Putative Binding Research Articles

Regulation of 1-aminocyclopropane-1-carboxylate oxidase gene expression during leaf ontogeny in white clover

The expression of two 1-aminocyclopropane-1-carboxylate (ACC) oxidase (E.C. 1.4.3)(ACO) genes, TR-ACO2 and TR-ACO3 from white clover has been examined in leaf tissue in response to physiological stimuli. In detached mature-green leaves, the expression of TR-ACO3 (a leaf-senescence-associated ACC oxidase) is induced over a 6 h time-course, while expression of TR-ACO2 (expressed constitutively in mature-green leaf tissue) decreased over the same time-course. An almost identical pattern of expression was observed with detached mature-green leaves treated with ethylene, while wounding also did not influence the timing of TR-ACO3 induction, significantly. No increase in overall ACO enzyme activity was detectable in crude extracts from wounded and detached leaf tissue, but after separation of these extracts using hydrophobic interaction column chromatography, two ACO isoforms were identified in non-wounded mature-green leaves (designated MGI and MGII), and the specific activity of one of these (MGII) increased in wounded tissues. In attached leaf tissue, TR-ACO2, but not TR-ACO3 expression was induced by treatment with ethylene; an induction that was not observed with prior treatment of plants with 1-MCP. This induction in response to ethylene was also observed at the translational level using antibodies to TR-ACO2. Analysis, in silico, of the 5′ flanking sequences to identify putative transcriptional binding domains reveals that both the TR-ACO2 and TR-ACO3 5′ flanking sequence contain domains that are associated with an ethylene-response. The significance of these observations in terms of how the expression of these ACO genes is regulated during leaf ontogeny in vivo is discussed.

Caveolin-1 influences human influenza A virus (H1N1) multiplication in cell culture

BackgroundThe threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication.ResultsUsing a motif-based search strategy for antiviral targets we identified caveolin-1 (Cav-1) as a putative cellular interaction partner of human influenza A viruses, including the pandemic influenza A virus (H1N1) strains of swine origin circulating from spring 2009 on. The influence of Cav-1 on human influenza A/PR/8/34 (H1N1) virus replication was determined in inhibition and competition experiments. RNAi-mediated Cav-1 knock-down as well as transfection of a dominant-negative Cav-1 mutant results in a decrease in virus titre in infected Madin-Darby canine kidney cells (MDCK), a cell line commonly used in basic influenza research as well as in virus vaccine production. To understand the molecular basis of the phenomenon we focussed on the putative caveolin-1 binding domain (CBD) located in the lumenal, juxtamembranal portion of the M2 matrix protein which has been identified in the motif-based search. Pull-down assays and co-immunoprecipitation experiments showed that caveolin-1 binds to M2. The data suggest, that Cav-1 modulates influenza virus A replication presumably based on M2/Cav-1 interaction.ConclusionAs Cav-1 is involved in the human influenza A virus life cycle, the multifunctional protein and its interaction with M2 protein of human influenza A viruses represent a promising starting point for the search for antiviral agents.

Subcellular localization of Mayven following expression of wild type and mutant EGFP tagged cDNAs

BackgroundProcess formation by glial cells is crucial to their function. Mayven, an actin binding, multi-domain polypeptide, and member of the BTB-BACK-Kelch family have been shown to be important in oligodendrocyte process extension. To assess the role of Mayven in neural cell process extension we have tracked the subcellular distribution of exogenous Mayven following expression of a rat Mayven -EGFP cDNA in a variety of neural cell backgrounds and specifically in OEC tranfectants following drug treatment to disrupt the integrity of the cytoskeleton. A comparison was made between the subcellular localization following transient transfection of OECs with full-length Mayven cDNA and a series of mutant domain constructs.ResultsThe subcellular location of Mayven in OEC transfectants showed a characteristic distribution with intense foci of staining towards the process tips corresponding to regions of accumulated Mayven overlapping in part with lammelipodial actin and was absent from the filipodia and the outer membrane. This signature pattern was also observed in Schwann cells, Oli-Neu cells, astrocytes and the neuroblastoma cell line B104 transfectants and resembled the exogenous and endogenous Mayven distribution in oligodendrocytes. This contrasted with the localization pattern in non-neural cells. There was a re-localization of Mayven in OEC transfectants following drug treatment to challenge the integrity of the actin cytoskeleton while breakdown of the microtubular component had no discernible impact on the accumulation of Mayven in the process tips. Deletion of the first three amino acids of the SH3 motif of the putative Fyn Kinase binding domain at the amino terminus significantly compromised this signature pattern as did the removal of the last Kelch repeat unit of six unit Kelch domain comprising the carboxyl terminus. In addition, there was a reduction in process length in mutant transfectants. Co-expression studies with a haemagglutinin (HA) tagged wild type Mayven cDNA and EGFP tagged mutant cDNAs suggested a homomeric interaction mediated by the BTB/POZ domain.ConclusionsExogenous Mayven is transported to the lamellipodia in neural transfectants associating with the actin cytoskeletal network. In addition to the importance of the internal BTB/POZ domain, this subcellular distribution pattern is dependent on the presence of an intact amino and carboxyl terminus.

S1703 The Mechanism of Transfomer 2-β Gene Expression in Response to Oxidative Stress in Human Colon Epithelial Cells

significantly. Lung apelin mRNA levels were unaffected. Exp 2: In Vitro hyperoxia reduced ileal cell apelin mRNA levels by 50% (P < 0.05). Exp 3: Overexpression of NF-κB reduced rat apelin core promoter activity by 60-80%. Mutation of putative NF-κB binding sites impaired NF-κB-induced reduction in apelin core promoter activity. Conclusions and Discussion. Our findings demonstrate that hyperoxia decreases intestinal apelin expression in the rat. NF-κB may play a role in the hyperoxia-induced reduction in intestinal apelin expression since NF-κB is activated by hyperoxia and overexpression of NF-κB downregulates apelin transcriptional activity. More importantly, these findings suggest that a reduced apelin expression may influence organogenesis in infants exposed to postnatal hyperoxia. It is well known that hypoxia upregulates apelin expression though HIF. This is the first demonstration of down-regulated apelin expression by hyperoxia.

S1635 Loss of Bile Acid Responsive Gene Retinoic Acid Receptor-Related Orphan Receptor A (Rora) in Barrett's Metaplasia, Implications for Refluxed Induced Signaling in Esophageal Cells

Introduction The involvement of gastro-oesophageal-reflux-disease in the development of Barrett9s metaplasia (BM) is well established. Recent evidence has begun to implicate bile acids such as the secondary bile acid deoxycholic acid (DCA) in BM and subsequent carcinogenesis. Our laboratory has identified the transcription factor retinoic acid receptor-related orphan receptor A (RORA) as a novel bile acid responsive gene with altered expression in BM through a novel integrative transcriptomic profiling approach. RORA, a putative tumour suppressor gene, has no established target hormone and is postulated to act independently from putative hormone binding. We now aim to validate RORA expression, induction and implications to oesophageal cell signalling. Methods Oesophageal cell lines HET-1A (normal-squamous), QhTRT (metaplastic), GohTRT (dysplastic) and SKGT4(adenocarcinoma) were utilised. Expression of RORA was determined by semi-quantitative real-time RT-PCR (ABI) in BM (n=16) and control patients (n=14) and the above cell lines. RORA protein was examined by Western blotting utilising a polyclonal antibody specific to RORA (Abcam). Results A significant reduction in RORA levels, as determined by real-time RT-PCR, was observed in a cohort of BM patients (p Conclusion This study for the first time demonstrates a loss of a critical regulator of cell growth, circadian rhythm and inflammation, RORA, in BM. Additional work described the ability of refluxed agents, such as DCA, to induce RORA in oesophageal cells. It is likely that RORA loss in BM may allow for dysregulated cellular growth, impact upon responses to nocturnal reflux through alterations in circadian rhythm and for resistance to refluxed induced damage in BM.

Abstract 4723: Common low penetrance allelic variants for chronic lymphocytic leukemia identified in a genome-wide association study also predict outcome

Abstract B-cell chronic lymphocytic leukemia (CLL) is the most common lymphoid malignancy in the western world. Although strong evidence for an inherited susceptibility is provided by first-degree relatives of CLL patients having &amp;gt;5-fold increased risk of themselves developing CLL, the genetic basis of CLL predisposition is principally unknown. Low penetrance risk alleles for CLL have been identified at 11q24.1 (rs735665, GRAMD1B), 15q23 (rs7176508), 2q37.1 (rs13397985, SP140), 2q37.3 (rs757978, FARP2), 2q13 (rs17483466, ACOXL) and 6p25.3 (rs872071, IRF4). The strongest evidence for association was for a single nucleotide polymorphism (rs872071) in the 3′ UTR of the gene encoding the interferon regulatory factor-4 (IRF4) transcription factor. Given a role in determining risk of disease it is plausible that these allelic variants also play a role in disease progression and overall survival. To test this hypothesis, polymorphic status was determined in a large case series of 403 patients diagnosed with CLL recruited via 5 UK clinical centers, and correlated with established prognostic markers and clinical outcome. Variant IRF4 (defined by rs872071) predicts for a more aggressive disease course; carriers of the disease-associated allele at the IRF4 locus (G/G and G/A genotypes) had a significantly shorter treatment-free survival (TFS) compared to non-carriers (A/A genotype). The risk allele was also significantly associated with increased frequency of CD38-positive CLL. We have also examined IRF4 SNPs in linkage disequilbrium with rs872071 and these remain prognostically informative. IRF4 is expressed in germinal centre and post-germinal centre B-cells committed to plasma cell differentiation, and has a complex role in regulating B-cell maturation and homeostasis. Function of the rs870271 polymorphic variant remains undetermined, although given its location in the 3′UTR we speculate that it may affect gene expression, RNA stability or translation efficiency. The 3′ UTR of IRF4 includes several established and putative miRNA binding sites; at least two of these are bound by miRNAs giving rise to a reduction in IRF4 protein expression in germinal centre B-cells. In summary, this study identifies a common variant in IRF4, previously associated with increased risk of developing CLL, as a predictor of a more aggressive disease course and reduced time to first treatment. The strong association with CD38 expression and the identification of a putative IRF4 binding site in the CD38 gene immediately upstream of the transcriptional start site suggests a plausible mechanism by which IRF4 status could affect prognosis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4723.

Abstract 260: Elucidating the roles of the alternatively spliced transcripts of Septin 9

Abstract The septins are a family of 14 genes, the products of which are thought to function primarily as scaffolds which recruit other proteins to particular cellular locations. This gene family is highly conserved and encodes proteins involved in a variety of cellular functions including cytokinesis, apoptosis and vesicle trafficking. Septin colocalisation with actin and tubulin has also been reported. Previous work from this laboratory mapped the SEPT9 gene to a region of allelic imbalance on chromosome 17q in sporadic epithelial ovarian tumours. SEPT9 undergoes extensive alternate splicing at both the 5’ (v1, v2 v3, v4, v4* and v5) and 3’ (a, b and c) ends, with SEPT9_v4 and _v4* differing only in their 5’UTRs, therefore encoding the same polypeptide. We have shown perturbed expression of SEPT9 transcripts in neoplasia, specifically up-regulation of SEPT9_v1 and _v4* transcripts. In a range of normal tissues we noted three alternatively spliced 3’ transcripts (a, b &amp; c) were expressed. However, in ovarian tumour development expression of 2 of these (b &amp; c) is decreased. This suggests that expression of the various SEPT9 transcripts is independently regulated. We therefore sought to gain some insight into control of expression of these various splice variants and why specific patterns of deregulation might contribute to neoplasia. With respect to the cell cycle, we observed by immunofluoresence that SEPT9 protein is visible around the cell membrane during anaphase and into telophase. During cytokinesis the SEPT9 protein localises to the cleavage furrow and is part of the ‘ring’ structure of dividing cells. We also used a double thymidine block to synchronise HeLa cells but there was no alteration in levels of transcripts with specific 5’ or 3’ ends in these cells. Next we looked at expression of the SEPT9 5’ and 3’ ends in response to cellular stress, in particular DNA damage following doxorubicin treatment. We observe an up-regulation of the 5’ v4* transcript and 3’ c variant after doxorubicin treatment in a p53 wild type background but not in p53 null cells. We then used a model of ovarian surface epithelial cells immortalised with telomerase and a temperature sensitive SV40 large T antigen which proliferate at 330C but senesce when shifted to the non-permissive temperature (370C). We see that SEPT9_v2 and v4 mRNA levels increase significantly as cells undergo senescence. Finally, we note that the b and c variants share the same short 3’UTR (57 bases) while the a variant has a long 3’UTR (1.938kb). Bioinformatics of both 3’UTRs identified several putative microRNA binding sites present in the 3’UTR of the a variant but only one in that of b and c. We show that the 3’UTR of a drives a reporter gene when cloned upstream of luciferase and in a sequence dependant manner. In summary, our data supports the hypothesis that the alternatively spliced transcripts of SEPT9 play individual roles within the cell and that the 3’UTRs appear to be important in the regulation of SEPT9. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 260.

Abstract 1951: MicroRNA-mediated regulation of the Fanconi anemia-BRCA pathway

Abstract The Fanconi anemia (FA)-BRCA pathway is a DNA damage response/repair pathway critical for genomic stability, tumor suppression and cellular resistance to interstrand DNA crosslinking agents. At the subcellular level, the FA-BRCA pathway is required for the localization of DNA response/repair proteins (e.g. FANCD2, RAD51) to sites of DNA damage where they form nuclear foci. While several mechanisms of pathway regulation have been identified (e.g. post-translational modifications), regulation of the FA-BRCA pathway by microRNAs is currently unknown. MicroRNAs are short non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. MicroRNAs have been implicated in the regulation of several biological processes and are often deregulated in diseases such as cancer. We hypothesize that the FA-BRCA pathway is regulated by microRNAs, based on our finding that several microRNAs inhibit ionizing radiation (IR)-induced nuclear foci formation of FANCD2 and/or RAD51. We developed a cell-based screen to identify microRNAs that negatively regulate the FA-BRCA pathway. Using IR-induced FANCD2/RAD51 nuclear foci formation as a readout, we screened the human miRIDIAN microRNA Mimics library (Dharmacon), which contains over 400 human microRNA mimics. HeLa cells were transfected with microRNA mimics, irradiated and immunostained for FANCD2/RAD51 foci formation. MicroRNA mimics that decreased IR-induced FANCD2/RAD51 foci formation were scored as positives. Through the initial screen, we identified 45 microRNAs that inhibited IR-induced FANCD2 and/or RAD51 foci formation in HeLa cells. Seven of these microRNAs were found to inhibit foci formation without significantly altering cell cycle progression. We focused on two of these microRNAs, miR-103 and miR-107, because they are overexpressed in a subset of human cancers. We found that miR-103 and miR-107 inhibited IR-induced RAD51 foci formation, reduced the efficiency of homologous recombination repair, and sensitized tumor cell lines to cisplatin. Furthermore, miR-103 and miR-107 downregulated protein levels of RAD51 itself and those of RAD51D, a RAD51 paralog required for RAD51 foci formation, in several cell lines. Both RAD51 and RAD51D contain putative miR-103 and miR-107 binding sites in the 3’-UTRs of their respective transcripts and may therefore be direct targets of these microRNAs. In conclusion, our study identified 7 microRNAs that inhibited IR-induced FANCD2 and/or RAD51 foci formation without altering cell cycle progression. We further show that two of these microRNAs, miR-103 and miR-107, regulate homologous recombination repair and chemosensitivity to interstrand cross-linking agents. Furthermore, miR-103 and miR-107 were found to regulate RAD51 and RAD51D expression. These findings are consistent with the hypothesis that microRNAs regulate the FA-BRCA pathway and promote chemosensitivity and genomic instability in cancer cells. 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 1951.

Abstract 4003: The role of ubiquitin-mediated binding in p210 BCR/ABL induced leukemogenesis

Abstract Chronic myelogenous leukemia (CML) is a malignant hematopoietic stem cell disorder that is invariably associated with a balanced reciprocal translocation between chromosomes 22 and 9. The major product of the rearrangement is a 210 kD in-frame fusion protein (p210 BCR/ABL) that contains amino-terminal sequences from BCR, and carboxy-terminal sequences from the non-receptor tyrosine kinase ABL. This fusion event leads to the constitutive activation of the ABL-encoded kinase activity, which is the principal driving force behind CML. Thus, inhibitors that target the kinase activity produce durable clinical remissions. Although the ABL-encoded kinase activity is essential for disease progression, several studies have shown that BCR encoded sequences are also necessary for BCR/ABL-mediated leukemogenesis. In this current study we have identified ubiquitin as a binding partner for BCR, and demonstrated that binding is retained in p210 BCR/ABL. We have localized the docking site for ubiquitin to residues 180-191 which is present in all fusion variants of BCR/ABL (p190, p210 and p230). This putative ubiquitin binding domain (UBD) does not conform to any known consensus sequence, and is unique to BCR. Deletion of the UBD does not impair the auto-or transkinase activity of p210 BCR/ABL, nor does it impair the interaction between p210 BCR/ABL and GRB2, or the ability of p210 BCR/ABL to activate ERK1/2. A mutation at residue tyr-177 of p210 BCR/ABL also does not impair the interaction with ubiquitin suggesting that the GRB2 and ubiquitin binding sites are adjacent, but separable. β-catenin has been identified as a binding partner for BCR and BCR/ABL, and the docking site has been mapped to a region of BCR/ABL that contains the UBD. Over-expression of p210 BCR/ABL, but not the ubiquitin binding mutant, leads to an accumulation of β-catenin that is phosphorylated on the serine residues that normally trigger ubiquitin-mediated turnover. This difference cannot be attributed to a difference in the activation status of GSK-3β or Pin-1. These results suggest that either β-catenin and ubiquitin share a docking site on p210 BCR/ABL, or that the interaction between β-catenin and p210 BCR/ABL is ubiquitination dependent. The latter possibility is consistent with a previous study which showed that glycine residue in β-catenin is required for Bcr interaction. We propose a model in which p210 BCR/ABL may influence the Wnt signaling pathway by binding ubiquitinated β-catenin, and stabilizing it against degradation. In a CML animal model, mice received the ubiquitin binding mutant have significantly increased the survival time compared with p210 BCR-ABL positive transplant mice. The role of ubiquitin mediated binding in p210 BCR/ABL-mediated leukemogenesis is currently being explored. 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 4003.

Abstract 4744: Sequence-specific p53-DNA binding to gene regulatory elements is altered by single-nucleotide polymorphisms

Abstract Single nucleotide polymorphisms (SNPs) may influence disease susceptibility by modifying cellular responses to environmental stresses. Regulatory SNPs occur in non-coding gene regions and can modify gene expression in an allele-specific manner by altering transcription factor binding. The tumor suppressor p53, a transcription factor, coordinates the expression of an intricate network of genes in response to DNA damage. Importantly, sequence-specific DNA binding is essential for p53 tumor suppressive activity. Unfortunately, lack of robust computational methods and laborious experimental assays have hampered the discovery and characterization of the effects of SNPs in p53 response elements (p53REs). Therefore, we developed a refined bioinformatics approach to identify SNPs in bona fide and putative p53-DNA binding sites. We also developed the microsphere assay for protein-DNA binding (MAPD) to assess the impact of genetic variation on p53-RE binding following cellular exposure to DNA-damaging agents. Thirty-two SNPs (within 12 bona fide and 20 putative p53REs) identified by our bioinformatics approach were evaluated using the MAPD assay. We monitored p53-DNA binding in nuclear extracts from doxorubicin- or IR-treated human lymphoblastoid and U2OS osteosarcoma cells. Significant increases in p53 binding over the negative control were observed for the majority of the p53REs. p53 bound several candidate elements at levels comparable to the positive control (p21 p53RE). Alleles that were predicted to lower p53 binding indeed displayed reduced binding toward 23 of the 32 sequences. Of these alleles, 10 diminished p53 binding by ≥50%. Base substitutions at conserved C and G bases had the greatest impact on p53-DNA binding. In addition, five p53RE SNPs altered gene expression in a set of genotyped lymphoblastoid cell lines. Cellular binding of p53 to putative REs are currently being validated by ChIP-seq. These findings demonstrate that our bioinformatics approach combined with MAPD and gene expression analysis can identify SNPs that alter p53 activity in response to DNA damage and discover novel genes within the p53 transcriptional network. This strategy also can be employed to identify functional SNPs for future studies in human populations. 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 4744.

Abstract LB-332: MDM4 transcription is discordantly regulated by common variants of p53

Abstract The transcription factor p53 is a central mediator of the DNA damage response programs controlling cell cycle arrest and apoptosis that are critical to the prevention of oncogenesis. Although p53 has been subject to intense study, the functional consequences of the common proline to arginine coding polymorphism at codon 72 have yet to be fully elucidated. We hypothesized that the previously reported difference in apoptotic potential between cells expressing the two common p53 alleles may be due in part to differences in each variant's ability to transcriptionally regulate downstream target genes. To test this, we used a combination of expression profiling and ChIP-on-chip techniques to identify differences in the transcriptional networks regulated by the alternate alleles of p53 in isogenic cell lines. Our results indicate that although the majority of p53 transcriptional targets are equivalently regulated by both allelic variants, a small number of genes are specifically regulated by the respective versions of p53. Surprisingly, among the genes differentially induced by alternate p53 variants is MDM4 (HDMX), a well-characterized regulator of p53 activity. We subsequently used luciferase expression assays to identify a functional site containing a putative p53 binding element within the MDM4 promoter that discordantly interacts with the two p53 variants. These data provide the first evidence that MDM4 transcription is directly mediated by p53, and suggest a mechanism that could contribute to previously-reported differences in apoptotic potential between the common p53 alleles. 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 LB-332.

Abstract 1947: Identification of microRNAs that regulate DNA damage response

Abstract Mammalian cells have developed an elaborate DNA damage response system and DNA repair machinery, which play a critical role in the prevention of cancer and development of resistance to anti-cancer DNA damaging agents. Many proteins involved in DNA damage response, such as phosphorylated histone H2AX (γH2AX) and 53BP1, form nuclear foci at sites of DNA damage and facilitate DNA damage response and repair. The precise regulation of DNA damage response is crucial for cellular survival after DNA damage and its abrogation often results in genomic instability. MicroRNAs (miRNAs) are short, non-coding RNA molecules, which post-transcriptionally regulate gene expression by repressing translation and/or degrading mRNA. Some miRNAs function as oncogenes or tumor suppressors. However, the role of miRNAs in DNA damage response remains largely unknown. Here, we identified miRNAs that regulate DNA damage response, especially γH2AX/53BP1 foci formation, and modulate chemo-/radio-sensitivity of tumor cells. We developed a cell-based screening assay utilizing ionizing radiation (IR)-induced γH2AX/53BP1 foci formation in a human osteosarcoma cell line, U2OS, as a readout. The human miRNA Mimics and Inhibitors Libraries (Dharmacon), consisting of 810 miRNA mimics or inhibitors, were transfected in U2OS cells and screened with an automated high-throughput fluorescence microscope and an automatic foci counter. We found that two and five miRNA mimics reproducibly inhibited IR-induced γH2AX and 53BP1 foci formation, respectively, while none of the miRNA inhibitors inhibited their foci formation. Among these hits, we focused on miR-138, because miR-138 is reported to be deregulated in a subset of cancer and may be involved in tumorigenesis and chemosensitivity of cancer. We found that miR-138 decreased the expression of H2AX and reduced H2AX phosphorylation upon DNA damage. Consistent with a defect in DNA damage response, miR-138 sensitized U2OS cells to DNA damaging agents, such as IR and cisplatin. Importantly, the 3′UTR of H2AX contains a putative miR-138 binding site, suggesting that H2AX may be a direct target of miR-138. Taken together, we have identified some miRNAs which inhibit IR-induced foci formation of γH2AX/53BP1. In particular, miR-138 strongly reduced H2AX-mediated DNA damage response and enhanced the chemo-/radio-sensitivity of cancer cells, suggesting that miR-138 might serve as an important regulator of genome instability and a possible therapeutic target for cancer treatment. 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 1947.

Abstract 2960: TRIM29 is a novel biomarker for basal-like breast cancer

Abstract Basal-like breast cancer is a subtype of tumors associated with aggressive clinical behavior and poor prognosis. The genes responsible for their aggressive phenotype have not been well characterized and may provide an opportunity for targeted therapies. Tripartite motif-containing 29 (TRIM29) is a member of the TRIM protein family. Microarray and RT-qPCR analyses shows TRIM29 overexpressed in Basal-like breast tumors and it is highly correlated to other basal tumor markers such as CK5/6, CK17, EGFR, and FOXC1. We recently observed using immunohistochemistry that TRIM29 predominantly localizes to the nucleus in basal tumor epithelial cells, in contrast to a cytoplasmic localization seen in ER-positive tumors. Thus, TRIM29 may act as a transcriptional regulatory factor involved in carcinogenesis. In order to investigate the role of TRIM29 in the initiation and progression of Basal-like breast cancer, we employed chromatin immunoprecipitation and massively parallel sequencing (ChIP-seq) to create a genome-wide profile of in vivo TRIM29-binding sites in MDA-MB-468 cells, a human breast cancer cell line. ChIP-seq analysis revealed 94 putative TRIM29 binding regions in the cancer genome of MDA-MB-468 cells with an estimated false discovery rate of less than 5%. One TRIM29 binding motif was found in the TRIM29 binding regions with a core consensus sequence of CTGTCTCC. We also identified some known cancer genes in close proximity to TRIM29 binding sites, including JUND, FBXO31, CCND3, EGFR, and FOXC1. In addition to mapping TRIM29-DNA binding sites, we used co-immunoprecipitation (co-IP) coupled with tandem mass spectrometry to identify proteins that interact with TRIM29 in MDA-MB-468 cells. Proteins that co-precipitated with TRIM29 included splicing factor proline/glutamine-rich (SFPQ, a transcriptional co-repressor), poly(A) binding protein (PABPC4); FUS and TAF15 RNA polymerase II (both are proto-oncoproteins and transcriptional activators), and A kinase anchor protein 13 (AKAP13, an adaptor protein involved in protein kinase A signaling pathway). Confirmation of the initial co-IP results was achieved through several independent techniques, including detection of binding partners in TRIM29 precipitates by Western blot and reciprocal co-IP using antibodies to partner proteins and detection of TRIM29 in the precipitates. Overall, results from our studies indicated that besides functioning as a transcription factor, TRIM29 may also participate in signal transduction pathways by forming protein complexes. Our studies have identified new biomarkers for basal-like breast cancers and revealed kinases that could potentially be targeted by biologic therapies. 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 2960.

Abstract 1239: The transcription factor Kaiso negatively regulates the hypoxia inducible factor-1 alpha

Abstract Introduction: Hypoxia (reduced oxygen levels) is a common characteristic of solid tumors, and is strongly correlated with poor prognosis and resistance to treatment. In response to hypoxia, cells initiate a cascade of transcriptional events regulated by the Hypoxia Inducible Factor 1 (HIF-1) heterodimer. During hypoxia, the oxygen sensitive HIF-1α subunit is stabilized and translocates to the nucleus where it interacts with the HIF-1β subunit to form a functional HIF-1 transcription factor that regulates genes to facilitate cellular adaptation to hypoxia. To date, while the mechanisms governing HIF-1α stabilization and function have been well studied, those governing HIF-1α gene expression are not fully understood. Recent studies have revealed that the transcription factor Kaiso, a member of the POZ-ZF family of transcription factors implicated in tumorigenesis, is aberrantly expressed and mislocalized at the hypoxic core of various tumors. Mounting evidence suggests that, like other POZ-ZF proteins, Kaiso has a role in tumorigenesis. More importantly, the HIF-1α promoter contains several copies of the consensus Kaiso Binding Site (KBS) suggesting that HIF-1α may be a Kaiso target gene. Since HIF-1α is a key regulator of many hypoxia-induced pathways, and Kaiso is aberrantly expressed in hypoxia, some feed-back mechanism may exist between Kaiso and HIF-1α. These studies seek to elucidate the relationship between Kaiso and HIF-1α during the adaptive response to hypoxia. Methods: Western blot analysis was performed on whole cell lysates harvested from cultured cells that were incubated in hypoxia for varying time periods to compare Kaiso expression levels in these cells. Electrophoretic Mobility Shift Assays (EMSAs) were used to determine the specificity of Kaiso binding to the HIF-1α promoter. To assess the effect of Kaiso on HIF-1α transcription, promoter-reporter assays were performed in cultured cells using a HIF-1α promoter-luciferase construct and a Kaiso expression plasmid. Results: During hypoxia, Kaiso protein levels steadily decline and are significantly reduced after 24 hours in the MCF7 tumor epithelial cell line. Interestingly, the decrease in Kaiso protein levels is less apparent in the non-tumor MCF10A breast cell line. Kaiso binds 3 of the 6 putative Kaiso binding sites as well as a CpG rich region in the HIF-1α promoter in a methylation-dependent manner. Furthermore, Kaiso represses transcription of the HIF-1α promoter in MCF7, MCF10A and HCT116 cell lines. Conclusion: These results indicate that Kaiso negatively regulates HIF-1α and thus implicates HIF-1α as a Kaiso target gene. The fluctuation of Kaiso expression during hypoxia suggests that HIF-1 may also regulate Kaiso and that a feedback mechanism may exist between Kaiso and HIF-1α. Ongoing studies are being performed to elucidate the functional interaction between HIF-1α and Kaiso. 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 1239.

957 INHIBITION OF HEPATIC STELLATE CELL ACTIVATION FOLLOWING ANTI-IGFBPRP1 ADMINISTRATION TO THIOACETAMIDE-TREATED MICE

Background and Aim: HSC have been taken centre stage of liver fibrogenesis due to their remarkable synthesis of extracellular matrix proteins and secretion of profibrogenic mediators. MicroRNAs (miRNAs) are small noncoding RNAs, that posttranscriptionally inhibit gene expression by targeting the untranslated region (UTR) of various transcripts. Recently, we have shown that miR-29a and miR-29b reduce collagen expression in HSC. In the present study, we now investigated the role of miR-29 during the expression of profibrogenic growth factors. Methods: miR-29 expression was analysed by Real Time PCR in HSC after stimulation with Hepatocyte Growth Factor (HGF) and in rat liver after four weeks bile-duct obstruction (N=22 BDL and N=13 sham). In order to identify fibrogenic mediators as putative targets of miR-29a and miR-29b, different databases were screened. The putative targets were subsequently analysed by reporter assays. For this purpose the psiCheck vector providing endogenous normalisation of the luciferase reporter activity was used and putative miR-29 binding sites of the identified UTRs were cloned downstream to the luciferase reporter gene. Ago-miR-29, mimicking the miR-29a, were transfected into HSC and synthesis of identified targets were determined. Results: miR-29a and miR-29b were downregulated in rat livers suffering from cholestasis induced experimental fibrosis. Searching of different databases revealed that transcripts of IGF-I and members of the PDGF/VEGF family are putative miR-29 target sequences. Transfection of Ago-miR-29 did not affect reporter expression of PDGF-B and PDGF-B receptor. However, reporter assays demonstrate functional miR-29 binding to the 3′-UTR of the PDGF-C, IGF-I and VEGF-A mRNA but not to the corresponding mutated sequences taken as negative controls. Furthermore, we were able to show that miR-29 was markedly upregulated after stimulation of HSC with the antifibrogenic mediator HGF. Conclusion: miR-29, that is downregulated during experimental fibrosis, but upregulated by HGF is postulated to be involved in regulation of extracellular matrix synthesis as well as in secretion of the profibrogenic mediators IGF-I, VEGF-A and PDGF-C. Therefore, miR-29, are antifibrogenic miRNAs not only by targeting collagen biosynthesis but also by interfering with the profibrogenic cell communication.

Differential expression of citA gene encoding the mitochondrial citrate synthase of Aspergillus nidulans in response to developmental status and carbon sources

As an extension of our previous studies on the mitochondrial citrate synthase of Aspergillus nidulans and cloning of its coding gene (citA), we analyzed differential expression of citA in response to the progress of development and change of carbon source. The cDNA consisted of 1,700 nucleotides and was predicted to encode a 474-amino acid protein. By comparing the cDNA sequence with the corresponding genomic sequence, we confirmed that citA gene contains 7 introns and that its transcription starts at position -26 (26-nucleotide upstream from the initiation codon). Four putative CreA binding motifs and three putative stress-response elements (STREs) were found within the 1.45-kb citA promoter region. The mode of citA expression was examined by both Northern blot and confocal microscopy using green fluorescent protein (sGFP) as a vital reporter. During vegetative growth and asexual development, the expression of citA was ubiquitous throughout the whole fungal body including mycelia and conidiophores. During sexual development, the expression of citA was quite strong in cleistothecial shells, but significantly weak in the content of cleistothecia including ascospores. Acetate showed a strong inductive effect on citA expression, which is subjected to carbon catabolite repression (CCR) caused by glucose. The recombinant fusion protein CitA(40)::sGFP (sGFP containing the 40-amino acid N-terminal segment of CitA) was localized into mitochondria, which supports that a mitochondrial targeting signal is included within the 40-amino acid N-terminal segment of CitA.

The bacterial virulence factor NleA's involvement in intestinal tight junction disruption during Enteropathogenic E. coli infection is independent of its putative PDZ binding domain

Enteropathogenic Escherichia coli (EPEC) is an enteric pathogen able to cause severe diarrhea. Once adhered to the small intestine, EPEC disrupts tight junctions that are important for intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Recently we have shown that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. NleA has a predicted PDZ-binding domain at its C-terminus which is proposed to be involved in protein interactions with PDZ domain containing proteins. Since several PDZ-domain-containing proteins localize to tight junctions, we hypothesized that the PDZ-binding domain of NleA might be important for its role in tight junction disruption. However, here we show that a molecular variant of NleA lacking the PDZ-binding domain behaves indistinguishably from the wild-type protein with respect to disruption of tight junctions.

Characterization of a new dehydration responsive element binding factor in central arctic cowberry

The C-repeat binding factor/dehydration responsive element binding factor 1 (CBF/DREB1) is a critical regulator of the development of tolerance to abiotic stress in plants. Here we report the cloning of the homologous gene, VviDREB1, from a central arctic plant, the cowberry Vaccinium vitis-idaea L., using rapid amplification of cDNA ends and genome walking techniques. The cDNA and genomic DNA are 887 and 3790 bp long, respectively. Alignment and phylogenetic tree analysis suggested that VviDREB1 should be classified into the A-1 group of the DREB subfamily. The 1522-bp-long promoter contains putative cis-acting elements and transcription factor binding motifs, including elements predicted to respond to phytohormones, abiotic stress, and light. The deduced amino acid sequence of the VviDREB1 cDNA contains a predicted DNA-binding domain with a three-stranded anti-parallel β-sheet and an α-helix, similar to that of the Arabidopsis thaliana ethylene-responsive-element-binding factor AtERF1, suggesting that VviDREB1 might bind to the dehydration responsive and ethylene responsive element. The expression of VviDREB1 was mainly localized to the nuclei of onion epidermal cells. Importantly, VviDREB1 expression in the cowberry was up-regulated by cold, high-salt, and abscisic acid treatment. Our findings may provide a novel basis for further studies of the acclimation and tolerance of cowberry to abiotic stresses.

DOCKING AND MD SIMULATIONS OF THE INTERACTION OF THE POTASSIUM-SPARING DIURETIC AGENT AMILORIDE WITH THE hASIC1a CHANNEL USING A HOMOLOGY MODEL

The interaction of the K+ -sparing agent amiloride — a synthetic chlorinated pyrimidine derivative — with the hASIC1a ion channel is investigated here along homology modeling of the pore region (using the crystal structure of the cASIC1 channel as a template and the known sequence of hASIC1a), automated docking (using the NMR solution structure of amiloride and its conjugated acid, refined by computations), and molecular dynamics simulations. This represents the first modeling and computational chemistry of the pore region of ASIC/DEG/ENaCs/FaNaCh channels. The results agree with the putative amiloride binding site for alphaENaC channel chimeras once the amiloride free base is considered, while its conjugated acid — in contrast with literature beliefs — is poorly scored on a nearby protein pocket. Different protonation conditions of the pore region are irrelevant because histidine residues are far from the binding sites. Mapping the amino acids of the homology model closest to amiloride can have heuristic value in stimulating in silico search of new pore-blocking agents, experimental studies of ASIC channels themselves, and development of code for constant-pH MD simulations.

Analysis of a promoter polymorphism of the GLUT1 gene in patients with hepatocellular carcinoma

The glucose transporter isoform 1 (GLUT1) is a key rate-limiting factor in the transport and metabolism of glucose in cancer cells. Recently, we found that GLUT1 expression is increased in hepatocellular carcinoma (HCC) and promotes tumorigenicity of HCC cells. Hypoxia further increased GLUT1 expression in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor (HIF)-1alpha. The promoter region of the GLUT1 gene harbors a single nucleotide polymorphism (SNP; Rs710218; A to T at -2841) closely positioned to a putative HIF-1alpha binding site, and recently, this SNP was found to be more frequent in patients with renal cell carcinoma. In the present study, the A-2841T genotype distribution did not differ significantly between HCC patients (n = 95; AA: 60%; AT 36% and TT: 4%) and healthy controls (n = 127; AA: 50%; AT 41% and TT: 9%). However and noteworthy, non-carriers of the T allele had higher GLUT1 expression levels in cancerous hepatic tissue, and tended to reveal a more aggressive tumour growth. These data indicate that the SNP Rs710218 is not associated with a higher risk for HCC but rather for HCC progression, potentially via HIF-1alpha mediated increased GLUT1 expression.