Global Gene Expression Studies Research Articles

Bile Acids Promote Diethylnitrosamine‐induced Hepatocellular Carcinoma via Increased Inflammatory Signaling.

Hepatocellular carcinoma (HCC) is the most common hepatic malignancy and the third leading cause of cancer related deaths. Previous studies have implicated bile acids in pathogenesis of HCC but the mechanisms are not known. We investigated the mechanisms of bile acid‐mediated promotion of HCC using a diethylnitrosamine (DEN)‐mediated initiation‐cholic acid (CA)‐induced tumor promotion protocol in mice. The data show that 0.2% CA treatment resulted in 3‐fold increase in number and size of DEN‐induced liver tumors. All tumors observed in DEN‐treated mice were well‐differentiated HCCs. Microarray based global gene expression studies combined with Ingenuity Pathway Analysis revealed NF‐κB pathway as the main signaling pathways activated in the DEN‐treated 0.2% CA‐fed livers. Further studies showed significantly higher TNFα, IL‐1β, MCP‐1 mRNA, a significant increase in total and phosphorylated‐p65 and phosphorylated IκBa (degradation form) and higher EGR1 mRNA and protein in livers of DEN‐treated 0.2% CA‐fed mice. Interestingly, total bile acid quantification revealed increase in hepatic and serum bile acids in both DEN‐treated and DEN‐treated 0.2% CA‐fed mice, which was higher in the latter group. Finally, quantification of total serum bile acids in normal, cirrhotic and HCC human samples revealed increased bile acids in serum of cirrhotic and HCC patients. Taken together, these data indicate that bile acids are mechanistically involved pathogenesis of HCC and may promote HCC formation via activation of inflammatory signaling.

Transcriptional profiling and biological pathway analysis of human equivalence PCB exposure in vitro: Indicator of disease and disorder development in humans

Background and aimsOur earlier gene-expression studies with a Slovak PCBs-exposed population have revealed possible disease and disorder development in accordance with epidemiological studies. The present investigation aimed to develop an in vitro model system that can provide an indication of disrupted biological pathways associated with developing future diseases, well in advance of the clinical manifestations that may take years to appear in the actual human exposure scenario. MethodsWe used human Primary Blood Mononuclear Cells (PBMC) and exposed them to a mixture of human equivalence levels of PCBs (PCB-118, -138, -153, -170, -180) as found in the PCBs-exposed Slovak population. The microarray studies of global gene expression were conducted on the Affymetrix platform using Human Genome U133 Plus 2.0 Array along with Ingenuity Pathway Analysis (IPA) to associate the affected genes with their mechanistic pathways. High-throughput qRT-PCR Taqman Low Density Array (TLDA) was done to further validate the selected 6 differentially expressed genes of our interest, viz., ARNT, CYP2D6, LEPR, LRP12, RRAD, TP53, with a small population validation sample (n=71). ResultsOverall, we revealed a discreet gene expression profile in the experimental model that resembled the diseases and disorders observed in PCBs-exposed population studies. The disease pathways included endocrine system disorders, genetic disorders, metabolic diseases, developmental disorders, and cancers, strongly consistent with the evidence from epidemiological studies. InterpretationThese gene finger prints could lead to the identification of populations and subgroups at high risk for disease, and can pose as early disease biomarkers well ahead of time, before the actual disease becomes visible.

Mycobacterium tuberculosis effectors involved in host–pathogen interaction revealed by a multiple scales integrative pipeline

BackgroundMycobacterium tuberculosis (Mtb) has evolved multiple strategies to counter host immunity. Proteins are one important player in the host–pathogen interaction. A comprehensive list of such proteins will benefit our understanding of pathogenesis of Mtb. MethodsA genome-scale dataset was created from different sources of published data: global gene expression studies in disease models; genome-wide insertional mutagenesis defining gene essentiality under different conditions; genes lost in clinical isolates; subcellular localization analysis and non-homology analysis. Using data mining and meta-analysis, expressed proteins critical for intracellular survival of Mtb are first identified, followed by subcellular localization analysis, finally filtering a series of subtractive channel of analysis to find out promising drug target candidates. ResultsThe analysis found 54 potential candidates essential for the intracellular survival of the pathogen and non-homologous to host or gut flora, and might be promising drug targets. ConclusionBased on our meta-analysis and bioinformatics analysis, 54 hits were found from Mtb around 4000 open reading frames. These hits can be good candidates for further experimental investigation.

Discovery of novel vitamin D-regulated proteins in INS-1 cells: a proteomic approach.

Experimental evidence indicates that vitamin D may have a beneficial role in pancreatic β-cell function. Global gene expression studies have shown that the active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3 ] modulates genes involved in ion transport, lipid metabolism and insulin secretion. We employed stable isotope labelling by amino acids in cell culture in combination with liquid chromatography-tandem mass spectrometry to quantitatively assess the impact of two vitamin D metabolites, 1,25-(OH)2 D3 and 25-hydroxyvitamin D3 [25-(OH)D3 ], on global protein expression on a model rat β-cell line, insulinoma-derived INS-1 cells. Although treatment with 1,25-(OH)2 D3 resulted in 31 differentially expressed proteins, 25-(OH)D3 had no impact on protein expression. Of these 31 proteins, 29 were upregulated, whereas two showed a decrease in abundance. Proteins whose expression levels markedly increased in the presence of 1,25-(OH)2 D3 included Crat, Hmgn2, Protein Tmsbl1 and Gdap1. One of the most important findings in this study is upregulation of proteins implicated in insulin granule motility and insulin exocytosis, suggesting a positive effect on insulin secretion. Moreover, modulation of several membrane transport proteins suggests that 1,25-(OH)2 D3 has an impact on the homeostatic regulation of ions, which is critical for most functions in the β-cell. In this study, we discovered a number of novel 1,25-(OH)2 D3 -regulated proteins, which may contribute to a better understanding of the reported beneficial effects of vitamin D on pancreatic β-cells. All in all, our findings should pave the way for future studies providing insights into molecular mechanisms by which 1,25-(OH)2 D3 regulates protein expression in pancreatic β-cells.

Transcriptional profiling of adult neural stem-like cells from the human brain.

There is a great potential for the development of new cell replacement strategies based on adult human neural stem-like cells. However, little is known about the hierarchy of cells and the unique molecular properties of stem- and progenitor cells of the nervous system. Stem cells from the adult human brain can be propagated and expanded in vitro as free floating neurospheres that are capable of self-renewal and differentiation into all three cell types of the central nervous system. Here we report the first global gene expression study of adult human neural stem-like cells originating from five human subventricular zone biopsies (mean age 42, range 33–60). Compared to adult human brain tissue, we identified 1,189 genes that were significantly up- and down-regulated in adult human neural stem-like cells (1% false discovery rate). We found that adult human neural stem-like cells express stem cell markers and have reduced levels of markers that are typical of the mature cells in the nervous system. We report that the genes being highly expressed in adult human neural stem-like cells are associated with developmental processes and the extracellular region of the cell. The calcium signaling pathway and neuroactive ligand-receptor interactions are enriched among the most differentially regulated genes between adult human neural stem-like cells and adult human brain tissue. We confirmed the expression of 10 of the most up-regulated genes in adult human neural stem-like cells in an additional sample set that included adult human neural stem-like cells (n = 6), foetal human neural stem cells (n = 1) and human brain tissues (n = 12). The NGFR, SLITRK6 and KCNS3 receptors were further investigated by immunofluorescence and shown to be heterogeneously expressed in spheres. These receptors could potentially serve as new markers for the identification and characterisation of neural stem- and progenitor cells or as targets for manipulation of cellular fate.

Functional study of the Hap4-like genes suggests that the key regulators of carbon metabolism HAP4 and oxidative stress response YAP1 in yeast diverged from a common ancestor.

The transcriptional regulator HAP4, induced by respiratory substrates, is involved in the balance between fermentation and respiration in S. cerevisiae. We identified putative orthologues of the Hap4 protein in all ascomycetes, based only on a conserved sixteen amino acid-long motif. In addition to this motif, some of these proteins contain a DNA-binding motif of the bZIP type, while being nonetheless globally highly divergent. The genome of the yeast Hansenula polymorpha contains two HAP4-like genes encoding the protein HpHap4-A which, like ScHap4, is devoid of a bZIP motif, and HpHap4-B which contains it. This species has been chosen for a detailed examination of their respective properties. Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not. Moreover HpHAP4-B is able to complement the H2O2 hypersensitivity of the ScYap1 deletant, YAP1 being, in S. cerevisiae, the main regulator of oxidative stress. Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4. Deletion of these two genes in their natural host, H. polymorpha, confirms that HpHAP4-A participates in the control of the fermentation/respiration balance, while HpHAP4-B is involved in oxidative stress since its deletion leads to hypersensitivity to H2O2. These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.

Gene expression profiling in adipose tissue from growing broiler chickens

In this study, total RNA was collected from abdominal adipose tissue samples obtained from ten broiler chickens at 3, 4, 5, and 6 weeks of age and prepared for gene microarray analysis with Affymetrix GeneChip Chicken Genome Arrays (Affymetrix) and quantitative real-time PCR analysis. Studies of global gene expression in chicken adipose tissue were initiated since such studies in many animal species show that adipose tissue expresses and secretes many factors that can influence growth and physiology. Microarray results indicated 333 differentially expressed adipose tissue genes between 3 and 6 wk, 265 differentially expressed genes between 4 and 6 wk and 42 differentially expressed genes between 3 and 4 wk. Enrichment scores of Gene Ontology Biological Process categories indicated strong age upregulation of genes involved in the immune system response. In addition to microarray analysis, quantitative real-time PCR analysis was used to confirm the influence of age on the expression of adipose tissue CC chemokine ligands (CCL), toll-like receptor (TLR)-2, lipopolysaccharide-induced TNF factor (LITAF), chemokine (C-C motif) receptor 8 (CCR8), and several other genes. Between 3 and 6 wk of age CCL5, CCL1, and CCR8 expression increased (P = 0.0001) with age. Furthermore, TLR2, CCL19, and LITAF expression increased between 4 and 6 wk of age (P = 0.001). This is the first demonstration of age related changes in CCL, LITAF, and TLR2 gene expression in chicken adipose tissue. Future studies are needed to elucidate the role of these adipose tissue genes in growth and the immune system.

Identification of smokers that are responsive to cessation by global gene expression analysis

Approximately 5600 constituents have been identified in cigarette smoke (Perfetti & Rodgman 2011). A number of these compounds are known to have specific toxicological properties. In order to demonstrate any reduction in the impact of these tobacco smoke toxicants on smokers, a number of research tools are employed. We describe here the results of a global gene expression study as part of a clinical study designed to assess reversibility of biomarkers in a cohort of smokers participating in a smoking cessation program. The aim of this study was to examine gene expression profiles before and after smoking cessation and identify candidate biomarkers responsive to reduction in cigarette smoke exposure.

Histone Lysine Methyltransferase SDG8 Is Involved in Brassinosteroid-Regulated Gene Expression in Arabidopsis thaliana

The plant steroid hormones, brassinosteroids (BRs), play important roles in plant growth, development, and responses to environmental stresses. BRs signal through receptors localized to the plasma membrane and other signaling components to regulate the BES1/BZR1 family of transcription factors, which modulates the expression of thousands of genes. How BES1/BZR1 and their interacting proteins function to regulate the large number of genes are not completely understood. Here we report that histone lysine methyltransferase SDG8, implicated in histone 3 lysine 36 di- and trimethylation (H3K36me2 and me3), is involved in BR-regulated gene expression. BES1 interacts with SDG8, directly or indirectly through IWS1, a transcription elongation factor involved in BR-regulated gene expression. The knockout mutant sdg8 displays a reduced growth phenotype with compromised BR responses. Global gene expression studies demonstrated that, while BR regulates about 5000 genes in wild-type plants, the hormone regulates fewer than 700 genes in sdg8 mutant. In addition, more than half of BR-regulated genes are differentially affected in sdg8 mutant. A Chromatin Immunoprecipitation (ChIP) experiment showed that H3K36me3 is reduced in BR-regulated genes in the sdg8 mutant. Based on these results, we propose that SDG8 plays an essential role in mediating BR-regulated gene expression. Our results thus reveal a major mechanism by which histone modifications dictate hormonal regulation of gene expression.

Genome activation in bovine embryos: Review of the literature and new insights from RNA sequencing experiments

Maternal-to-embryonic transition (MET) is the period in early embryonic development when maternal RNAs and proteins stored in the oocyte are gradually degraded and transcription of the embryonic genome is activated. First insights into the timing of embryonic genome activation (EGA) came from autoradiographic analyses of embryos following incorporation of [3H]uridine. These studies identified the eight- to 16-cell stage of bovine embryos as the period of major EGA, but detected first transcriptional activity already in one-cell embryos. Subsequent studies compared the transcriptome profiles of untreated embryos and of embryos incubated with the transcription inhibitor α-amanitin to reveal transcripts of embryonic origin. In addition, candidate gene-based and global gene expression studies over several stages of early development were performed and characteristic profiles were revealed. However, the onset of embryonic transcription was obscured by the presence of maternal transcripts and could only be determined for genes which are not expressed in oocytes. Using RNA sequencing of bovine germinal vesicle and metaphase II oocytes, and of four-cell, eight-cell, 16-cell and blastocyst stage embryos, we established the most comprehensive transcriptome data set of bovine oocyte maturation and early development. EGA was analyzed by (i) detection of embryonic transcripts which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. Using these three approaches we were able to map the onset of embryonic transcription for almost 7400 genes. Genes activated at the four-cell stage or before were functionally related to RNA processing, translation, and transport, preparing the embryo for major EGA at the eight-cell stage, when genes from a broad range of functional categories were found to be activated. These included transcriptional and translational functions as well as protein ubiquitination. The functions of the genes activated at the 16-cell stage were consistent with ongoing transcription and translation, while the genes activated in blastocysts included regulators of early lineage specification. Fine mapping of EGA provides a new layer of information for detecting disturbances of early development due to genetic, epigenetic, and environmental factors.

The influences of parental diet and vitamin E intake on the embryonic zebrafish transcriptome

The composition of the typical commercial diet fed to zebrafish can dramatically vary. By utilizing defined diets we sought to answer two questions: 1) How does the embryonic zebrafish transcriptome change when the parental adults are fed a commercial lab diet compared with a sufficient, defined diet (E+)? 2) Does a vitamin E-deficient parental diet (E−) further change the embryonic transcriptome? We conducted a global gene expression study using embryos from zebrafish fed a commercial (Lab), an E+ or an E− diet. To capture differentially expressed transcripts prior to onset of overt malformations observed in E− embryos at 48h post-fertilization (hpf), embryos were collected from each group at 36hpf. Lab embryos differentially expressed (p<0.01) 946 transcripts compared with the E+ embryos, and 2656 transcripts compared with the E− embryos. The differences in protein, fat and micronutrient intakes in zebrafish fed the Lab compared with the E+ diet demonstrate that despite overt morphologic consistency, significant differences in gene expression occurred. Moreover, functional analysis of the significant transcripts in the E− embryos suggested perturbed energy metabolism, leading to overt malformations and mortality. Thus, these findings demonstrate that parental zebrafish diet has a direct impact on the embryonic transcriptome.

Primitive and definitive erythropoiesis in mammals

Red blood cells (RBCs), which constitute the most abundant cell type in the body, come in two distinct flavors- primitive and definitive. Definitive RBCs in mammals circulate as smaller, anucleate cells during fetal and postnatal life, while primitive RBCs circulate transiently in the early embryo as large, nucleated cells before ultimately enucleating. Both cell types are formed from lineage-committed progenitors that generate a series of morphologically identifiable precursors that enucleate to form mature RBCs. While definitive erythroid precursors mature extravascularly in the fetal liver and postnatal marrow in association with macrophage cells, primitive erythroid precursors mature as a semi-synchronous cohort in the embryonic bloodstream. While the cytoskeletal network is critical for the maintenance of cell shape and the deformability of definitive RBCs, little is known about the components and function of the cytoskeleton in primitive erythroblasts. Erythropoietin (EPO) is a critical regulator of late-stage definitive, but not primitive, erythroid progenitor survival. However, recent studies indicate that EPO regulates multiple aspects of terminal maturation of primitive murine and human erythroid precursors, including cell survival, proliferation, and the rate of terminal maturation. Primitive and definitive erythropoiesis share central transcriptional regulators, including Gata1 and Klf1, but are also characterized by the differential expression and function of other regulators, including myb, Sox6, and Bcl11A. Flow cytometry-based methodologies, developed to purify murine and human stage-specific erythroid precursors, have enabled comparative global gene expression studies and are providing new insights into the biology of erythroid maturation.

Rosiglitazone Induces Cardiotoxicity by Accelerated Apoptosis

Present investigation explores the cardiotoxicity of rosiglitazone (ROSI) using rat heart cardiomyocytes and db/db mice. In H9c2 cells, ROSI at 50 and 60 μM induced an increase in the percentage of apoptotic cells and superoxide generation, along with an increase in the expression of various subunits of NADPH oxidase and nitric oxide synthases, confirmed that ROSI-induced apoptosis in H9c2 cells is by ROS generation. The increase in the expression of the antioxidants like superoxide dismutase (SOD), catalase, glutathione reductase (GR), glutathione-S-transferase (GST), and glutathione peroxidase (GPx) further confirmed this notion. Heme oxygenase-1, having an important role in cell protection against oxidative stress, was found to be increased along with induction of nuclear translocation of NF-E2-related factor and increased protein kinase C δ expression. Moreover, in db/db mice, oral administration of ROSI (10 mg/kg) for 10 days induced an increase in serum creatinine kinase-MB, tissue antioxidants like SOD, catalase, GR, GST, GPx expression, cardiac troponin T, and inducible nitric oxide synthase protein expression strongly support the in vitro findings. Furthermore, global gene expression studies also showed the perturbation of oxidative phosphorylation, fat cell differentiation, and electron transport chain following ROSI treatment in vivo. These results suggested that ROSI-induced cardiac damage is due to accelerated apoptosis both in vitro and in vivo.

Microrna Expression Profiling In Pediatric Acute Myeloid Leukemia Reveals a Tumor-Suppressive Role Of Mir-9 Associated With Translocation (8;21)

S.E. and J.K.K. as well as J.H.K. and M.M.H.E. contributed equally to this study.MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and in the development of leukemia. In addition, modulation of miRNA expression can be exploited therapeutically. To identify tumor suppressive miRNAs in pediatric acute myeloid leukemia (AML), we performed a large-scale miRNA expression profiling in 90 cytogenetically characterized, de novo AML cases using a RT-qPCR platform.In total, 253 miRNAs were significantly differentially expressed between patients with MLL rearrangements, t(8;21), inv(16), t(7;12), and t(15;17). Hierarchical clustering of patient samples using these sets of miRNA values showed that t(15;17) samples clearly cluster away from the other pediatric AML samples while t(7;12) patients cluster closely to core binding factor AMLs, (t(8;21) and inv(16). These three groups largely cluster away from the majority of MLL rearranged samples. Eight miRNAs specifically downregulated in MLL rearranged, t(8;21) and inv(16) AMLs were functionally evaluated in vitro using three cell lines representing those cytogenetic groups: THP-1 (MLL rearranged), KASUMI-1 (t[8;21]) and ME-1 (inv[16]). Two of two miRNAs tested in KASUMI-1 cells (miR-9 and miR-582), two of three miRNAs tested in ME-1 (miR-192/194 bicistron and miR-660) and one of three miRNAs tested in THP-1 (miR-181a/b bicistron) reduced cell growth and colony-forming capacity upon ectopic expression. In KASUMI-1 cells, one miRNA was identified, miR-9, that not only reduced cell growth and colony forming capacity but also strongly induced monocytic differentiation in concert with calcitriol without affecting apoptosis. During normal hematopoiesis miR-9 is only expressed in macrophages.The effects on cell growth, colony-forming capacity and differentiation were confirmed in a second AML cell line with t(8;21), SKNO-1. The differentiation induction was restricted to t(8;21) leukemic cell lines, while its growth inhibitory function was also evident in normal CD34+ hematopoietic stem and progenitor cells. Most strikingly, miR-9 exerted a tumor suppressive function in primary leukemic blasts from patients with t(8;21) (n=2), but not in patients with MLL rearrangements (n=3). Using global gene expression studies upon ectopic miR-9 expression, we identified and validated LIN28B and HMGA2 as high fidelity target genes of miR-9 by RT-qPCR, western blotting and luciferase reporter assays. LIN28B is known to suppress let-7 processing. Indeed, miR-9 overexpresion increased the levels of mature let-7 family members, also leading to HMGA2 downregulation. ShRNA-mediated downregulation of LIN28B or HMGA2 partially recapitulated the effects of miR-9 on proliferation and differentiation of t(8;21) cell lines.Thus, miR-9 is a tumor suppressor-miR in t(8;21) de novo pediatric AML, that acts in a stringent cell context dependent manner in concert with let-7 family members by repressing the oncogenic LIN28B/HMGA2 axis.This work was supported by grants to J.H.K. from the German National Academic Foundation (KL-2374/2-1) and to J.K.K., L.V., A.D.vO, C.M.Z. and M.M.H.-E. from the Children Cancer Free Foundation (KIKA, project 49). Disclosures:No relevant conflicts of interest to declare.

Gene Expression Profiles in Human and Mouse Primary Cells Provide New Insights into the Differential Actions of Vitamin D3 Metabolites

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) had earlier been regarded as the only active hormone. The newly identified actions of 25-hydroxyvitamin D3 (25(OH)D3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) broadened the vitamin D3 endocrine system, however, the current data are fragmented and a systematic understanding is lacking. Here we performed the first systematic study of global gene expression to clarify their similarities and differences. Three metabolites at physiologically comparable levels were utilized to treat human and mouse fibroblasts prior to DNA microarray analyses. Human primary prostate stromal P29SN cells (hP29SN), which convert 25(OH)D3 into 1α,25(OH)2D3 by 1α-hydroxylase (encoded by the gene CYP27B1), displayed regulation of 164, 171, and 175 genes by treatment with 1α,25(OH)2D3, 25(OH)D3, and 24R,25(OH)2D3, respectively. Mouse primary Cyp27b1 knockout fibroblasts (mCyp27b1 −/−), which lack 1α-hydroxylation, displayed regulation of 619, 469, and 66 genes using the same respective treatments. The number of shared genes regulated by two metabolites is much lower in hP29SN than in mCyp27b1 −/−. By using DAVID Functional Annotation Bioinformatics Microarray Analysis tools and Ingenuity Pathways Analysis, we identified the agonistic regulation of calcium homeostasis and bone remodeling between 1α,25(OH)2D3 and 25(OH)D3 and unique non-classical actions of each metabolite in physiological and pathological processes, including cell cycle, keratinocyte differentiation, amyotrophic lateral sclerosis signaling, gene transcription, immunomodulation, epigenetics, cell differentiation, and membrane protein expression. In conclusion, there are three distinct vitamin D3 hormones with clearly different biological activities. This study presents a new conceptual insight into the vitamin D3 endocrine system, which may guide the strategic use of vitamin D3 in disease prevention and treatment.

Understanding the Tumor Microenvironment and Radioresistance by Combining Functional Imaging With Global Gene Expression

The objective of this review is to present an argument for performing joint analyses between functional imaging with global gene expression studies. The reason for making this link is that tumor microenvironmental influences on functional imaging can be uncovered. Such knowledge can lead to (1) more informed decisions regarding how to use functional imaging to guide therapy and (2) discovery of new therapeutic targets. As such, this approach could lead to identification of patients who need aggressive treatment tailored toward the phenotype of their tumor vs those who could be spared treatment that carries risk for more normal tissue complications. Only a handful of papers have been published on this topic thus far, but all show substantial promise.

Functional characterization of an abiotic stress-inducible transcription factor AtERF53 in Arabidopsis thaliana

AP2/ERF proteins play crucial roles in plant growth and development and in responses to biotic and abiotic stresses. ETHYLENE RESPONSE FACTOR 53 (AtERF53) belongs to group 1 in the ERF family and is induced in the early hours of dehydration and salt treatment. The functional study of AtERF53 is hampered because its protein expression in Arabidopsis is vulnerable to degradation in overexpressed transgenic lines. Taking advantage of the RING domain ligase1/RING domain ligase2 (rglg1rglg2) double mutant in which the AtERF53 can express stably, we investigate the physiological function of AtERF53. In this study, we demonstrate that expression of AtERF53 in wild-type Arabidopsis was responsive to heat and abscisic acid (ABA) treatment. From results of the cotransfection experiment, we concluded that AtERF53 has positive transactivation activity. Overexpression of AtERF53 in the rglg1rglg2 double mutant conferred better heat-stress tolerance and had resulted in higher endogenous ABA and proline levels compared to rglg1rglg2 double mutants. AtERF53 also has a function to regulate guard-cell movement because the stomatal aperture of AtERF53 overexpressed in rglg1rglg2 double mutant was smaller than that in the rglg1rglg2 double mutant under ABA treatment. In a global gene expression study, we found higher expressions of many stress-related genes, such as DREB1A, COR15A, COR15B, PLC, P5CS1, cpHSC70s and proline and ABA metabolic-related genes. Furthermore, we identified several downstream target genes of AtERF53 by chromatin immunoprecipitation assay. In conclusion, the genetic, molecular and biochemical result might explain how AtERF53 serving as a transcription factor contributes to abiotic stress tolerance in Arabidopsis.

Abstract 5368: Epigenetic changes in breast cancer cells associated with acquired Tamoxifen resistance.

Abstract Estrogen receptor (ER) status remains one of the most important breast cancer diagnostic and prognostic biomarkers. Roughly two-thirds of all breast cancers are ER-positive and can be treated with the anti-estrogen, Tamoxifen, however resistance occurs in 33% of women who take the drug for more than 5 years. MCF-7 breast cancer cells were previously cultured with 1 uM Tamoxifen, and after 6 months the clonal lines TMX2-11 and TMX2-28 with acquired resistance were isolated. TMX2-28 lost ER-α gene expression while a second clonal line, TMX2-11, did not. TMX2-28 cells are highly invasive in vitro, in contrast to TMX2-11 and the parent cell line, MCF-7. Studies of endocrine resistance and global gene expression in Tamoxifen-resistant cell cultures and human tumors have detected alterations in numerous pathways including ER-signaling, growth factor receptor signaling, cell cycle, and apoptosis. Aberrant DNA promoter methylation is a major epigenetic mechanism affecting gene expression in cancer. Our focus is on its role in Tamoxifen resistance. Hypermethylation of normally unmethylated cytosines in CpG-rich promoter regions frequently results in gene silencing, while hypomethylation in CpGs outside promoter regions leads to genetic instability. To develop a broad understanding of Tamoxifen resistance and to identify novel pathways and targets of aberrant methylation, DNA from MCF-7, TMX2-11, and TMX2-28 was analyzed using the Illumina HumanMethylation450 BeadChip. The three cell lines have similar hypermethylation patterns in 306,000 CpG sites and similar hypomethylation patterns in 132,000 CpG sites across the genome. A subset of CpG sites was identified where the two Tamoxifen-selected lines share a methylation pattern distinct from the MCF-7 parent line. When normalized to MCF-7, TMX2-11 and TMX2-28 share 722 hypermethylated and 65 hypomethylated CpG sites in the promoter region encompassing 539 and 55 genes respectively. Pathway analysis indicates the hypermethylated genes are involved in processes relevant to acquired Tamoxifen resistance. These pathways include cell signaling, adhesion, transcriptional activation and repression, differentiation, proliferation, and apoptosis. Ongoing studies on a subset of the genes found differentially methylated between parental and Tamoxifen-selected cell lines are aimed at confirming methylation findings and examining gene expression. Additionally, the cells will be treated with the demethylating agent 5-aza-deoxycitidine to determine whether reversal of the hypermethylation restores Tamoxifen sensitivity. Greater knowledge of the molecular modifications accompanying Tamoxifen-resistance is needed, as it will lead to discovery of new therapeutic targets and improved treatment, possibly allowing prediction of resistance from biopsy DNA. Citation Format: Kristin E. Williams, Douglas L. Anderton, Maxwell P. Lee, Brian T. Pentecost, Kathleen F. Arcaro. Epigenetic changes in breast cancer cells associated with acquired Tamoxifen resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5368. doi:10.1158/1538-7445.AM2013-5368

Abstract 776: Functional analysis of BRCA1 associated protein-1 (ubiquitin carboxy-terminal hydrolase) in melanoma.

Abstract In the US alone, 44,250 men and 32,000 women are expected to be diagnosed with melanoma in 2012. Metastatic melanoma is the leading cause of skin cancer-related deaths worldwide. In the metastatic cutaneous melanoma (CM), anti-BRAF and anti-MEK agents have recently been shown to attenuate progression of the disease, though durable responses are still rare (NEJM articles). For metastatic ocular melanoma (OM), there is currently no established treatment strategy. Thus, one major unmet need in the field is the identification of novel “driver” physiologies which underpin the progression of disease and which can be leveraged for therapeutic gain. One new component in the ubiquitination pathway that has been recently implicated in the biology of both CM and OM is BRCA1 associated protein-1 (BAP1). Preliminary data from our laboratory showed that germline inactivating mutations of BAP1 predispose individuals to both CM and metastatic OM along with other neoplasms. In this study we examined the effects of BAP1 knockdown and overexpression on melanoma cell lines and primary melanocytes. BAP1 gene is mutated in melanoma and reported to be tumor suppressor, however suppression of BAP1 led to paradoxical growth inhibition, suggesting that the effects of BAP1 may be context dependent. Currently we are performing global gene expression and allele specific functional studies in an effort to identify novel substrates and other pathways impacted by BAP1. These findings may provide mechanistic insight into the role of BAP1 in melanoma pathogenesis. Citation Format: Raj Kumar, Ching-Ni Njauw, Zhenyu Ji, Benchun Miao, Michael Taylor, Anpuchchelvi Rajadurai, Durga Udayakumar, Hensin Tsao. Functional analysis of BRCA1 associated protein-1 (ubiquitin carboxy-terminal hydrolase) in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 776. doi:10.1158/1538-7445.AM2013-776

Regulatory T cells get nervous in the melanoma microenvironment

Regulatory T cells get nervous in the melanoma microenvironment