Set Of Novel Genes Research Articles

New Levels of Transcriptome Complexity at Upper Thermal Limits in WildDrosophilaRevealed by Exon Expression Analysis

While the cellular heat-shock response has been a paradigm for studying the impact of thermal stress on RNA metabolism and gene expression, the genome-wide response to thermal stress and its connection to physiological stress resistance remain largely unexplored. Here, we address this issue using an array-based exon expression analysis to interrogate the transcriptome in recently established Drosophila melanogaster stocks during severe thermal stress and recovery. We first demonstrated the efficacy of exon-level analyses to reveal a level of thermally induced transcriptome complexity extending well beyond gene-level analyses. Next, we showed that the upper range of both the cellular and physiological thermal stress response profoundly affected message expression and processing in D. melanogaster, limiting expression to a small subset of transcripts, many that share features of known rapidly responding stress genes. As predicted from cellular heat-shock research, constitutive splicing was blocked in a set of novel genes; we did not detect changes to alternative splicing during heat stress, but rather induction of intronless isoforms of known heat-responsive genes. We observed transcriptome plasticity in the form of differential isoform expression during recovery from heat shock, mediated by multiple mechanisms including alternative transcription and alternative splicing. This affected genes involved in DNA regulation, immune response, and thermotolerance. These patterns highlight the complex nature of innate transcriptome responses under stress and potential for adaptive shifts through plasticity and evolved genetic responses at different hierarchical levels.

Targeting HRASV12G Expression to the Zebrafish Early Hemogenic Progenitors Induces a Myeloproliferative Disorder by Repressing the Notch Pathway

AbstractAbstract 4676 IntroductionMyeloproliferative diseases (MPDs) are a group of haematological disorders characterized by the hyper proliferation of different blood cells in peripheral blood and other hematopoietic organs. The clinical heterogenity of these neoplasms reflects the different gene pathways involved, most of which are only partially known. Given the genetic homology and the physiological similarity to mammals, zebrafish has emerged as an ideal model to study human normal and malignant haematopoiesis. In the last decade several oncogenes involved in the development of hematopoietic neoplasms have been used to model leukemia in zebrafish with the aim to discovery new molecular pathways involved in malignant transformation. Despite the first encouraging results these experimental models failed to fully recapitulate human myeloproliferative disorders. MethodsWe took advantage of the Gal4/UAS binary system to induce the expression of human oncogenic HRASV12G in the zebrafish hematopoietic compartment. We used a specific transgenic line that drives oncogene expression in zebrafish early hematopoietic progenitors under control of the FLI.1 (Friend Leukemia virus Integration 1) promoter. ResultsWe observed the development of a myelo-erythroid proliferative disease in few days in zebrafish transgenic larva. The pathological phenotype is characterized by the expansion of the hematopoietic tissue, an increased expression of myelo-erythroid specific genes (PU.1, gata1, mpx, c-mpl) associated with a slight increase of staminality markers (lmo2, scl, c-myb, runx.1), and a higher number of l-plastin expressing cells. Moreover blood smear of pathological larva displayed leukemic blasts and the arrest of erythrocyte differentiation whereas kidney marrow of juvenile fish displayed abnormal myelopoiesis characterized by the increase of erythro-myeloid progenitors.We found that the pathological phenotype is associated with a down regulation of the Notch pathway as shown by the decreased gene expression of notch pathways target genes (notch1, notch3, her6). Furthermore we discovered a novel set of genes involved in neoplastic transformation induced by HRASV12 expression through RNA-Seq analysis of pathological larva. ConclusionsThe expansion of the zebrafish hematopoietic compartment characterized by the hyper-proliferation of the myelo-erythroid progenitors that we found in this model reproduces some of the pathological features of human myeloproliferative disorders. This study showed that forcing oncogene expression in the hemogenic endothelial cells induces the transdifferentiation of the early hemogenic pluripotent stem cells into abnormal myeloerythoid progenitors by repressing the Notch pathways. Transcriptome analysis identified a number of potential effectors of this transformation. Disclosures:No relevant conflicts of interest to declare.

Identification of a novel set of genes reflecting different in vivo invasive patterns of human GBM cells.

BackgroundMost patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting.We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type).MethodsWe determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis.ResultsHere, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion.ConclusionsThis study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting.Massimiliano Monticone and Antonio Daga contributed equally to this work

Activin and BMP4 Synergistically Promote Formation of Definitive Endoderm in Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) herald tremendous promise for the production of clinically useful cell types for the treatment of injury and disease. Numerous reports demonstrate their differentiation into definitive endoderm (DE) cells, the germ layer from which pancreatic β cells and hepatocytes arise, solely from exposure to a high dose of recombinant Activin/Nodal. We show that combining a second related ligand, BMP4, in combination with Activin A yields 15%-20% more DE as compared with Activin A alone. The addition of recombinant BMP4 accelerates the downregulation of pluripotency genes, particularly SOX2, and results in upregulation of endogenous BMP2 and BMP4, which in turn leads to elevated levels of phospho-SMAD1/5/8. Combined Activin A and BMP4 treatment also leads to an increase in the expression of DE genes CXCR4, SOX17, and FOXA2 when compared with Activin A addition alone. Comparative microarray studies between DE cells harvested on day 3 of differentiation further reveal a novel set of genes upregulated in response to initial BMP4 exposure. Several of these, including APLNR, LRIG3, MCC, LEPREL1, ROR2, and LZTS1, are expressed in the mouse primitive streak, the site of DE formation. Thus, this synergism between Activin A and BMP4 during the in vitro differentiation of hESC into DE suggests a complex interplay between BMP and Activin/Nodal signaling during the in vivo allocation and expansion of the endoderm lineage.

DNA methylation regulates lineage-specifying genes in primary lymphatic and blood endothelial cells

During embryonic development, the lymphatic system emerges by transdifferentiation from the cardinal vein. Although lymphatic and blood vasculature share a close molecular and developmental relationship, they display distinct features and functions. However, even after terminal differentiation, transitions between blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) have been reported. Since phenotypic plasticity and cellular differentiation processes frequently involve epigenetic mechanisms, we hypothesized that DNA methylation might play a role in regulating cell type-specific expression in endothelial cells. By analyzing global gene expression and methylation patterns of primary human dermal LEC and BEC, we identified a highly significant set of genes, which were differentially methylated and expressed. Pathway analyses of the differentially methylated and upregulated genes in LEC revealed involvement in developmental and transdifferentiation processes. We further identified a set of novel genes, which might be implicated in regulating BEC-LEC plasticity and could serve as therapeutic targets and/or biomarkers in vascular diseases associated with alterations in the endothelial phenotype.

Oncogene Cooperativity Analysis Reveals a Novel Set of Genes That Regulate the In Vivo Growth and Survival of Leukemia Stem Cells

Abstract 553In order to increase our understanding of key biological properties governing the development of leukemia stem cells (LSCs), we employed a novel gene identification strategy based on cooperation between initiating oncogenes. Previous studies have demonstrated that genes whose expression is regulated in a synergistic manner as a consequence of two cooperating oncogenes (termed “cooperativity response genes”, or CRGs) are highly enriched for activity in tumor formation. Further, in contrast to the thousands of genes identified by differential expression analyses of normal vs. leukemic cell populations, CRGs represent a much smaller subset of targets; thereby, providing a defined set of genes to investigate. We adapted the CRG strategy to identify synergistically regulated genes in primitive leukemic cells. Using a mouse model of myeloid blast crisis leukemia induced through the cooperation of BCR-ABL and NUP98-HOXA9, we performed genome-wide transcriptional profiling comparing hematopoietic cells expressing each translocation alone or in combination. Using this system, we were able to model the genetic alterations induced as normal cells progressed towards LSC transformation, identifying 72 CRGs (50 aberrantly up-regulated and 22 down-regulated) with potential importance in leukemia development. To investigate the relevance of these CRGs in leukemia biology, an RNAi screen approach was employed. Primary leukemic progenitors were purified and transduced with a custom lentiviral RNAi library and subsequently transplanted into recipient animals to assess the engraftment potential upon perturbation of the individual CRGs. Our findings demonstrate that knock-down of expression in 35 of 50 (70%) leukemia CRGs reduced in vivo growth of primitive leukemia, a finding that was independently validated through single gene perturbation of several genes that scored in the RNAi screen (GJB3, EphA3, PMP22, Serinc2, SerpinB2, and CP). In particular, serpinB2, a gene that scored strongly in the RNAi analysis, was shown to directly effect the frequency of LSC in vivo. Given that the cooperative gene signature represented genes with many distinct cellular functions, we hypothesized that the CRG expression profile represents a key regulatory network in leukemia survival. To investigate our hypothesis we utilized the Broad Institute's Connectivity Map (CMAP) to identify pharmacological compounds with the ability to modulate multiple CRGs simultaneously. This analysis revealed that both Tyrophostin AG-825 (AG825) and 4-hydroxy-2-nonenol (4HNE) were predicted to reverse the gene expression induced as a consequence of leukemic transformation. To test the effect of these agents as selective toxicants to leukemia, we treated both normal and leukemia murine bone marrow cells with each compound. Both bulk and phenotypically primitive leukemic cells were eradicated in dose-responsive fashion upon treatment with either AG825 or 4HNE, while normal cells showed significantly reduced sensitivity. Progenitor function as measured by colony forming assays also showed a selective reduction in leukemia colony formation, suggesting that both these compounds are toxic to the majority of leukemic cell types. Interestingly, similar results were obtained when human normal and leukemic bone marrow specimens were treated with both drugs, suggesting the CRG signature represents an important class of genes with conserved function across species. To determine the level of conservation of the leukemia CRG signature between murine and human leukemia, we profiled eight normal and leukemic patient specimens for expression of the CRG signature. Of the 39 evaluable human CRG orthologs, 13 showed similar expression trends in human leukemia samples relative to normal controls. Intriguingly, both AG825 and 4HNE were predicted to inhibit this 13-gene signature by the CMAP database, suggesting that the compounds may act through these genes to influence leukemia cell death. Taken together, our findings demonstrate the importance of cooperative gene regulation in leukemogenesis and provide a novel platform for future research toward more effective therapeutic strategies to treat leukemia. Disclosures:No relevant conflicts of interest to declare.

A Functional Genomic Screen Combined with Time-Lapse Microscopy Uncovers a Novel Set of Genes Involved in Dorsal Closure of Drosophila Embryos

Morphogenesis, the establishment of the animal body, requires the coordinated rearrangement of cells and tissues regulated by a very strictly-determined genetic program. Dorsal closure of the epithelium in the Drosophila melanogaster embryo is one of the best models for such a complex morphogenetic event. To explore the genetic regulation of dorsal closure, we carried out a large-scale RNA interference-based screen in combination with in vivo time-lapse microscopy and identified several genes essential for the closure or affecting its dynamics. One of the novel dorsal closure genes, the small GTPase activator pebble (pbl), was selected for detailed analysis. We show that pbl regulates actin accumulation and protrusion dynamics in the leading edge of the migrating epithelial cells. In addition, pbl affects dorsal closure dynamics by regulating head involution, a morphogenetic process mechanically coupled with dorsal closure. Finally, we provide evidence that pbl is involved in closure of the adult thorax, suggesting its general requirement in epithelial closure processes.

AP-1 protein induction during monopoiesis favors C/EBP: AP-1 heterodimers over C/EBP homodimerization and stimulates FosB transcription

AP-1 proteins heterodimerize via their LZ domains to bind TGACGTCA or TGACTCA, whereas C/EBPs dimerize to bind ATTGCGCAAT. We demonstrate that intact C/EBPα also heterodimerizes with c-Jun or c-Fos to bind a hybrid DNA element, TGACGCAA, or more weakly to TGATGCAA. A 2:1 ratio of c-Jun:C/EBPα or c-Fos:C/EBPα was sufficient for preferential binding. Semiquantitative Western blot analysis indicates that the summation of c-Jun, JunB, and c-Fos levels in differentiating myeloid cells is similar to or exceeds the entirety of C/EBPα and C/EBPβ, indicating the feasibility of heterodimer formation. Induction of AP-1 proteins during monocytic differentiation favored formation of C/EBP:AP-1 heterodimers, with C/EBPα homodimers more evident during granulopoiesis. Approximately 350 human and 300 murine genes contain the TGACGCAA motif between -2 kb and +1 kb of their transcription start sites. We focused on the murine Fosb promoter, which contains a C/EBP:AP-1 cis element at -56 and -253, with the hFOSB gene containing an identical site at -253 and a 1-bp mismatch at -56. C/EBPα:AP-1 heterodimers bound either site preferentially in a gel-shift assay, C/EBPα:c-Fos ER fusion proteins induced endogenous Fosb mRNA but not in the presence of CHX, C/EBP and AP-1 proteins bound the endogenous Fosb promoter, mutation of the -56 cis element reduced reporter activity fivefold, and endogenous FosB protein was expressed preferentially during monopoiesis versus granulopoiesis. Increased expression of Jun/Fos proteins elevates C/EBP:AP-1 heterodimer formation to potentially activate novel sets of genes during monopoiesis and potentially during other biologic processes.

Defence at dawn

A remarkable example has been discovered of a plant tuning its immune defence against a pathogen. The tuning consists of maximal expression of the relevant genes at the time of day when attack is most likely. See Letter p.110 Circadian rhythms regulate a wide variety of developmental and metabolic processes resulting in enhanced fitness. This study of plant defence against the fungal pathogen Hyaloperonospora arabidopsidis, which causes downy mildew disease in Arabidopsis plants, reveals a link between the plant innate immune responses and the clock. The central circadian regulator CCA1 is shown to regulate a novel set of genes involved in R-gene-mediated defence. The mechanism allows plants to 'anticipate' infection at dawn when the pathogen normally disperses its spores.

A comparative analysis of DNA methylation across human embryonic stem cell lines

BackgroundWe performed a comparative analysis of the genome-wide DNA methylation profiles from three human embryonic stem cell (HESC) lines. It had previously been shown that HESC lines had significantly higher non-CG methylation than differentiated cells, and we therefore asked whether these sites were conserved across cell lines.ResultsWe find that heavily methylated non-CG sites are strongly conserved, especially when found within the motif TACAG. They are enriched in splice sites and are more methylated than other non-CG sites in genes. We next studied the relationship between allele-specific expression and allele-specific methylation. By combining bisulfite sequencing and whole transcriptome shotgun sequencing (RNA-seq) data we identified 1,020 genes that show allele-specific expression, and 14% of CG sites genome-wide have allele-specific methylation. Finally, we asked whether the methylation state of transcription factor binding sites affects the binding of transcription factors. We identified variations in methylation levels at binding sites and found that for several transcription factors the correlation between the methylation at binding sites and gene expression is generally stronger than in the neighboring sequences.ConclusionsThese results suggest a possible but as yet unknown functional role for the highly methylated conserved non-CG sites in the regulation of HESCs. We also identified a novel set of genes that are likely transcriptionally regulated by methylation in an allele-specific manner. The analysis of transcription factor binding sites suggests that the methylation state of cis-regulatory elements impacts the ability of factors to bind and regulate transcription.

Subtypes of cervical adenosquamous carcinomas classified by EpCAM expression related to radiosensitivity

Adenosquamous carcinoma (ASC) is a relatively uncommon histological subtype of cervical cancer (CC). A point of controversy is the relative prognosis of ASC compared to squamous cell carcinoma (SCC). We hypothesized that ASC could be classified into two intrinsic molecular subtypes with different outcomes. We examined 143 biopsy samples of CC patients to identify a molecule for classification using microarray expression analysis and immunohistochemical analysis (IHA). We found specific expression profiles of candidate genes that distinguished two clusters. All adenocarcinoma (AC) patients were classified into one cluster, and most SCC patients fell into the other cluster. ASC patients were classified across the two clusters, which showed significantly different prognoses. The SCC-like expression signature comprised ANXA8, CK5, IFI16, and nectin-1; and the AC-like signature comprised EpCAM, and TMEM98. These signature-specific genes hypothetically indicated specific pathways by ontological analysis. The AC-like signature suggested an epithelial-mesenchymal transition and activated β-catenin pathway, while the SCC-like signature suggested keratinocyte differentiation, HPV infection, and p53-mediated apoptosis. IHA revealed that positive expression of the most promising protein, EpCAM, was significantly associated with poor prognosis. In addition, the inhibition of EpCAM expression using siRNA significantly increased radiation-induced cell death in the cervical cell line, ME-180. In conclusion, we identified two possible ASC subtypes associated with different expression profiles and different prognoses. This work provides a novel set of genes that could be used as independent prognostic markers and therapy targets.

Using the Phenogen website for ‘in silico’ analysis of morphine-induced analgesia: identifying candidate genes

The identification of genes that contribute to polygenic (complex) behavioral phenotypes is a key goal of current genetic research. One approach to this goal is to combine gene expression information with genetic information, i.e. to map chromosomal regions that regulate gene expression levels. This approach has been termed 'genetical genomics', and, when used in conjunction with the identification of genomic regions (QTLs) that regulate the complex physiological trait under investigation, provides a strong basis for candidate gene discovery. In this paper, we describe the implementation of the genetical genomic/phenotypic approach to identify candidate genes for sensitivity to the analgesic effect of morphine in BXD recombinant inbred mice. Our analysis was performed 'in silico', using an online interactive resource called PhenoGen (http://phenogen.ucdenver.edu). We describe in detail the use of this resource, which identified a set of candidate genes, some of whose products regulate the cellular localization and activity of the mu opiate receptor. The results demonstrate how PhenoGen can be used to identify a novel set of genes that can be further investigated for their potential role in pain, morphine analgesia and/or morphine tolerance.

A genetic screen identifies the Triple T complex required for DNA damage signaling and ATM and ATR stability

In response to DNA damage, cells activate a complex signal transduction network called the DNA damage response (DDR). To enhance our current understanding of the DDR network, we performed a genome-wide RNAi screen to identify genes required for resistance to ionizing radiation (IR). Along with a number of known DDR genes, we discovered a large set of novel genes whose depletion leads to cellular sensitivity to IR. Here we describe TTI1 (Tel two-interacting protein 1) and TTI2 as highly conserved regulators of the DDR in mammals. TTI1 and TTI2 protect cells from spontaneous DNA damage, and are required for the establishment of the intra-S and G2/M checkpoints. TTI1 and TTI2 exist in multiple complexes, including a 2-MDa complex with TEL2 (telomere maintenance 2), called the Triple T complex, and phosphoinositide-3-kinase-related protein kinases (PIKKs) such as ataxia telangiectasia-mutated (ATM). The components of the TTT complex are mutually dependent on each other, and act as critical regulators of PIKK abundance and checkpoint signaling.

Abstract 4593: Molecular characterization of the androgen metabolite 3βAdiol as a partial agonist of ERα

Abstract Previously we have shown that androgens induce the growth of estrogen-dependent, estrogen receptor (ER)-positive breast cancer cell lines via metabolism to estrogen-like steroids, independent of CYP19 aromatase. One pathway involves the 3β-hydroxysteroid dehydrogenase (3β-HSD) mediated metabolism of 5α-dihydrotestosterone (DHT) to 5α-androstane-3β,17β-diol (3βAdiol). 3βAdiol induces the growth of ER+ breast cancer cells by directly activating ER. However, in MCF-7 cells, maximal growth induction by 3βAdiol was only ∼75% of maximal growth induced by 17β-estradiol (E2), suggesting that 3βAdiol acts as a weak or partial agonist of ERα. In this study, we characterize 3βAdiol as a partial agonist of ERα using cell growth assays and an in vitro receptor binding assay. We use cDNA microarrays to examine gene expression induced by the partial agonist 3βAdiol vs. the full agonist E2. We tested if 3βAdiol as a partial agonist could antagonize E2-induced breast cancer cell growth. MCF-7 cells in estrogen-free conditions were treated with 100pM E2 (∼EC90) and increasing concentrations of 3βAdiol. Increasing 3βAdiol suppressed growth induced by E2 to the maximal level induced by 3βAdiol alone. These data confirm that 3βAdiol acts as a partial agonist of ERα. Additionally, we performed an in vitro, florescence polarization-based receptor binding assay to determine the relative affinities of 3βAdiol and E2 for ERα. Based on their ability to compete with a labeled ligand for binding to ERα, 3βAdiol bound ERα with an approximately 10-fold lower affinity than E2. This decreased affinity of 3βAdiol for ERα is consistent with the reduced potency of 3βAdiol versus E2 observed in growth assays. To compare changes in gene expression induced by either the full agonist E2 or partial agonist 3βAdiol, samples were analyzed with Affymetrix HG-U133 Plus 2.0 microarrays. MCF-7 cells were treated for 24 hours with 10nM of steroid or vehicle control. To evaluate overall changes in gene expression induced by the steroids, probe sets were selected that had >2-fold change in expression versus the control array, with at least one sample having an expression value >26. 1273 probe sets were differentially expressed between the E2 and control arrays, and 1318 between the 3βAdiol and control arrays, however, 1030 of these were common to both steroids. Genes in canonical estrogen signaling pathways were similarly regulated by both E2 and 3βAdiol, including GREB1, Bcl-2, TFF1 and CXCL12. Only 45 probe sets were differentially expressed between the E2 and 3βAdiol arrays. These data demonstrate that 3βAdiol is a partial agonist of ERα in our breast cancer cell model. The majority of changes in gene expression are similar between the full and partial agonist. These data suggest that the differential growth induction by the full and partial agonist are likely due to differential levels or time courses of gene induction, rather than the induction of a novel set of genes. 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 4593.

Abstract 5186: Identification of novel cell-surface proteins associated with homotypic adhesion in human colon cancer cell lines

Abstract The loss of homotypic cell adhesion is one of several prerequisites for metastasis. This study aimed to characterize this process by comparing the adhesive properties and E-cadherin expression of a panel of eight human colon cancer cell lines. Homotypic adhesion was determined by cell dissociation assays. E-cadherin is generally considered the primary mediator of cell-cell adhesion in epithelial cells and epithelial-derived tumors, but E-cadherin mRNA and protein expression did not correlate with homotypic adhesion across the cell lines. Consequently, the mediation of adhesion in these cell lines must be more complex. To characterize this additional complexity, gene expression profiling was performed and a set of genes encoding transmembrane proteins was identified that correlated with adhesion across the cell lines. The functional relevance of these correlated genes in adhesion was confirmed by siRNA knockdown in SW480 colon cancer cells. Knockdown of either CD99 or CD99L2 led to a loss of homotypic adhesion, down-regulation of E-cadherin mRNA and protein levels and up-regulation of vimentin mRNA and protein levels. The expression patterns of E-cadherin and vimentin following gene knockdown is consistent with epithelial-mesenchymal transition (EMT). EMT is a transcriptional program active in both development and in tumor progression, which causes cells to have decreased adhesion, increased invasion, and increased motility. In contrast, knockdown of the correlated gene LRRC8B in SW480 cells led to a loss of homotypic adhesion but had no effect on the expression of either E-cadherin or vimentin, suggesting an EMT-independent mechanism. The cell-surface molecules CD99 and CD99L2 appear to be involved in a signaling and/or transcriptional network that helps maintain an epithelial phenotype, and loss of this network may be associated with EMT and cancer progression. To characterize this network, gene expression profiling was performed in SW480 cells following siRNA-mediated knockdown of either CD99 or CD99L2. Because these two cell-surface proteins are structurally related and displayed similar functional roles in adhesion, we hypothesized that they would share overlapping downstream transcriptional effects. Analysis of the gene expression data has identified a shared set of transcription factors whose expression is affected by the knockdown of either CD99 or CD99L2. The approach of correlating the adhesion characteristics and gene expression of a panel of colon cancer cell lines has identified a set of novel genes which are functionally involved in adhesion. Further investigation will help to identify the contributions of CD99, CD99L2 and LRRC8B to the homotypic adhesion process and clarify the downstream transcriptional networks involved in this facet of tumor progression. 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 5186.

Combined High Resolution Genomic and Expression Profiles Microarray Analysis in Sezary Syndrome.

Abstract 3238Poster Board III-175We have used single nucleotide polymorphism (SNP) and comparative genomic hybridization array (aCGH) to study DNA copy number (CN) changes and loss of heterozygosity (LOH) in a series of 28 patients affected by Sézary syndrome (SS), a rare form of cutaneous-T-cell lymphoma (CTCL). Our data identified, further confirming previous studies, recurrent losses of 17p13.2-p11.2 and 10p12.1-q26.3 occurring in 71 and 68% of cases respectively; common gains were detected for 17p11.2-q25.3 (64%) and chromosome 8/8q (50%). Moreover, we identified novel genomic lesions recurring in more than 30% of tumours: loss of 9q13-q21.33 and gain of 10p15.3-10p12.2. Individual chromosomal aberrations did not show a significant correlation with prognosis, however when more than three recurrent chromosomal alterations (gain or loss) were considered, a statistical association was observed using Kaplan-Meier survival analysis. Crossing genomic data mapping with those obtained from gene expression profiles of matching samples we were able to identify a total of 113 deregulated transcripts in aberrant chromosomal regions that included cancer related genes such as members of the NF-kB pathway (NKIRAS2, PSMD3, BAG4, BTRC, TRAF2) that might explain its constitutive activation in CTCL. Matching this list of genes with those discriminating patients with different survival times we identify several common candidates that might exert critical roles in SS, like PIP5K1B and BUB3. Taken together this study confirms and maps more precisely the regions of gain and loss and, combined to transcriptional profiles, suggest a novel set of genes of potential interest in SS. DisclosuresNo relevant conflicts of interest to declare.

Identification of Key Regions and Genes Important in the Pathogenesis of Sézary Syndrome by Combining Genomic and Expression Microarrays

In this study, we used single nucleotide polymorphism and comparative genomic hybridization array to study DNA copy number changes and loss of heterozygosity for 28 patients affected by Sézary syndrome (SS), a rare form of cutaneous T-cell lymphoma (CTCL). Our data identified, further confirming previous studies, recurrent losses of 17p13.2-p11.2 and 10p12.1-q26.3 occurring in 71% and 68% of cases, respectively; common gains were detected for 17p11.2-q25.3 (64%) and chromosome 8/8q (50%). Moreover, we identified novel genomic lesions recurring in >30% of tumors: loss of 9q13-q21.33 and gain of 10p15.3-10p12.2. Individual chromosomal aberrations did not show a significant correlation with prognosis; however, when more than three recurrent chromosomal alterations (gain or loss) were considered, a statistical association was observed using Kaplan-Meier survival analysis. Integrating mapping and transcriptional data, we were able to identify a total of 113 deregulated transcripts in aberrant chromosomal regions that included cancer-related genes such as members of the NF-kappaB pathway (BAG4, BTRC, NKIRAS2, PSMD3, and TRAF2) that might explain its constitutive activation in CTCL. Matching this list of genes with those discriminating patients with different survival times, we identify several common candidates that might exert critical roles in SS, such as BUB3 and PIP5K1B. Altogether, our study confirms and maps more precisely the regions of gain and loss and, combined to transcriptional profiles, suggests a novel set of genes of potential interest in SS.

Reprogramming after Chromosome Transfer into Mouse Blastomeres

It is well known that oocytes can reprogram differentiated cells, allowing animal cloning by nuclear transfer. We have recently shown that fertilized zygotes retain reprogramming activities, suggesting that such activities might also persist in cleavage-stage embryos. Here, we used chromosome transplantation techniques to investigate whether the blastomeres of two-cell-stage mouse embryos can reprogram more differentiated cells. When chromosomes from one of the two blastomeres were replaced with the chromosomes of an embryonic or CD4(+) T lymphocyte donor cell, we observed nuclear reprogramming and efficient contribution of the manipulated cell to the developing blastocyst. Embryos produced by this method could be used to derive stem cell lines and also developed to term, generating mosaic "cloned" animals. These results demonstrate that blastomeres retain reprogramming activities and support the notion that discarded human preimplantation embryos may be useful recipients for the production of genetically tailored human embryonic stem cell lines.

Biosynthesis of an antifungal oligopeptide in Burkholderia contaminans strain MS14

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. When compared with the wild type strain, antifungal activities of mutants MS14MT13 and MS14MT18 against Geotrichum candidum were eliminated, and the activity of mutant MS14MT15 was reduced by approximately 80%. Sequence analysis of a 22.7-kb DNA segment revealed the presence of six open-reading frames (ORFs), including the genes disrupted in the mutants. The wild type level of antifungal activity was observed for mutant MS14MT15 ( ambR2::Tn 5) complemented in trans with the intact ambR2 gene, which encodes a LuxR type regulator. AmbR2 positively regulates the transcription of the other five ORFs identified, which including two nonribosomal peptide synthetase genes required for biosynthesis of antifungal compound. The compound consists of amino acid residues having a molecular weight of 1200.6 Da. These results show that strain MS14 possesses a novel set of genes dedicated to the biosynthesis of an antifungal oligopeptide.

Identification of Novel Epigenetic Markers for Clear Cell Renal Cell Carcinoma

We identified significantly hypermethylated genes in clear cell renal cell carcinoma. We previously identified a set of under expressed genes in renal cell carcinoma tissue through transcriptional profiling and a robust computational screen. We selected 19 of these genes for hypermethylation analysis using a rigorous search for the best candidate regions, considering CpG islands and transcription factor binding sites. The genes were analyzed for hypermethylation in the DNA of 38 matched clear cell renal cell carcinoma and normal samples using matrix assisted laser desorption ionization time-of-flight mass spectrometry. The significance of hypermethylation was assessed using 3 statistical tests. We validated the down-regulation of significantly hypermethylated genes at the RNA and protein levels in a separate set of patients using reverse transcriptase-polymerase chain reaction, immunohistochemistry and Western blots. We found 7 significantly hypermethylated regions from 6 down-regulated genes, including SFRP1, which was previously shown to be hypermethylated in renal cell carcinoma and other cancer types. To our knowledge we report for the first time that another 5 genes (SCNN1B, SYT6, DACH1, and the tumor suppressors TFAP2A and MT1G) are hypermethylated in renal cell carcinoma. Robust computational screens and the high throughput methylation assay resulted in an enriched set of novel genes that are epigenetically altered in clear cell renal cell carcinoma. Overall the detection of hypermethylation in these highly down-regulated genes suggests that assaying for their methylation using cells from urine or blood could provide the basis for a viable diagnostic test.