Threshold Cycle Method Research Articles

Association of Higher Defensin β-4 Genomic Copy Numbers with Behçet's Disease in Iraqi Patients.

Behçet's disease (BD) is an immune-mediated small vessel systemic vasculitis. Human β-defensins are antimicrobial peptides associated with many inflammatory diseases and are encoded by the β-defensin family of multiple-copy genes. However, their role in BD necessitates further investigation. The aim of the present study was to investigate the possible association of BD in its various clinical forms with defensin β-4 (DEFB4) genomic copy numbers. This case-control study was conducted from January to September 2011 and included 50 control subjects and 27 unrelated Iraqi BD patients registered at Baghdad Teaching Hospital, Bagdad, Iraq. Copy numbers of the DEFB4 gene were determined using the comparative cycle threshold method by duplex real-time polymerase chain reaction technology at the Department of Dermatology of Jena University Hospital, Jena, Germany. DEFB4 genomic copy numbers were significantly higher in the BD group compared to the control group (P = 0.010). However, no statistically significant association was found between copy numbers and clinical variables within the BD group. The DEFB4 copy number polymorphism may be associated with BD; however, it is not associated with different clinical manifestations of the disease.

Differential Expression of Ribosomal Genes in Brain and Blood of Alzheimer's Disease Patients.

Changes in rRNA and rDNA expression have been associated with cellular and organism aging and have been linked to Alzheimer's disease (AD) pathogenesis. In this study, we investigated the mRNA expression of ribosomal genes (28S/18S) and β-amyloid precursor protein (APP) in different post mortem brain tissue regions (the entorhinal and auditory cortices and the hippocampus) of AD patients and elderly control subjects and also evaluated the extent of expression in peripheral blood from young, healthy, elderly, and Alzheimer's disease patients in order to investigate whether these individuals experienced the effects of aging. The comparative threshold cycle (CT) method via Real Time Polymerase Chain Reaction and the Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) were used to analyze gene expression and the Apolipoprotein E (APOE) genotype, respectively. When the brain areas were analyzed collectively, we observed a significant decrease in APP expression and a significant increase in levels of mRNA of 18S and 28S in Alzheimer's disease patients compared to healthy elderly individuals. Furthermore, there was a significant upregulation of 28SrRNA in the entorhinal cortex and hippocampus, but not in the auditory cortex of patients with AD. On the other hand, tests of blood samples verified a decreased expression of 28S rRNA in patients with AD. These results support the hypothesis that changes in rRNA are present in AD patients, are tissue-specific, and seem to occur independently and differently in each tissue. However, the next challenge is to discover the mechanisms responsible for the differences in expression observed in the blood and the brain in both healthy elderly individuals and Alzheimer's disease patients, as well as the impact of these genes on AD pathogenesis.

Investigation of fasciculation and elongation protein ζ-1 (FEZ1) in peripheral blood reveals differences in gene expression in patients with schizophrenia.

Schizophrenia (SZ) is a chronic neuropsychiatric disorder characterized by affective, neuromorphological and cognitive impairment, deteriorated social functioning and psychosis with underlying molecular abnormalities, including gene expression changes. Observations have suggested that fasciculation and elongation protein ζ-1 (FEZ1) may be implicated in the pathogenesis of schizophrenia. Nevertheless, our current knowledge of the expression of FEZ1 in peripheral blood of schizophrenia patients remains unclear. The purpose of this study was to identify the characteristic gene expression patterns of FEZ1 in peripheral blood samples from schizophrenia patients. We performed quantitative reverse-transcriptase (qRT-PCR) analysis using peripheral blood from drug-free schizophrenia patients (n = 29) and age and gender-matched general population controls (n = 24). For the identification of FEZ1 gene expression patterns, we applied a comparative threshold cycle (CT) method. A statistically significant difference of FEZ1 mRNA level was revealed in schizophrenia subjects compared to healthy controls (p = 0.0034). To the best of our knowledge, this study is the first describing a down-regulation of FEZ1 gene expression in peripheral blood of patients with schizophrenia. Our results suggested a possible functional role of FEZ1 in the pathogenesis of schizophrenia and confirmed the utility of peripheral blood samples for molecular profiling of psychiatric disorders including schizophrenia. The current study describes FEZ1 gene expression changes in peripheral blood of patients with schizophrenia with significantly down-regulation of FEZ1 mRNA. Thus, our results provide support for a model of SZ pathogenesis that includes the effects of FEZ1 expression.

In-Cell Detection By Flow Cytometry of JAK2 V617F Mutation Using Gold Nanoflares

Today’s conventional technology has to overcome many challenging aspects for integrating expressional analysis of a mutation into the cellular function of a living cell. It is very striking to detect the expressional status of a mutation in a living cell without compromising its cellular function as well as its viability that allows to perform subsequent functional analysis. This has been a long time goal for most of the biological studies.Myeloproliferative neoplasms (MPN) originate at the level of hematopoietic stem cell with a single nucleotide mutation in the cytosolic tyrosine kinase, Janus kinase 2 (JAK2) named as JAK2V617F which facilitates the constitutive intracellular activation of JAK2 and found in 95% of Polycythemia Vera (PV) patients. Detection of this mutation is a major criteria for diagnosis of the malignancy and changes in the expression levels of JAK2V617F is valued for the prognosis as well as response to the treatment.Here, we have utilized the nanotechnology’s new development, entitled as "multiplexed nanoflares", a gold nanoparticle agent that is capable of simultaneously detecting two distinct mRNA targets inside a living cell. These probes are highly oriented oligonucleotide sequences, which are hybridized to a reporter dye such as Cy3 and Cy5 and each complementary to its corresponding mRNA target. When multiplexed nanoflares are exposed to their targets, they provide a sequence specific signal in both extra and intracellular environments. Thus, this signal could be easily detected by flow cytometry or confocal microscopy.We have employed JAK2 V617F specific nanoflare probes labeled with Cy3 (JAK2V617F-Cy3) and JAK2 wild type (wtJAK2) nanoflare probe labeled with Cy5 (JAK2-Cy5) including negative and positive control probes to distinguish and isolate mRNA’s of JAK2V617F and wtJAK2 in live cells by measuring subsequent fluorescence with flow cytometry. The initial testing of the nanoflares has been performed by treating cell lines such as K562 (wtJAK2), SET-2 (JAK2V617F heterozygote), HEL (JAK2V617F homozygote) cells with nanoflare probes which did not alter cell viability and morphology with the applied concentrations during 18 hours of cell culture. We measured the levels of two mRNA targets, JAK2V617F and JAK2, and detected 90±6 % positivity with JAK2V617F-Cy3 treated HEL cells and no positivity in K562 cells, meanwhile wtJAK2 probe treated cells succeeded staining of SET and K562 cells. Real time reverse transcription PCR (RT-PCR) was performed on the same cells to compare the relative amounts of target RNA in each cell type by calculating relative changes in mRNA expression using the ΔΔCt (cycle threshold) method and comfirmed the results of flow cytometry with sensitivity of 0.001. Subsequently, we have tested mononuclear cells (MNC) received from peripheral blood of 5 patients with PV, one of which has JAK2V617F heterozygote allele (patient 5) and 4 patients had JAK2V617F homozygote allele (patients 1-4)which were tested with nested allel specific PCR. Flow cytomeyric analysis of the patients‘ MNC treated with the wtJAK2 and JAK2V617F probes demonstrated distinct populations of wtJAK2+ and JAK2V617F+. Populations of wtJAK2+/JAK2V617F+ (2.9%), wtJAK2-/JAK2V617F- ( 21.4%), wtJAK2+/JAK2V617F-( 0%), wtJAK2-/JAK2V617F+ ( 79%) of patient 5 and populations of JAK2V617F+ (36,8%± 5 ), JAK2V617F- of patients 1-4 were sorted by cell sorter with ~ 90% purity following detection and analysis of each populations for relative changes in mRNA expressions . The data revealed distinct ΔCt values between each populations.In this study, we have aimed for a new approach by utilizing nanotechnology aligned with the conventional techniques in order to save time, money and outcome. The capability of in-cell detection and isolation of the JAK2V617F and wtJAK2 first time by performing the expression analysis, changes the routine protocols backwards, enables the detecting direct effect of the mutation to the cells as well as other genes. This study creates a platform where functional analysis regarding to the more detailed understanding of the initiation of malignancy as well as responses to the therapies could be performed. DisclosuresNo relevant conflicts of interest to declare.

Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis (Bangiales, Rhodophyta) by quantitative real-time PCR

Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes (18S rRNA (18S), ubiquitin-conjugating enzyme (UBC), actin (ACT), β-tubulin (TUB), elongation factors 2 (EF2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold (C t) method and by two different software packages: geNorm and Norm-Finder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.

Expression of Eph A4, Eph B2 and Eph B4 receptors in AML.

Eph receptors represent the largest subfamily of receptor tyrosine kinases (RTKs). The up- regulation of Eph receptors has been documented in various solid tumors, where it often correlates with poor prognosis. Their significance in hematologic malignancies is still unclear. This study aimed to investigate the expression of Eph A4, Eph B2, and Eph B4 mRNA in non - M3 AML patients and determine their prognostic significance. Bone marrow samples from 101 newly diagnosed non - M3 AML patients and 26 healthy controls for comparison were quantified by real time reverse transcriptase polymerase chain reaction (RT-PCR), and the comparative cycle threshold (Ct) method was used to determine their relative expression levels to GUS control gene. The results showed that expression of all selected Eph receptors was significantly lower in AML patients comparing to controls. It also differed according to FAB subtypes. The decreased expression levels of Eph A4 were associated with higher leukocytes (p = 0.022) and blast cell counts (p = 0.001), and unfavorable FLT3-ITD mutation. Our study revealed significant correlation between lower EphB2 expression levels, and higher complete remission rate (p = 0.009724) and longer overall survival. Additionally, we found that patients with shorter RFS had decreased EphB4 expression (p = 0.00). In conclusion, the results suggest the prognostic impact of decreased expression levels of some Eph receptors in AML patients.

Identification of Ligand Specificity Determinants in Lantibiotic Bovicin HJ50 and the Receptor BovK, a Multitransmembrane Histidine Kinase

Lantibiotic bovicin HJ50 is produced by Streptococcus bovis HJ50 and acts as the extracellular signal to autoregulate its own biosynthesis through BovK/R two-component system. Bovicin HJ50 shows a linear N-terminal and glubolar C-terminal structure, and the sensor histidine kinase BovK contains eight transmembrane segments lacking any extensive surface-exposed sensory domain. The signal recognition mechanism between bovicin HJ50 and BovK is still unknown. We performed saturated alanine scanning mutagenesis and other amino acid substitutions on bovicin HJ50 using a semi-in vitro biosynthesis. Results of the mutants inducing activities indicated that several charged and hydrophobic amino acids in ring B of bovicin HJ50, as well as two glycines were key residues to recognize BovK. Circular dichroism analyses indicated that both glycines contributed to bovicin HJ50 structural changes in the membrane. Biotin-labeled bovicin HJ50 could interact with the N-terminal sensor of BovK, and several charged residues and a conserved hydrophobic region in the N-terminal portion of BovK sensor domain were important for interacting with the signal bovicin HJ50. By combining the results, we suggested a mechanism of bovicin HJ50 recognizing and activating BovK mainly through electrostatic and hydrophobic interactions.

PU.1 promotes cell cycle exit in the murine myeloid lineage associated with downregulation of E2F1

Acute myeloid leukemia (AML) is characterized by increased proliferation and reduced differentiation of myeloid lineage cells. AML is frequently associated with mutations or chromosomal rearrangements involving transcription factors. PU.1 (encoded by Sfpi1) is an E26 transformation-specific family transcription factor that is required for myeloid differentiation. Reduced PU.1 levels, caused by either mutation or repression, are associated with human AML and are sufficient to cause AML in mice. The objective of this study was to determine whether reduced PU.1 expression induces deregulation of the cell cycle in the myeloid lineage. Our results showed that immature myeloid cells expressing reduced PU.1 levels (Sfpi1(BN/BN) myeloid cells) proliferated indefinitely in cell culture and expanded invivo. Transplantation of Sfpi1(BN/BN) cells induced AML in recipient mice. Cultured Sfpi1(BN/BN) cells expressed elevated messenger RNA transcript and protein levels of E2F1, an important regulator of cell cycle entry. Restoration of PU.1 expression in Sfpi1(BN/BN) myeloid cells blocked proliferation, induced differentiation, and reduced E2F1 expression. Taken together, these data show that PU.1 controls cell cycle exit in the myeloid lineage associated with downregulation of E2F1 expression.

Real-time quantitative PCR assays for evaluation of soybean varieties for resistance to the stem and root rot pathogen Phytophthora sojae

Phytophthora root and stem rot caused by Phytophthora sojae is one of the most destructive disease of soybeans in the world. Effective management of the disease depends on selection and use of soybean varieties resistant to the disease. Fast and reliable procedures are vital to screen soybean varieties against the pathogen. Novel real-time quantitative (qPCR) assays were developed for both absolute and relative quantification of P. sojae in infected root tissues. QPCR assays were based on the detection of the internal transcribed spacer (ITS) gene of the pathogen and 18S ribosomal gene of the host plant. Absolute qPCR allowed the detection of as low as 10 femtograms (fg) of P. sojae DNA in soybean roots. Relative qPCR, employing the comparative threshold cycle (Ct) method, was effective and reliable for quantification of P. sojae DNA normalized to plant DNA in infected soybean root tissues. P. sojae DNA quantities detected in both qPCR assays had high correlations with disease severity index (DSI) ratings of soybean varieties. QPCR assays developed in this study were useful for determination of the levels of P. sojae DNA in different varieties of soybean and for evaluation of them for relative resistance to the pathogen.

A New Method for Quantitative Real-Time Polymerase Chain Reaction Data Analysis

Quantitative real-time polymerase chain reaction (qPCR) is a sensitive gene quantification method that has been extensively used in biological and biomedical fields. The currently used methods for PCR data analysis, including the threshold cycle method and linear and nonlinear model-fitting methods, all require subtracting background fluorescence. However, the removal of background fluorescence can hardly be accurate and therefore can distort results. We propose a new method, the taking-difference linear regression method, to overcome this limitation. Briefly, for each two consecutive PCR cycles, we subtract the fluorescence in the former cycle from that in the latter cycle, transforming the n cycle raw data into n-1 cycle data. Then, linear regression is applied to the natural logarithm of the transformed data. Finally, PCR amplification efficiencies and the initial DNA molecular numbers are calculated for each reaction. This taking-difference method avoids the error in subtracting an unknown background, and thus it is more accurate and reliable. This method is easy to perform, and this strategy can be extended to all current methods for PCR data analysis.

A gene expression profile indicative of early stage HER2 tyrosine kinase inhibitor response.

e11536 Background: Lapatinib, afatinib and neratinib are tyrosine kinase inhibitors (TKIs) of HER2 and EGFR growth receptors. A panel of breast cancer cell lines was treated with these agents and gefintib (EGFR inhibitor) and the expression pattern of a specific panel of genes investigated as a potential marker of early drug response. Methods: RNA was extracted from breast cancer cell lines (BT474, SKBR3 and MDAMB453) with differing HER2 expression patterns and sensitivity to lapatinib before and 12hrs after treatment with 1 µM of lapatinib, 150nM of afatinib, 150nM of neratinib or 1µM of gefitinib. Gene expression changes were measured by Taqman RT-PCR and RQ values were determined using the comparative cycle threshold (Ct) method. Results: The expression of RB1CC1, ERBB3, FOXO3a, NR3C1 was directly correlated with the degree of sensitivity of the cell line to lapatinib and was observed to “switch” from up-regulated to down-regulated in the HER2 expressing lapatinib insensitive cell line (MDAMD453). The CCND1 gene (functionally linked to the other four genes) demonstrated an inversely proportional response to drug exposure; showing a trend of strong down-regulation in lapatinib-sensitive lines. A similar expression pattern was observed following the treatment with both neratinib and afatinib. In contrast, gefitinib treatment, resulted in a completely different expression pattern change. Conclusions: In these HER2-expressing cell models, lapatinib, neratinib and afatinib treatment generated a common, characteristic and specific gene expression response, proportionate to the sensitivity of the cell lines to the HER2 inhibitor. Characterisation of changes in these genes shortly after drug treatment may therefore give a valuable predictor of the likely extent and specificity of tumour HER2 inhibitor response in patients, potentially guiding more specific use of these agents.

Distinct Renal Pathology and a Chemotactic Phenotype after Enterohemorrhagic Escherichia coli Shiga Toxins in Non-Human Primate Models of Hemolytic Uremic Syndrome

Enterohemorrhagic Escherichia coli cause approximately 1.5 million infections globally with 176,000 cases occurring in the United States annually from ingesting contaminated food, most frequently E. coli O157:H7 in ground beef or fresh produce. In severe cases, the painful prodromal hemorrhagic colitis is complicated by potentially lethal hemolytic uremic syndrome (HUS), particularly in children. Bacterial Shiga-like toxins (Stx1, Stx2) are primarily responsible for HUS and the kidney and neurologic damage that ensue. Small animal models are hampered by the inability to reproduce HUS with thrombotic microangiopathy, hemolytic anemia, and acute kidney injury. Earlier, we showed that nonhuman primates (Papio) recapitulated clinical HUS after Stx challenge and that novel therapeutic intervention rescued the animals. Here, we present detailed light and electron microscopic pathology examination of the kidneys from these Stx studies. Stx1 challenge resulted in more severe glomerular endothelial injury, whereas the glomerular injury after Stx2 also included prominent mesangiolysis and an eosinophilic inflammatory infiltration. Both toxins induced glomerular platelet-rich thrombi, interstitial hemorrhage, and tubular injury. Analysis of kidney and other organs for inflammation biomarkers showed a striking chemotactic profile, with extremely high mRNA levels for IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1α and elevated urine chemokines at 48hours after challenge. These observations give unique insight into the pathologic consequences of each toxin in a near human setting and present potential pathways for therapeutic intervention.

206 EFFECT OF OVARIAN HYPERSTIMULATION ON CIRCULATORY microRNA PROFILE IN BOVINE FOLLICULAR FLUID AND BLOOD PLASMA

MicroRNAs (miRNA) are noncoding small RNA that are known to play a role in posttranscriptional regulation of genes involved in various physiological processes including disease and reproduction. The circulatory forms of miRNA, which are present in body fluids, are reported to be used as biomarkers for disease and pregnancy. This study was conducted to investigate the effect of ovarian hyperstimulation on the expression pattern of circulatory miRNA in blood plasma and follicular fluid. For this, one group of Simmental heifers (n = 6) was hyper-stimulated using a superovulation (SO) protocol of 8 consecutive FSH injections over 4 days in decreasing doses, and others (n = 6) were synchronized (SY) using a standard synchronization protocol. Following this, the blood samples were collected at Day 0 (onset of oestrus) and Day 7, and follicular fluid was collected from both groups at Day 0 of the oestrous cycle. Total RNA, including small RNA, was then isolated from follicular fluid and blood plasma using a miRNeasy mini kit (Qiagen, Valencia, CA, USA) and subsequently used for circulatory miRNA expression studies using the human miRCURY LNA™ Universal RT miRNA PCR array system (Exiqon, Woburn, MA, USA). Following the miRNA PCR array run, data analysis was performed using a delta threshold cycle (ΔCT) method. Results showed that 23 miRNAs were found to be differentially expressed in blood plasma (fold change ≥2 and P ≤ 0.05) between SO and SY groups. Among these, 8 miRNAs including miR-127-3p, miR-494, miR-147, miR-134, and miR-153 were downregulated and 15 miRNAs including miR-34a, miR-103, miR-181c, miR-24-2-3p, miR-18a-3p, miR-20b-3p, and miR-708-3p were found to be upregulated in SO groups. Similarly, in follicular fluid, 71 miRNAs were found to be differentially expressed between the SO and SY groups. Of these, 33 miRNA including miR-100, miR-877, miR-659, miR-200c, miR-29b-2-3p, miR-361-5p, and miR-145 were downregulated, whereas 38 miRNAs including miR-374a, miR-720, miR-155-3p, miR-202-3p, miR-33b, miR-12-3p, and miR-224-3p were upregulated in follicular fluid aspirated from SO cows compared with the SY animals. Ingenuity pathway analysis of predicted target genes of miRNA, which are dysregulated due to ovarian hyperstimulation, showed the dominance of pathways related to neuregulin signalling, axonal guidance signalling, GNRH signalling, and Wnt β-catenin signalling pathways. In conclusion, this study revealed alternation in circulatory miRNA expression profile both in blood plasma and follicular fluid as a result of ovarian hyperstimulation.

207 CIRCULATORY microRNA SIGNATURES IN FOLLICULAR FLUID IN RELATION TO THE GROWTH STATUS OF BOVINE OOCYTES

Cell-to-cell communication within the follicle involves many signalling molecules, and this process is believed to be mediated by secretion and uptake of exosomes that contained several bioactive molecules including circulatory microRNAs (miRNA). The present study was conducted to investigate the circulating miRNA expression pattern in exosome and nonexosomal portion of follicular fluid (FF) in follicles with fully grown or growing oocytes. For this, the FF and cumulus–oocyte complexes (COC) were retrieved from 5- to 8-mm individual follicles from ovaries obtained from local abattoir. Then, the oocytes were subjected to brilliant cresyl blue (BCB) staining and classified as BCB+ (fully grown oocytes) and BCB– (growing oocytes). Accordingly, the corresponding FF was classified as BCB+ and BCB– based on their oocyte source. Following this, the exosomes were trapped from each FF categories using ExoQuick™ (SBI System Bioscience). Thus, total RNA was isolated from exosomal and nonexosomal portion of the FF using miRNeasy mini kit (Qiagen) and subjected to miRNA expression studies. The human miRCURY LNA™ Universal RT miRNA PCR array system (Exiqon) was used for miRNA expression profiling. Data analysis was performed using a comparative threshold cycle (ΔCT) method. The results revealed that 26 miRNAs were found to be differentially expressed (fold change ≥2 and P < 0.05) between the exosomal portion of FF from fully grown and growing oocyte groups. Among these, 17 miRNA including miR-608, miR-654-5p, miR-640, miR-582-5p, miR-449b, miR-155 were upregulated and 9 miRNA including miR-373, miR-526b*, miR-33a*, miR-30b, miR-29a* were downregulated in exosomal portion of FF of growing oocyte groups. The ingenuity pathway analysis of genes predicted to be targeted by those miRNAs were found to be involved in WNT/β catenine, purine metabolism, protein ubiquitination, and cAMP-mediated signalling pathways. Similarly, 36 miRNA were differentially expressed between the non-exosomal portion of FF of fully grown and growing oocytes. From those, 27 miRNA including let-7i*, miR-328, miR-223, miR-19b-1*, miR-423-5p, miR-29c, miR-659 were upregulated, whereas the expression level of 9 miRNA including miR-381, miR-18a*, miR-30e*, miR-934, and miR-302c was downregulated in the nonexosomal portion of FF of growing oocyte groups. In addition, the ingenuity pathway analysis indicated that the genes predicted to be targeted by these miRNA were found to be involved in NRF2-mediated oxidative stress response, tight junction signalling, and protein ubiquitination pathways. In conclusion, this study detected the presence of exosome or non-exosome-mediated circulation of miRNA in the bovine follicular fluid and oocyte growth-dependent variation of circulatory miRNA in the follicular environment.

ANTI-Leukemic Activity of DNA Methyltransferase Inhibitors Azacitidine and Decitabine On AML1-ETO Positive CELLS Is Evident At LOW Doses and Selectively Mediated by Epigenetic Mechanisms

AbstractAbstract 4615 Introduction.Alterations in chromatin organization are a common mechanism of leukemogenesis. Drugs affecting epigenetics have been proposed as therapy of hematopoietic neoplasms. We have previously shown selective activity of histone deacetylase (HDAC) inhibitors on AML1/ETO positive cells (Barbetti, et al- Oncogene, 2007). The AML1/ETO multiprotein complex induces and stabilizes the local DNA methylation pattern by recruiting HDACs and DNA methyl-transferase-1 (DNMT1), resulting in the stable silencing of AML1-controlled genes, like IL3. Our hypothesis is that DNMT inhibitors (DNMTi) 5-Azacytidine (AZA) and Decitabine (DAC) exert a targeted action on AML1/ETO positive leukemic cells. Methods.We used U937-A/E-9/14/18 cell line, carrying a ponasterone-A inducible construct of AML1/ETO (kindly provided by M. Luebbert). We analyzed the effects of AZA and DAC at different doses (0.01, 0.1, 1 and 10μM), evaluating cell viability and apoptosis (by Annexin V assay, and by WB caspase 9, 8, 3 -cleavage).Cells were then treated with AZA 0.1–1μM or DAC 0.1–0.01μM for 24h. We determined by chromatin immunoprecitation (ChIP) assay the association of the IL3 promoter with H4acetylated, H3K4me3, H3K9me2 and H3K27me3 and the quantitative expression of IL3 by Q-RT PCR, calculated by a comparative threshold cycle method, using the formula 2′(−ΔΔCt). Other promoters controlled by AML1/ETO, like those of GM-CSF, MPO and CEBPalpha were also evaluated. Methylation of IL3 promoter was evaluated by Methylation Specific PCR (MSP).Confirmatory experiments were carried out in the human leukemic cell line Kasumi-1 naturally expressing AML1/ETO. Results.Although both DNMTi were active on leukemic cells, irrespective of the presence of AML1/ETO expression, the dose at which this effect was evident was differing of one logarithm. AZA 0.1μM significantly increased the percentage of apoptotic cells only in U937 AML1/ETO positive cell line. We observed that untreated apoptotic cells percentage was 4.72% (±0.55 Standard Deviation: SD) while after AZA 0.1μM was 5.70% (± 0.24 SD) (P<0.01). In AML1/ETO negative cells AZA 0.1μM did not induce a significant increase of apoptosis (P>0.05). These data were confirmed by the induction of cleavage of caspase 9, 8 and 3. DAC 0.01μM significantly induced apoptosis cells only in U937 AML1/ETO positive cells (NT= 6.27% ±0.24 SD; DAC 0.01μM = 7.50% ±0.57 SD; P<0.01).Given these results, we investigated whether this different response was correlated with chromatin rearrangements directly involving the AML1/ETO gene.We evaluated IL3 expression after AZA and DAC treatments, which induced a significant increase, higher in AML1/ETO-expressing than in non-expressing U937 cells. In fact, in AML1/ETO positive cells the amount of IL3 mRNA 2′(−ΔΔCt) after AZA and DAC respect to NT was 10.5 ± 0.3 DS and 6.1± 0.5 SD, respectively. In AML1/ETO negative cells, after AZA was 5.70 ±2.7 SD (P>0.05) and after DAC 2.28 ± 0.4 SD (P>0.05), both respect to NT.By ChIP assay we showed that in untreated leukemic cells, both in U937 AML1/ETO-positive and negative cells, at baseline H3K4me3 was not associated with the IL3 promoter, while H3K9me2 was associated with it. H4-acetylated became associated to the IL3 promoter after AZA and DAC treatments but exclusively in U937 cells expressing AML1/ETO. On the other hand, H3K27me3 was co-immunoprecipitated with the IL3 promoter in U937 AML1/ETO negative and in U937 AML1/ETO positive untreated cells. AZA and DAC treatments led to the dissociation of this modified histone from the IL-3 promoter indicating the modification of chromatin rearrangement (Fig 1). Conclusions.Our observations demonstrate that AML1/ETO positive cells are more sensitive to DNMTi than cells not carrying such chimeric gene, in terms of biological effects like cytotoxicity and induction of apoptosis. The dissociation of the IL3 promoter from H3K27me3 and its association with H4acetylated upon DNMTi exposure is observed exclusively in U937 AML1/ETO-positive cells and clearly indicates that the epigenetic mechanism of action of these drugs is exerted selectively on cells harbouring the chimeric protein. This result could support a working hypothesis to develop a treatment for AML1/ETO-positive leukaemias including DNMTi in the therapeutic armamentarium. [Display omitted] Disclosures:Santini:Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria.

Role of transcriptional corepressor ETO2 in erythroid cells

Transcriptional corepressor ETO2 is a component of a protein complex containing master regulators of hematopoiesis, including GATA-1, SCL/TAL1, LMO2, and LDB1. To elucidate the role of ETO2 during erythroid differentiation, including the effects of ETO2 on GATA-1 targets, we performed gene expression profiling using K562 cells overexpressed with ETO2. The analysis demonstrated that 667 and 598 genes were upregulated and downregulated (more than twofold), respectively, in ETO2-overexpressing cells. ETO2-repressed genes included those encoding prototypical erythroid proteins. To test what percentages of ETO2-repressed genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n=5,749), demonstrating that 23.1% of ETO2-repressed genes contained significant GATA-1 in their loci. However, there was no significant enrichment of PU.1 target genes among ETO2-repressed genes. Gene ontology analysis among ETO2-repressed genes revealed significant enrichment of genes related to "oxygen transporter," corresponding to globin genes. Quantitative chromatin immunoprecipitation and ETO2 knockdown analyses confirmed that ETO2 directly regulates globin genes in K562 cells. Next, we evaluated the role of ETO2 in human primary erythroblasts, derived from cord blood CD34-positive cells. In an exvivo model of erythroid differentiation from CD34-positive cells, ETO2 protein level peaked at day 2-4 and almost diminished at the later stage of differentiation. Furthermore, short hairpin RNA-mediated knockdown and retroviral vector-mediated overexpression of ETO2 in primary erythroblasts suggested that ETO2 significantly represses HBB, HBA, and ALAS2 expression. In summary, ETO2 regulates GATA-1 target genes critical for erythroid differentiation, and the decrease of ETO2 levels during erythroid differentiation would contribute to the activation of these targets.

L-Dopa decarboxylase (DDC) constitutes an emerging biomarker in predicting patients’ survival with stomach adenocarcinomas

Stomach adenocarcinoma represents a major health problem and is regarded as the second commonest cause of cancer-associated mortality, universally, since it is still difficult to be perceived at a curable stage. Several lines of evidence have pointed out that the expression of L-Dopa decarboxylase (DDC) gene and/or protein becomes distinctively modulated in several human neuroendocrine neoplasms as well as adenocarcinomas. In order to elucidate the clinical role of DDC on primary gastric adenocarcinomas, we determined qualitatively and quantitatively the mRNA levels of the gene with regular PCR and real-time PCR by using the comparative threshold cycle method, correspondingly, and detected the expression of DDC protein by immunoblotting in cancerous and normal stomach tissue specimens. A statistically significant association was disclosed between DDC expression and gastric intestinal histotype as well as tumor localization at the distal third part of the stomach (p = 0.025 and p = 0.029, respectively). Univariate and multivariate analyses highlighted the powerful prognostic importance of DDC in relation to disease-free survival and overall survival of gastric cancer patients. According to Kaplan-Meier curves, the relative risk of relapse was found to be decreased in DDC-positive (p = 0.031) patients who, also, exhibited higher overall survival rates (p = 0.016) than those with DDC-negative tumors. This work is the first to shed light on the potential clinical usefulness of DDC, as an efficient tumor biomarker in gastric cancer. The provided evidence underlines the propitious predictive value of DDC expression in the survival of stomach adenocarcinoma patients.

Concentration-dependent effects of tiludronate on equine articular cartilage explants incubated with and without interleukin-1β

To determine concentration-dependent effects of tiludronate on cartilage explants incubated with or without recombinant equine interleukin-1β (rEq IL-1). Articular cartilage explants from the femorotibial joints of 3 young adult horses. Cartilage explants were incubated with 1 of 6 concentrations (0, 0.19, 1.9, 19, 190, or 1,900 mg/L) of tiludronate and with or without rEq IL-1 (0.01 ng/mL) for 96 hours. Prostaglandin E(2) (PGE(2)) concentrations in culture medium and explant digests were analyzed via PGE(2) enzyme immunoassay. Sulfated glycosaminoglycan (sGAG) concentrations in culture medium were quantified via 1,9-dimethylmethylene blue assay. Chondrocyte apoptosis in paraffin embedded explant sections was measured via terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Relative gene expression of matrix metalloproteinases (MMPs), interleukin (IL)-6, and IL-8 was determined via the comparative cycle threshold method. rEq IL-1 increased PGE(2) concentration, sGAG release from explants, chondrocyte apoptosis, and MMP gene expression. Lower tiludronate concentrations reduced rEq IL-1-induced sGAG release and chondrocyte apoptosis, whereas the higher tiludronate concentrations increased sGAG release and chondrocyte apoptosis. At the highest tiludronate concentration evaluated, IL-8 gene expression was increased independent of whether rEq IL-1 was present. Tiludronate had biphasic concentration-dependent effects on cartilage explants that were independent of PGE(2) secretion or MMP gene expression. Low tiludronate concentrations had some chondroprotective effects, whereas high tiludronate concentrations were detrimental to equine articular cartilage. Administration of tiludronate intra-articularly to horses may be detrimental, dependent on the dose used. In vivo studies are needed before intra-articular tiludronate administration to horses can be recommended.

Some Novel Insights on HPV16 Related Cervical Cancer Pathogenesis Based on Analyses of LCR Methylation, Viral Load, E7 and E2/E4 Expressions

This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1∧E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1∧E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 CT/E4 CT. Relative quantification based on comparative CT (theshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 CT (p = 0.007) and E2 CT (p<0.0001), respectively, each normalized with ACTB CT, among episomal cases only. The k-means clustering analysis considering E7 CT from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.

P118 IFN-A transcriptional response in endothelial cells: Key modulators and gene regulatory modules

Introduction Inference of gene regulation from expression data may help to unravel regulatory mechanisms involved in complex diseases or in the action of specific drugs. Interferon-α (IFN-α) is a pleiotropic cytokine endowed with potent biological activities, achieved through the up-regulation of hundreds of interferon-stimulated genes (ISGs). Aim of this study was to extend previous work on IFN-α transcriptional response in endothelial cells, by identifying IFN-α modulators and inferring putative gene regulatory modules involving them. Methods Primary endothelial cells (HUVEC) were pulsed in vitro with human recombinant IFN-α under different perturbation conditions: siRNA inactivation of target genes. A panel of 96 transcripts, including ISGs belonging to IFN-α top signature and genes related to the IFN-α signalling pathway, were screened by custom TaqMan Low-Density Arrays. Stealth siRNA were used for RNAi-mediated knockdown of seven candidate IFN-α modulators: STAT1, IRF1, IRF7, GBP1, OAS2, IFIH1 and USP18. The effects of each siRNA were evaluated with respect to a calibrator siRNA using the comparative threshold cycle method (ΔΔCT method). A selection procedure, testing the null hypothesis of no difference in the effects of a siRNA targeting a candidate modulator and the calibrator siRNA, was used to assign a significance value (p-value) to each regulation. Significant regulations were defined fixing a cut-off of 0.05 on the Bonferroni corrected p-values. Regulatory modules, built by different feed-forward loops (FFLs), the basic three-node building blocks of biological networks, were then inferred by combining the significant regulations, induced by different couples of IFN-α modulators. Results Our analysis showed both mainly positive (STAT1, IFIH1) and mainly negative (USP18, GBP1, IRF1) modulators of IFN-α transcriptional response, while IRF7 and OAS2 were found to act equally in both directions. STAT1 was confirmed as the primary positive modulator of IFN-α; following its silencing several genes were significantly down-regulated and only few genes up-regulated. Conversely, USP18 was found to be the strongest negative modulator and its silencing caused a massive up-regulation of genes, including some involved in cell-to-cell adhesion. An interesting regulatory module in which STAT1 activates IFIH1 and they both regulate six genes, including IFNα-R1, was reconstructed by our inference method. Some of the inferred FFLs are currently being validated. Conclusion The investigation of IFN-α transcriptional modulators through our method has led to a clear picture, depicting the transcriptional impact of each considered modulator in terms of sign of regulation, strength and target genes. The inferred gene regulatory modules, involving key modulators, may help to unravel the mechanisms of action of IFN-α and discover future target of intervention in knock-out experiments.