCR7 is highly expressed in human breast, lung, and prostate cancers. Using quantitative reverse transcription-PCR analyses, we demonstrated that CXCR7 was repressed in U2OS cells overexpressing HIC1. Inversely, inactivation of endogenous HIC1 by RNA interference in normal human WI38 fibroblasts results in up-regulation of CXCR7 and SIRT1. In silico analyses followed by deletion studies and luciferase reporter assays identified a functional and phylogenetically conserved HIC1-responsive element in the human CXCR7 promoter. Moreover, chromatin immunoprecipitation (ChIP) and ChIP upon ChIP experiments demonstrated that endogenous HIC1 proteins are bound together with the C-terminal binding protein corepressor to the CXCR7 and SIRT1 promoters in WI38 cells. Taken together, our results implicate the tumor suppressor HIC1 in the transcriptional regulation of the chemokine receptor CXCR7, a key player in the promotion of tumorigenesis in a wide variety of cell types.

ZAC1 Is Up-regulated by Hypertonicity and Decreases Sorbitol Dehydrogenase Expression, Allowing Accumulation of Sorbitol in Kidney Cells

Affymetrix GeneChip technology was employed to detect differentially expressed genes in inner medullary collecting duct (IMCD3) cells grown under isotonic and hypertonic conditions. A marked up-regulation was found for the zinc-finger protein ZAC1 under hypertonic stress (219-fold, p < 0.001). Changes in expression for ZAC1 were verified by quantitative PCR for message and Western blotting for protein. In mouse and human kidney tissues, ZAC1 expression was substantial in the papilla and was absent in the cortex. Furthermore, ZAC1 expression significantly increased in the papilla of mice following 36 h of fluid restriction and decreased in polyuric mice consuming sucrose in water. Because ZAC1 has been described to be a potential negative regulator of sorbitol dehydrogenase (SDH) in hippocampal cells, we examined whether this relationship also occurs in kidney cells under hypertonic stress. We found that stable IMCD3 clones silenced for ZAC1 to varying levels demonstrated an inverse effect on SDH expression. ZAC1 binds to a consensus repression site within the promoter of SDH, pointing to a mechanism whereby ZAC1 acts by repressing SDH transcriptional activity during hypertonic conditions. Taken together, these data strongly suggest that ZAC1 is up-regulated under hypertonic stress and negatively regulates expression of SDH, allowing for accumulation of sorbitol as a compatible organic osmolyte.

Effect of the Methoxychlor Metabolite HPTE on the Rat Ovarian Granulosa Cell Transcriptome In Vitro

Ovarian granulosa cells play a central role in steroidogenesis, which is critical for female reproduction. Follicle-stimulating hormone (FSH) promotes cyclic adenosine monophosphate (cAMP)-mediated signaling to regulate granulosa cell steroidogenesis. We have shown previously that 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) inhibits FSH- and dibutyryl cAMP-stimulated steroidogenesis and affects the messenger RNA levels of steroidogenic pathway enzymes in rat granulosa cells. However, HPTE showed a differential effect in FSH- and cAMP-stimulated cells in that HPTE more completely blocked FSH- when compared to cAMP-driven steroidogenesis. The objective of this study was to analyze the effects of HPTE on global gene expression profiles in untreated granulosa cells and those challenged with FSH or cAMP. Granulosa cells from immature rats were cultured with 0, 1, 5, or 10 microM HPTE in the presence or absence of either 3 ng FSH/ml or 1mM cAMP for 48 h. Total RNA was isolated for real-time quantitative PCR and microarray analysis using the GeneChip Rat Genome 230 2.0 and ArrayAssist Microarray Suite. An investigation of changes in gene expression across all HPTE treatments showed that HPTE altered more genes in FSH- (approximately 670 genes) than in cAMP-stimulated cells (approximately 366 genes). Analysis confirmed that HPTE more effectively inhibited FSH- than cAMP-induced steroid pathway gene expression and steroidogenesis. Furthermore, expression patterns of novel genes regulating signal transduction, transport, cell cycle, adhesion, differentiation, motility and growth, apoptosis, development, and metabolism were all altered by HPTE. This study further established that HPTE exerts differential effects within the granulosa cell steroidogenic pathway and revealed that these effects include broader changes in gene expression.

The Bimodality Index: A criterion for Discovering and Ranking Bimodal Signatures from Cancer Gene Expression Profiling Data

Identifying genes with bimodal expression patterns from large-scale expression profiling data is an important analytical task. Model-based clustering is popular for this purpose. That technique commonly uses the Bayesian information criterion (BIC) for model selection. In practice, however, BIC appears to be overly sensitive and may lead to the identification of bimodally expressed genes that are unreliable or not clinically useful. We propose using a novel criterion, the bimodality index, not only to identify but also to rank meaningful and reliable bimodal patterns. The bimodality index can be computed using either a mixture model-based algorithm or Markov chain Monte Carlo techniques. We carried out simulation studies and applied the method to real data from a cancer gene expression profiling study. Our findings suggest that BIC behaves like a lax cutoff based on the bimodality index, and that the bimodality index provides an objective measure to identify and rank meaningful and reliable bimodal patterns from large-scale gene expression datasets. R code to compute the bimodality index is included in the ClassDiscovery package of the Object-Oriented Microarray and Proteomic Analysis (OOMPA) suite available at the web site http;//bioinformatics.mdanderson.org/Software/OOMPA.

Muscle CD31(−) CD45(−) Side Population Cells Promote Muscle Regeneration by Stimulating Proliferation and Migration of Myoblasts

CD31(-) CD45(-) side population (SP) cells are a minor SP subfraction that have mesenchymal stem cell-like properties in uninjured skeletal muscle but that can expand on muscle injury. To clarify the role of these SP cells in muscle regeneration, we injected green fluorescent protein (GFP)-positive myoblasts with or without CD31(-) CD45(-) SP cells into the tibialis anterior muscles of immunodeficient NOD/scid mice or dystrophin-deficient mdx mice. More GFP-positive fibers were formed after co-transplantation than after transplantation of GFP-positive myoblasts alone in both mdx and NOD/scid muscles. Moreover, grafted myoblasts were more widely distributed after co-transplantation than after transplantation of myoblasts alone. Immunohistochemistry with anti-phosphorylated histone H3 antibody revealed that CD31(-) CD45(-) SP cells stimulated cell division of co-grafted myoblasts. Genome-wide gene expression analyses showed that these SP cells specifically express a variety of extracellular matrix proteins, membrane proteins, and cytokines. We also found that they express high levels of matrix metalloproteinase-2 mRNA and gelatinase activity. Furthermore, matrix metalloproteinase-2 derived from CD31(-) CD45(-) SP cells promoted migration of myoblasts in vivo. Our results suggest that CD31(-) CD45(-) SP cells support muscle regeneration by promoting proliferation and migration of myoblasts. Future studies to further define the molecular and cellular mechanisms of muscle regeneration will aid in the development of cell therapies for muscular dystrophy.

Effect of 1.8 GHz radiofrequency electromagnetic fields on gene expression of rat neurons

To investigate the changes of gene expression in rat neuron induced by 1.8 GHz radiofrequency electromagnetic fields (RF EMF) to screen for RF EMF-responsive genes and the effect of different exposure times and modes on the gene expression in neuron. Total RNA was extracted immediately and purified from the primary culture of neurons after intermittent exposed or sham-exposed to a frequency of 1.8 GHz RF EMF for 24 hours at an average special absorption rate (SAR) of 2 W/kg. Affymetrix Rat Neurobiology U34 array was applied to investigate the changes of gene expression in rat neuron. Differentially expressed genes (Egr-1, Mbp and Plp) were further confirmed by semi-quantitative revere transcription polymerase chain reaction (RT PCR). The expression levels of Egr-1, Mbp and Plp were observed at different exposure times (6, 24 h) and modes (intermittent and continuous exposure). Among 1200 candidate genes, 24 up-regulated and 10 down-regulated genes were found by using Affymetrix microarray suite software 5.0 which are associated with multiple cellular functions (cytoskeleton, signal transduction pathway, metabolism, etc.) after functional classification. Under 24 h and 6 h intermittent exposure, Egr-1 and Plp in experiment groups showed statistic significance (P < 0.05) compared with the control groups, while expression of Mbp did not change significantly (P > 0.05). After 24 h continuous exposure, Egr-1 and Mbp in experiment groups showed statistic significance (P < 0.05) compared with the control group, while expression of Plp did not change significantly (P > 0.05). Under the same exposure mode 6 h, expression of all the 3 genes did not change significantly. Different times (6, 24 h) and modes (intermittent and continuous exposure) of exposure exerted remarkable different influences on the expression of Egr-1, Mbp, Plp genes (P < 0.01). The changes of many genes transcription were involved in the effect of 1.8 GHz RF EMF on rat neurons; Down-regulation of Egr-1 and up-regulation of Mbp, Plp indicated the negative effects of RF EMF on neurons; The effect of RF intermittent exposure on gene expression was more obvious than that of continuous exposure; The effect of 24 h RF exposure (both intermittent and continuous) on gene expression was more obvious than that of 6 h (both intermittent and continuous).

Analysis of gene expression patterns of hepatocyte‐like cells derived from human bone marrow mesenchymal stem cells

The aim of the study was to determine the degree of differentiation of hepatocyte‐like cells derived from human mesenchymal stem cells (hMSC) by comparing global gene expression patterns of differentiated and undiffentiated hMSC from bone marrow (hBM‐MSC), adult liver and fetal hepatocytes.hMSC were isolated from bone marrow and differentiated into hepatocyte‐like cells. Microarray analysis was performed using the HG‐U133A array from Affymetrix. For comparative analysis of expression patterns published datasets and results obtained from this study were combined. All datasets were analysed by Microarray Suite 5.0 and Genesis software.In comparison to undifferentiated hBM‐MSC, hepatocyte differentiated hBM‐MSC showed a dramatic change of the gene expression profile. We found approximately 1000 genes upregulated and 1400 genes were downregulated in differentiated MSC with a fold change ≥3. Among these, around 60% and 40% showed similar expression levels as in adult liver, respectively. Expression of glutamine synthetase and ribonuclease 4 is significantly upregulated in hepatocyte differentiated MSC, typical for adult hepatocytes in the perivenous area of the liver lobule. Thus, hepatocyte‐like cells derived from hBM‐MSC feature human hepatocyte gene expression patterns, which encourages to step forward to use hBM‐MSC as a novel source for cell therapy of liver diseases.

Multiple pathways are involved in drug resistance to doxorubicin in an osteosarcoma cell line

Drug resistance continues to be a stumbling block in achieving a better cure rate in several cancers, including osteosarcoma. To understand this, we developed a doxorubicin drug-resistant osteosarcoma cell line (143B-DR-DOX). This cell line had an IC50 of 75 micromol/l compared with the parental 143B cell line's IC50 of 0.4 micromol/l. Using a 22000 70-mer oligomicroarray, gene expression studies were performed in four replicates. Data analysis was done using the TIGR Microarray suite. Seventy-four genes were found to be either upregulated (21) or downregulated (53). Real time quantitative-PCR was done on 21 genes, which confirmed the gene expression data for 11 genes. Choosing the significant fold change criteria of greater than 2-fold upregulation or downregulation, four genes including multidrug resistance 1, interleukin-8, Krüppel-like factor 2 and MGC4175 were found to be upregulated and seven genes including epidermal growth factor receptor-coamplified and overexpressed protein, uridine phosphorylase 1, a disintegrin and metalloproteinase domain 19, cytochrome C1, SEC, S-adenosyl homocysteine hydrolase and p53 were found to be downregulated. The data suggest that apart from the known gene alterations in doxorubicin resistance (multidrug resistance 1, topoisomerase IIbeta), others can also contribute to the drug-resistance phenotype. The involvement of interleukin-8 and Krüppel-like factor 2 suggests that the peroxisome proliferator-activated receptors gamma pathway may also be involved in doxorubicin drug resistance in the 143B-DR-DOX cell line.