Functional Annotation Clustering Research Articles

Microarray Analysis of Equine Endometrium at Days 8 and 12 of Pregnancy1

Establishment and maintenance of pregnancy in equids is only partially understood. To provide new insights into early events of this process, we performed a systematic analysis of transcriptome changes in the endometrium at Days 8 and 12 of pregnancy. Endometrial biopsy samples from pregnant and nonpregnant stages were taken from the same mares. Composition of the collected biopsy samples was analyzed using quantitative stereological techniques to determine proportions of surface and glandular epithelium and blood vessels. Microarray analysis did not reveal detectable changes in gene expression at Day 8, whereas at Day 12 of pregnancy 374 differentially expressed genes were identified, 332 with higher and 42 with lower transcript levels in pregnant endometrium. Expression of selected genes was validated by quantitative real-time RT-PCR. Gene set enrichment analysis, functional annotation clustering, and cocitation analysis were performed to characterize the genes differentially expressed in Day 12 pregnant endometrium. Many known estrogen-induced genes and genes involved in regulation of estrogen signaling were found, but also genes known to be regulated by progesterone and prostaglandin E2. Additionally, differential expression of a number of genes related to angiogenesis and vascular remodeling suggests an important role of this process. Furthermore, genes that probably have conserved functions across species, such as CRYAB, ERRFI1, FGF9, IGFBP2, NR2F2, STC1, and TNFSF10, were identified. This study revealed the potential target genes and pathways of conceptus-derived estrogens, progesterone, and prostaglandin E2 in the equine endometrium probably involved in the early events of establishment and maintenance of pregnancy in the mare.

Effects of Salmonella on spatial-temporal processes of jejunal development in chickens

To study effects of Salmonella enteritidis on morphological and functional changes in chicken jejunal development, we analysed gene expression profiles at seven points post-infection in 1–21 day-old broiler chickens. Nine clusters with different gene expression patterns were identified, and the genes in each cluster were further analyzed by a functional annotation clustering method (DAVID). Functional and morphological developmental processes dominated in all the nine clusters. Salmonella infection caused delays in several intestinal-morphological processes, whereas functional metabolic processes occurred in a similar spatial-temporal frame compared to normal jejunum development. A clear difference between normal developing- and Salmonella disturbed jejunum was the higher expression of genes involved in cell turn-over at early stages in the infected jejunum. Surprisingly, we found no clustered immune related processes in the infected birds. To compare the immunological processes between control and Salmonella infected chickens, the gene expression data was superimposed on known immunological KEGG pathways. Furthermore an in-depth analysis on the immune gene level was performed. As expected, we did find immunological processes in the Salmonella infected jejunum. Several of these processes could be verified by immunohistochemistry measurements of different immunological cell types. However, the well-ordered spatial-temporal development of the immune system, as observed in control non-infected animals, was completely abolished in the infected animals. Several immunological processes started much earlier in time, whereas other processes are disorganised. These data indicate that normal morphological and immunological development of jejunum is changed dramatically by a disturbance due to Salmonella infection. Due to the disturbance, the well-organized spatial-temporal development of morphological processes are delayed, those of the immunological development are scattered, whereas metabolic functional processes are almost not affected. This demonstrates the flexibility of developmental processes in the broiler chicken intestine.

Axonal Transport Proteomics Reveals Mobilization of Translation Machinery to the Lesion Site in Injured Sciatic Nerve

Investigations of the molecular mechanisms underlying responses to nerve injury have highlighted the importance of axonal transport systems. To obtain a comprehensive view of the protein ensembles associated with axonal transport in injured axons, we analyzed the protein compositions of axoplasm concentrated at ligatures following crush injury of rat sciatic nerve. LC-MS/MS analyses of iTRAQ-labeled peptides from axoplasm distal and proximal to the ligation sites revealed protein ensembles transported in both anterograde and retrograde directions. Variability of replicates did not allow straightforward assignment of proteins to functional transport categories; hence, we performed principal component analysis and factor analysis with subsequent clustering to determine the most prominent injury-related transported proteins. This strategy circumvented experimental variability and allowed the extraction of biologically meaningful information from the quantitative neuroproteomics experiments. 299 proteins were highlighted by principal component analysis and factor analysis, 145 of which correlate with retrograde and 154 of which correlate with anterograde transport after injury. The analyses reveal extensive changes in both anterograde and retrograde transport proteomes in injured peripheral axons and emphasize the importance of RNA binding and translational machineries in the axonal response to injury.

Abstract 4608: Overexpression of PKCα alters gene expression pattern in T47D breast cancer cells associated with hormone-independent growth and tamoxifen resistance

Abstract Background: Tamoxifen (TAM) resistant breast cancer is a major obstacle in the clinical management of breast cancer and the mechanisms are not fully understood. Our lab has previously shown that overexpression of protein kinase C α (PKCα) in T47D breast cancer cells leads to a hormone-independent and TAM-resistant phenotype (Chisamore et. al., 2001, Clin. Cancer Res.7:3156-3165). Furthermore, we found that PKCα overexpression correlates with TAM-resistance and disease recurrence in patients (Tonetti et. al., 2003, Br. J. Cancer 88:1400-1402). Taken together, these findings suggest overexpression of PKCα is important in the progression of breast cancer to a hormone-independent and TAM-resistant phenotype. Methods: To determine the mechanism whereby overexpression of PKCα can mediate this progression, gene expression profiles of T47D: A18/neo and T47D: A18/PKCα cells following treatment with 0.1% EtOH or 10 nM 17β-estradiol (E2) for 24 hours were compared utilizing the Affymetrix Human Gene ST 1.0 Array. Total RNA was harvested for cRNA labeling and hybridization to Affymetrix ST 1.0 GeneChips. Data was analyzed using Partek Genomics statistical package (Partek, Inc). ANOVA test was used to calculate significance of the differential expression. Gene Ontology (GO) term enrichment was carried out using the Functional Annotation Clustering tool in DAVID Bioinformatics Database. Results: The basal expression of more than one thousand genes are differently expressed in T47D cells just by overexpressing PKCα, including genes related to cell cycle progression, gene expression regulation, protein transport and localization and metabolic processes. Interestingly, many of the E2-regulated genes in T47D: A18/neo cells have similar expression patterns in T47D: A18/PKCα cells in the absence of E2 treatment. Thirty six genes are up regulated by E2 in T47D: A18/neo cells and they have higher basal expression levels in T47D: A18/PKCα cells compared with T47D: A18/neo cells, including TFF1, CXCL12, STC2 and MYC. On the other hand, seventy six genes are down regulated by E2 in T47D: A18/neo cells that also have lower basal expression levels in the PKCα-overexpressing cells compared to T47D: A18/neo, including ERBB2, TGFB2 and TGFB3. Furthermore, E2 does not have a great influence on the gene expression levels in T47D: A18/PKCα cells. Conclusion: These results suggest that PKCα overexpression may lead to estrogen-independent activation of ER target genes and result in a pattern of gene expression similar to that in T47D cells treated with E2. This altered activation of ER by PKCα may lead to a gene expression pattern that mediates the progression to the estrogen-independent growth and TAM resistance. Pharmacological inhibition of this progression by inhibiting PKCα may offer treatment options for TAM-resistant breast cancer. 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 4608.

Abstract 4924: Gene-specific hyper and hypomethylation in follicular lymphoma progression

Abstract Epigenetic mechanisms involved in the origin and transformation of indolent lymphomas to aggressive forms are poorly understood. Based on the comparison of methylation states of follicular lymphoma (FL) pre and post- transformation, it has been suggested that DNA methylation changes occur during the early stages of lymphomagenesis. To obtain a more complete understanding of methylation aberrations responsible for progression and transformation of FL, we analyzed 20 untreated FL biopsies (10 low grade, 10 high grade), 5 diffuse large B cell lymphoma (DLBCL) biopsies, and 5 benign reactive lymph node (RLN) biopsies by using methyl-DNA immunoprecipitation coupled with analysis with Agilent 244K human promoter ChIP-on-chip microarrays. Our analysis revealed statistically significant differences in methylation status for 120 genes between FL samples and RLN samples and for 23 genes between low and high grade FL samples. To investigate the biological significance of these findings, we used DAVID functional annotation clustering algorithms and Gominer software. We found that the most methylation-enriched functional groups were comprised of genes with roles in cell cycle control, adhesion and motility, transcription regulation, homeobox, and developmental genes. In order to validate the DNA methylation differences of the significant gene regions, we utilized the MassARRAY EpiTYPER assay. Our data identified 13 genes (CYP26B1, GRM7, HS3ST2, DOCK5, GRB10, SST, TAC1, PALM2-AKAP, FLJ40125, ST6GAL2, WDR35, NCAM1, SATB2) with increased levels of DNA methylation in FL samples compared to controls; 3 of these genes (ST6GAL2, WDR35, NCAM1) exhibited increased methylation in high grades FLs and 1 gene (SATB2) in low grades FLs. We are currently investigating gene specific DNA hypomethylation in low versus high grade FL and FL versus DLBCL. Our data are the first to characterize the global changes in methylation that occur in FL progression. Our findings suggest that high grade FL are associated with hypermethylation of a specific subset of genes compared to low grade subtypes. Ongoing efforts include investigating the functional relevance of these methylation changes by treating, in vitro, FL cell lines with demethylating agents to evaluate changes in gene expression levels and correlating with phenotype alterations. 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 4924.

Identification of Methylmercury Tolerance Gene Candidates in Drosophila

Methylmercury (MeHg) is a ubiquitous environmental contaminant that preferentially targets the developing nervous system. Variable outcomes of prenatal MeHg exposure within a population point to a genetic component that regulates MeHg toxicity. We therefore sought to identify fundamental MeHg tolerance genes using the Drosophila model for genetic and molecular dissection of a MeHg tolerance trait. We observe autosomal dominance in a MeHg tolerance trait (development on MeHg food) in both wild-derived and laboratory-selected MeHg-tolerant strains of flies. We performed whole-genome transcript profiling of larval brains of tolerant (laboratory selected) and nontolerant (control) strains in the presence and absence of MeHg stress. Pairwise transcriptome comparisons of four conditions (+/-selection and +/-MeHg) identified a "down-down-up" expression signature, whereby MeHg alone and selection alone resulted in a greater number of downregulated transcripts, and the combination of selection + MeHg resulted in a greater number of upregulated transcripts. Functional annotation cluster analyses showed enrichment for monooxygenases/oxidoreductases, which include cytochrome P450 (CYP) family members. Among the 10 CYPs upregulated with selection + MeHg in tolerant strains, CYP6g1, previously identified as the dichlorodiphenyl trichloroethane resistance allele in flies, was the most highly expressed and responsive to MeHg. Among all the genes, Turandot A (TotA), an immune pathway-regulated humoral response gene, showed the greatest upregulation with selection + MeHg. Neural-specific transgenic overexpression of TotA enhanced MeHg tolerance during pupal development. Identification of TotA and CYP genes as MeHg tolerance genes is an inroad to investigating the conserved function of immune signaling and phase I metabolism pathways in MeHg toxicity and tolerance in higher organisms.

317 EFFECT OF ATORVASTATIN ON THE BIOLOGY OF BLADDER CANCER CELLS TREATED WITH BACILLUS CALMETTE-GUERIN

You have accessJournal of UrologyUrothelial Cancer: Medical & Surgical Therapy1 Apr 2010317 EFFECT OF ATORVASTATIN ON THE BIOLOGY OF BLADDER CANCER CELLS TREATED WITH BACILLUS CALMETTE-GUERIN Tullika Garg, Guangjian Zhang, Fanghong Chen, Yanli Cao, and William See Tullika GargTullika Garg More articles by this author , Guangjian ZhangGuangjian Zhang More articles by this author , Fanghong ChenFanghong Chen More articles by this author , Yanli CaoYanli Cao More articles by this author , and William SeeWilliam See More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.381AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The commonly employed class of cholesterol lowering agents, statins, functions by inhibiting the HMG-CoA reductase enzyme which is critical to cholesterol biosynthesis. Early clinical reports suggest that BCG efficacy may be decreased in patients taking statins. This study examined the effect of BCG on enzymes in the cholesterol synthesis pathway, the impact of statins on the BCG induced signaling pathways and gene expression, as well as the impact of statins on the in vitro cytotoxicity of BCG on human urothelial carcinoma (UC) cell lines. METHODS The human UC cell lines 253J and T24 were exposed to BCG and analyzed for upregulation of enzymes in the cholesterol biosynthesis pathway using Affymetrix gene expression profiling. The effect of atorvastatin on NFkB signaling and downstream gene expression, alone and in combination with BCG, was compared to non-treated and BCG exposed controls. The two cell lines were utilized to determine the effect of a physiologic concentration of atorvastatin on BCG associated cytotoxicity. RESULTS Analysis of gene expression using functional annotation clustering demonstrated highly significant enrichment of steroid biosynthetic genes in 253J cells following BCG treatment (p = 1.4E-14). While noted, up-regulation of these genes was not significant in T24 cells (p = 0.3). Compared to both BCG exposed and untreated controls, the most pronounced effect of atorvastatin treatment was decreased expression of genes involved in cell cycle regulation (p < 1.0e-40). This effect was independent of whether atorvastatin was used along or in combination with BCG. Atorvastatin significantly increased BCG induced signaling through the NFkB pathway (p<0.001). Parodoxically, atorvastatin significantly decreased the downstream expression of a panel of NFkB dependent, BCG induced genes as measured by quantitative rtPCR (IL-6, IL-8, CCL20, CXC1, P<0.05). Exposure to BCG resulted in decreased proliferation of UC cells in both T24 and 253J cells lines. The addition of atorvastatin to BCG treated cells resulted in significantly decreased BCG cytotoxicity as compared to the BCG alone groups (ANOVA p = 0.001 and p = 0.003 for 253J and T24 respectively). CONCLUSIONS Physiologic concentrations of atorvastatin exert a direct effect on the biology of urothelial carcinoma cells. In the context of BCG exposure, atorvastatin alters BCG induced signaling, inflammatory gene expression, and cytotoxcity. Given a clear in-vitro effect clinicians should consider short term cessation of statins in patients failing to respond to an induction course of BCG. Milwaukee, WI© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e125 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tullika Garg More articles by this author Guangjian Zhang More articles by this author Fanghong Chen More articles by this author Yanli Cao More articles by this author William See More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Epigenetic Placental Programming of Preeclampsia

Preeclampsia (PE) affects 8–10% of women in the US and long‐term consequences include development of maternal hypertension and hypertension in offspring. As methylation patterns are established during fetal life, we focused on epigenetic alterations as a plausible explanation of the heritable development of hypertension resulting from maternal PE. In this proof‐of‐principle study we determined genome‐wide CpG DNA methylation patterns in placental tissue from women with normotensive pregnancy and PE (n=3/group) by using 2.1M microarrays (NimbleGen). These arrays tile 7000 bp upstream and 3000 bp downstream a promoter in 100 bp segments and cover 28000 CpG islands. Criteria for gene selection included methylation +/−1000 bp of the respective gene's transcription start site. We identified 163 genes methylated only in PE and uploaded them to DAVID (http://david.abcc.ncifcrf.gov) where we used a classification stringency of high to determine functional annotation clusters. Cluster 1had an enrichment score of 5.59; this cluster contains the GO Terms of anatomical structure formation, angiogenesis, blood vessel development, vasculature development, and blood vessel morphogenesis. There are 12 genes total in this cluster 10 of which are common to all 5 GO Terms. This work suggests that an epigenetic mechanism may be involved in PE.Robert Wood Johnson Nurse Faculty Scholar, Sigma Theta Tau International, ARS

Machine learning approach identifies new pathways associated with demyelination in a viral model of multiple sclerosis

Theiler’s murine encephalomyelitis is an experimentally virus-induced inflammatory demyelinating disease of the spinal cord, displaying clinical and pathological similarities to chronic progressive multiple sclerosis. The aim of this study was to identify pathways associated with chronic demyelination using an assumption-free combined microarray and immunohistology approach. Movement control as determined by rotarod assay significantly worsened in Theiler’s murine encephalomyelitis -virus-infected SJL/J mice from 42 to 196 days after infection (dpi). In the spinal cords, inflammatory changes were detected 14 to 196 dpi, and demyelination progressively increased from 42 to 196 dpi. Microarray analysis revealed 1001 differentially expressed genes over the study period. The dominating changes as revealed by k-means and functional annotation clustering included up-regulations related to intrathecal antibody production and antigen processing and presentation via major histocompatibility class II molecules. A random forest machine learning algorithm revealed that down-regulated lipid and cholesterol biosynthesis, differentially expressed neurite morphogenesis and up-regulated toll-like receptor-4-induced pathways were intimately associated with demyelination as measured by immunohistology. Conclusively, although transcriptional changes were dominated by the adaptive immune response, the main pathways associated with demyelination included up-regulation of toll-like receptor 4 and down-regulation of cholesterol biosynthesis. Cholesterol biosynthesis is a rate limiting step of myelination and its down-regulation is suggested to be involved in chronic demyelination by an inhibition of remyelination.

Screening for Beneficial Effects of Oral Intake of Sweet Corn by DNA Microarray Analysis

To identify novel functions of the oral intake of sweet corn, we performed DNA microarray analysis of the livers of sweet corn-fed mice. Functional annotation clustering 1600 genes with expression levels that were affected (more than 1.5-fold change) by dietary sweet corn indicated that both cell proliferation and programmed cell death were modulated by sweet corn intake. In the Wnt signaling pathway, which is involved in cell proliferation, the levels of Jun and beta-catenin expression were downregulated by dietary sweet corn. The mRNA levels of Rb and p53, negative regulators of the cell cycle, were increased in mice fed with sweet corn. Dietary corn upregulated expression levels of genes that regulate apoptosis positively (for example, BOK, BID, CASP4). These results suggested that sweet corn is a valuable food for suppressing cancer. Oral administration of sweet corn inhibited tumor growth (36.6% reduce in tumor weight, P < 0.05) in mice inoculated with Ehrlich tumor cells.

Insights into Mesenchymal Stem Cell Aging: Involvement of Antioxidant Defense and Actin Cytoskeleton

Progenitor cells such as mesenchymal stem cells (MSCs) have elicited great hopes for therapeutic augmentation of physiological regeneration processes, e.g., for bone fracture healing. However, regeneration potential decreases with age, which raises questions about the efficiency of autologous approaches in elderly patients. To elucidate the mechanisms and cellular consequences of aging, the functional and proteomic changes in MSCs derived from young and old Sprague-Dawley rats were studied concurrently. We demonstrate not only that MSC concentration in bone marrow declines with age but also that their function is altered, especially their migratory capacity and susceptibility toward senescence. High-resolution two-dimensional electrophoresis of the MSC proteome, under conditions of in vitro self-renewal as well as osteogenic stimulation, identified several age-dependent proteins, including members of the calponin protein family as well as galectin-3. Functional annotation clustering revealed that age-affected molecular functions are associated with cytoskeleton organization and antioxidant defense. These proteome screening results are supported by lower actin turnover and diminished antioxidant power in aged MSCs, respectively. Thus, we postulate two main reasons for the compromised cellular function of aged MSCs: (a) declined responsiveness to biological and mechanical signals due to a less dynamic actin cytoskeleton and (b) increased oxidative stress exposure favoring macromolecular damage and senescence. These results, along with the observed similar differentiation potentials, imply that MSC-based therapeutic approaches for the elderly should focus on attracting the cells to the site of injury and oxidative stress protection, rather than merely stimulating differentiation.

Genome-wide innate immune responses in HIV-1-infected macrophages are preserved despite attenuation of the NF-kappa B activation pathway.

Macrophages contribute to HIV-1 infection at many levels. They provide permissive cells at the site of inoculation, augment virus transfer to T cells, generate long-lived viral reservoirs, and cause bystander cell apoptosis. A body of evidence suggests that the role of macrophages in cellular host defense is also compromised by HIV-1 infection. In this respect, macrophages are potent cells of the innate immune system that initiate and regulate wide-ranging immunological responses. This study focuses on the effect of HIV-1 infection on innate immune responses by macrophages at the level of signal transduction, whole genome transcriptional profiling, and cytokine secretion. We show that in an ex vivo model, M-CSF-differentiated monocyte-derived macrophages uniformly infected with replicating CCR5-tropic HIV-1, without cytopathic effect, exhibit selective attenuation of the NF-kappaB activation pathway in response to TLR4 and TLR2 stimulation. However, functional annotation clustering analysis of genome-wide transcriptional responses to LPS stimulation suggests substantial preservation of gene expression changes at the systems level, with modest attenuation of a subset of up-regulated LPS-responsive genes, and no effect on a selection of inflammatory cytokine responses at the protein level. These results extend existing reports of inhibitory interactions between HIV-1 accessory proteins and NF-kappaB signaling pathways, and whole genome expression profiling provides comprehensive assessment of the consequent effects on immune response gene expression. Unexpectedly, our data suggest innate immune responses are broadly preserved with limited exceptions, and pave the way for further study of the complex relationship between HIV-1 and immunological pathways within macrophages.

Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources

DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.

Genome-Wide Profiling of Endothelial Progenitor Cells in Multiple Myeloma: Disease-Relevant Pathways and Overlaps with Common Cancer Biomarkers

Background: The current study is based on recent findings from our and other laboratories demonstrating endothelial progenitor cells (EPCs) in multiple myeloma (MM) to be a key component of the tumor microenvironment, integral to the neoplastic process. EPCs contribute to tumor neoangiogenesis, and their levels covary with MM progression. Furthermore, MM EPCs are genetically unstable, as evidenced by restricted X-chromosome inactivation patterns; by immunoglobulin rearrangements identical to those harbored by tumor cells; and by a range of shared chromosomal gains or losses with tumor cells, shown by array comparative genomic hybridization (aCGH). The present study further characterized the EPC genomic profile in MM by comparison of EPC gene expression with that of tumor cells and control ECs using a bioinformatics approach that integrated cancer gene databases to prioritize key molecular MM biomarkers.Methods: EPCs (> 98% vWF/CD133/KDR+/CD38–) from bone marrow aspirates of 22 untreated MM patients were outgrown on laminin-coated flasks. The fractions enriched for tumor cells were > 50% CD38+. For expression profiling, total RNA from EPCs, MM cells, and control ECs (EPCs and HUVECs) was hybridized to Affymetrix U133 Plus 2.0 arrays, and comparisons were made by ANOVA with corrections for multiple comparisons to achieve a false discovery rate < 5%. Functional enrichment of gene ontology (GO) categories was performed using DAVID (NIAID) and Ingenuity Systems software.Results: A total of 334 genes were differentially expressed in MM EPCs versus control ECs (> 1.5-fold difference and P <. 05). Of these, 81% were over-expressed in MM EPC. Functional annotation clustering of all 334 genes into GO categories by DAVID focused similar annotations together into 16 significant clusters (P < .01). This analysis revealed top biological clusters to be differentiation and development (P = 8×10−8, 159 genes); extracellular matrix adhesion (P = 2×109, 110 genes); bone formation (P = 4×10−5, 33 genes); and angiogenesis (P = 8×10−5, 35 genes). From these clusters, an expression profile was obtained consisting of the strongest 21 differentially expressed genes (versus control ECs; P < 1×10−5) characterized by highest expression of 4 genes associated with metastasis and tumor growth: COL1A1, LUM, CYP1B1, and GREM1. RT-PCR validation studies of these genes are ongoing. Moreover, there was a 34% greater overlap of MM EPC and tumor profiles versus control ECs and tumor profiles (P < .001). In comparing the 334-gene set to the tumor cell profile, 2 important EPC gene sets emerged. The first set included 158 MM EPC genes differing significantly from control ECs, but similar to tumor (Profile 1). We hypothesize that this profile is a consequence of the clonal identity previously reported between MM EPCs and tumor, and that these genes contribute to MM progression. The second profile included 39 EPC genes that differed significantly both from control ECs and tumor cells (Profile 2). This gene profile may confer a predisposition to clonal transformation of EPCs. When genes in both profiles were compared with published databases of cancer biomarkers (Campagne et al., BMC Bioinformatics 2006), significant overlaps (P < .05) were found. The largest similarities were observed with the HM200 gene list which comprises 200 genes most consistently differentially expressed in human/mouse cancers, and containing potentially key genes previously undescribed in MM, including FZD7, TWIST1, and CARD8. Current studies are aimed at integrating genes in EPC Profiles 1 and 2 with chromosomal copy number abnormalities found in EPCs, and with MM progression and survival.Conclusions: Our results strongly indicate that EPCs are an integral part of the neoplastic process in MM. Their altered genomic profile compared to control ECs indicates pathogenic functions critical for MM evolution, including angiogenesis, adhesion, bone turnover, and cell differentiation. The high degree of commonly expressed EPC genes compared with MM and other tumors permits prioritization of candidate MM-endothelial biomarkers not yet defined in this disease.

Regulation of the Hepatic Transcriptome by Estradiol and Progesterone in Cows.

The steroid hormones, estradiol (E2) and progesterone (P4), regulate reproductive function and gene expression in many tissues. These steroids are primarily metabolized in the liver where they can also regulate gene expression. Previous studies in a variety of tissues have reported antagonism by P4 of E2 actions; although in some circumstances E2 and P4 act synergistically. The study was designed to evaluate the effects of E2 and/or P4 on gene expression of the bovine liver using the microarray technique. Methods: A 4X4 Latin Square design was used with ovariectomized Holstein cows randomly assigned to 4 treatment groups (n≡4): 1) No hormone supplementation; 2) E2 treatment (ear implant delivering physiological E2 concentrations); 3) P4 treatment (2 intravaginal progesterone-releasing devices producing ≈ 3.5 ng/ml); 4) E2+P4. After 14 d of treatment, liver biopsies were collected and frozen. To avoid carry-over effects, there was a 28 d interval between periods. The mRNA concentrations in the liver biopsies were evaluated on bovine-specific arrays from Affymetrix (Bovine GeneChip Genome Array) that are claimed to represent over 23,000 transcripts (trp). Signal values on perfect-match and mismatch probes of each trp were combined into expression indexes using the MAS5.0 algorithm, and the Quantile procedure was used to normalize the data. The MIXED procedure of SAS was used to test for the main effects of E2, P4 and their interaction, followed by an analysis of overrepresented categories of differentially expressed genes using the DAVID functional annotation clustering analysis. Results: Treatment with E2 caused significant differential expression of 2841 trp: 1208 upregulated, 1633 downregulated. E2-induced upregulation was inhibited by P4 for 87 (7.2%) of these trp; whereas, E2-induced downregulation was inhibited by P4 for 203 trp (12.4%). Treatment with P4 caused significant differential expression of 2512 trp: 1100 upregulated, 1415 downregulated. P4-induced upregulation or downregulation was reversed by E2 for almost none of the trp (3 or 5 trp). Interestingly, most of the trp were upregulated by both P4 and E2 (683 trp) or downregulated by both E2 and P4 (759 trp). For some of these genes there was a synergistic effect when E2+P4 were combined (193 [13.6%] of downregulated trp; 61 [5.5%] of upregulated trp). Downregulated trp were surprisingly enriched with genes encoding SRC homology-3 domain proteins for all treatments. Those downregulated trp were also enriched in intracellular protein transport and calcium-binding proteins. In addition, E2-down-regulated trp were enriched in general receptor, signaling, and transport functions; whereas, P4-downregulated trp were clustered in EGF-related pathways. For all treatments, upregulated trp were enriched in proteins related to mitochondria processes (electron transport, ATP, cellular metabolism), chaperone and ubiquitin-protein ligase activity and NAD/oxidoreductase. Conclusions: Treatment for 14d with E2, P4, or E2+P4 regulated numerous hepatic genes with more downregulated than upregulated trp. There was some antagonism of E2-regulated genes by P4 but not P4-regulated genes by E2. One of the most remarkable findings was the overlap between the genes regulated by E2 and P4 with >50% of the genes found to be regulated by both steroids. This finding explains the similarities in the clustering results. Supported by CAPES fellowship-Brazil and Pfizer Global Academic Alliance.

Distinct genomic signatures of adaptation in pre- and postnatal environments during human evolution

The human genome evolution project seeks to reveal the genetic underpinnings of key phenotypic features that are distinctive of humans, such as a greatly enlarged cerebral cortex, slow development, and long life spans. This project has focused predominantly on genotypic changes during the 6-million-year descent from the last common ancestor (LCA) of humans and chimpanzees. Here, we argue that adaptive genotypic changes during earlier periods of evolutionary history also helped shape the distinctive human phenotype. Using comparative genome sequence data from 10 vertebrate species, we find a signature of human ancestry-specific adaptive evolution in 1,240 genes during their descent from the LCA with rodents. We also find that the signature of adaptive evolution is significantly different for highly expressed genes in human fetal and adult-stage tissues. Functional annotation clustering shows that on the ape stem lineage, an especially evident adaptively evolved biological pathway contains genes that function in mitochondria, are crucially involved in aerobic energy production, and are highly expressed in two energy-demanding tissues, heart and brain. Also, on this ape stem lineage, there was adaptive evolution among genes associated with human autoimmune and aging-related diseases. During more recent human descent, the adaptively evolving, highly expressed genes in fetal brain are involved in mediating neuronal connectivity. Comparing adaptively evolving genes from pre- and postnatal-stage tissues suggests that different selective pressures act on the development vs. the maintenance of the human phenotype.

Identification of differentially expressed proteins in spontaneous thymic lymphomas from knockout mice with deletion of p53

BackgroundKnockout mice with a deletion of p53 spontaneously develop thymic lymphomas. Two cell lines (SM5 and SM7), established from two independent tumours, exhibited about fifty to seventy two-fold differentially expressed proteins compared to wild type thymocytes by two-dimensional gel electrophoresis (2D-PAGE).ResultsProtein spots excised from 2D-PAGE gels, were subjected to in-gel tryptic digestion and identified by liquid chromatography – tandem mass spectrometry. A total of 47 protein spots were identified. Immunological verification was performed for several of the differentially regulated proteins where suitable antibodies could be obtained. Functional annotation clustering revealed similarities as well as differences between the tumours. Twelve proteins that changed similarly in both tumours included up-regulation of rho GDP-dissociation inhibitor 2, proteasome subunit α type 3, transforming acidic coiled-coil containing protein 3, mitochondrial ornithine aminotransferase and epidermal fatty acid binding protein and down-regulation of adenylosuccinate synthetase, tubulin β-3 chain, a 25 kDa actin fragment, proteasome subunit β type 9, cofilin-1 and glia maturation factor γ.ConclusionSome of the commonly differentially expressed proteins are also differentially expressed in other tumours and may be putative diagnostic and/or prognostic markers for lymphomas.

A Transcriptomal Profile for Predicting Complete Cytogenetic Response (CCR) in Chronic Phase CML Patients Treated with Imatinib.

Background: Patients with chronic phase CML who achieve a complete cytogenetic response (CCR) have a low risk of disease progression. Since patients unlikely to achieve CCR may benefit from more aggressive therapy up-front, prediction of response prior to therapy would be useful. Our previous work has demonstrated that the gene expression profiles of unselected leukemia cells from prospective cytogenetic responders and non-responders are similar (Crossman et al. Haematologica 90(4):459–64, 2005). Based on the hypothesis that cytogenetic refractoriness may be a property of leukemic progenitor rather than differentiated cells, we explored gene expression profiling of CD34+ cells as a tool for predicting CCR.Methods: Two independent data sets were generated. The first data set (learning set) was based on patients with CML who had either achieved a CCR within 1 year of imatinib therapy (R, n = 24), or remained at least 65% Ph+ (NR, n = 12). The prospectively collected, completely independent validation data set was based on 23 additional subjects using the same criteria (17 R and 6 NR). For the learning set CD34+ cells were isolated by FACS from bone marrow or peripheral mononuclear cells cryopreserved prior to imatinib therapy. For the validation set CD34+ were isolated from fresh cells using immunomagnetic columns. RNA was extracted and samples with ≥5ng of high quality total RNA were amplified and labelled with the Affymetrix two cycle cDNA synthesis and IVT labeling protocol using <20ng input RNA; 10μg of labelled target cRNA were hybridized to Affymetrix HG-U133 Plus 2.0 GeneChip® arrays. For both data sets, low-level analysis was done using Robust MultiArray Average (RMA) and gene-by-gene ANOVA was performed to determine differential expression between NR and R. Both statistical significance and effect size used to filter the ANOVA results. Parameters for the classification algorithms were chosen by nested cross-validation procedures. The classifier generated from the first study was then applied to the blinded validation set. Functional annotation clustering and over-representation analysis was also performed.Results: On the validation set, the classifier had an estimated accuracy rate of 85.7%. Based on transcript annotation analysis, there is significant over-representation of trancripts related to cell cycle, complement and coagulation, and apoptosis, among others. A functional cluster was identified related to regulation of transcription. Transcription factors up-regulated in cells from patients with subsequent cytogenetic refractoriness included ZNF168, MEIS1, KLF2, NFIB, MAF, FOSB and EGR1. In contrast, we found down-regulation of SERPINA1, THBD and PLAUR, a sub-network in the complement and coagulation cascades pathway that is associated with cell adhesion and migration.Conclusions:1.The transcriptional profiles of CD34+ cell from prospective cytogenetic responders and non-responders are distinct.2.A classifier based on the learning set predicted cytogenetic response in a totally independent validation set with ∼86% accuracy, making this one of the first prospectively validated classifiers.3.Examination of functional annotation for the transcripts in the classifier identified several functional clusters that are highly correlated with respect to direction of response (e.g. transcription factors) and may drive the biology of cytogenetic refractoriness.

OCPAT: an online codon-preserved alignment tool for evolutionary genomic analysis of protein coding sequences.

BackgroundRapidly accumulating genome sequence data from multiple species offer powerful opportunities for the detection of DNA sequence evolution. Phylogenetic tree construction and codon-based tests for natural selection are the prevailing tools used to detect functionally important evolutionary change in protein coding sequences. These analyses often require multiple DNA sequence alignments that maintain the correct reading frame for each collection of putative orthologous sequences. Since this feature is not available in most alignment tools, codon reading frames often must be checked manually before evolutionary analyses can commence.ResultsHere we report an online codon-preserved alignment tool (OCPAT) that generates multiple sequence alignments automatically from the coding sequences of any list of human gene IDs and their putative orthologs from genomes of other vertebrate tetrapods. OCPAT is programmed to extract putative orthologous genes from genomes and to align the orthologs with the reading frame maintained in all species. OCPAT also optimizes the alignment by trimming the most variable alignment regions at the 5' and 3' ends of each gene. The resulting output of alignments is returned in several formats, which facilitates further molecular evolutionary analyses by appropriate available software. Alignments are generally robust and reliable, retaining the correct reading frame. The tool can serve as the first step for comparative genomic analyses of protein-coding gene sequences including phylogenetic tree reconstruction and detection of natural selection. We aligned 20,658 human RefSeq mRNAs using OCPAT. Most alignments are missing sequence(s) from at least one species; however, functional annotation clustering of the ~1700 transcripts that were alignable to all species shows that genes involved in multi-subunit protein complexes are highly conserved.ConclusionThe OCPAT program facilitates large-scale evolutionary and phylogenetic analyses of entire biological processes, pathways, and diseases.