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Ribosome biogenesis requires prodigious transcriptional output in rapidly growing yeast cells and is highly regulated in response to both growth and stress signals. This minireview focuses on recent developments in our understanding of this regulatory process, with an emphasis on the 138 ribosomal protein genes (RPGs) themselves and a group of >200 ribosome biogenesis (RiBi) genes whose products contribute to assembly but are not part of the ribosome. Expression of most RPGs depends upon Rap1, a pioneer transcription factor (TF) required for the binding of a pair of RPG-specific TFs called Fhl1 and Ifh1. RPG expression is correlated with Ifh1 promoter binding, whereas Rap1 and Fhl1 remain promoter-associated upon stress-induced down regulation. A TF called Sfp1 has also been implicated in RPG regulation, though recent work reveals that its primary function is in activation of RiBi and other growth-related genes. Sfp1 plays an important regulatory role at a small number of RPGs where Rap1–Fhl1–Ifh1 action is subsidiary or non-existent. In addition, nearly half of all RPGs are bound by Hmo1, which either stabilizes or re-configures Fhl1–Ifh1 binding. Recent studies identified the proline rotamase Fpr1, known primarily for its role in rapamycin-mediated inhibition of the TORC1 kinase, as an additional TF at RPG promoters. Fpr1 also affects Fhl1–Ifh1 binding, either independently or in cooperation with Hmo1. Finally, a major recent development was the discovery of a protein homeostasis mechanism driven by unassembled ribosomal proteins, referred to as the Ribosome Assembly Stress Response (RASTR), that controls RPG transcription through the reversible condensation of Ifh1.

The role of three-dimensional genome organization as a critical regulator of gene expression has become increasingly clear over the last decade. Most of our understanding of this association comes from the study of long range chromatin interaction maps provided by Chromatin Conformation Capture-based techniques, which have greatly improved in recent years. Since these procedures are experimentally laborious and expensive, in silico prediction has emerged as an alternative strategy to generate virtual maps in cell types and conditions for which experimental data of chromatin interactions is not available. Several methods have been based on predictive models trained on one-dimensional (1D) sequencing features, yielding promising results. However, different approaches vary both in the way they model chromatin interactions and in the machine learning-based strategy they rely on, making it challenging to carry out performance comparison of existing methods. In this study, we use publicly available 1D sequencing signals to model chromatin interactions in two human cell lines and evaluate the prediction performance of 5 popular machine learning algorithms: decision trees, random forests, gradient boosting, support vector machines and multi-layer perceptron. Our approach accurately predicts long-range interactions and reveals that gradient boosting significantly outperforms the other four algorithms, yielding accuracies of ∼ 95%. We show that chromatin features in close genomic proximity to the anchors cover most of the predictive information. Moreover, we demonstrate that gradient boosting models trained with different subsets of chromatin features, unlike the other methods tested, are able to produce accurate predictions. In this regard, and besides architectural proteins, transcription factors are shown to be highly informative. Our study provides a framework for the systematic prediction of long-range chromatin interactions, identifies gradient boosting as the best suited algorithm for this task and highlights cell-type specific binding of transcription factors at the anchors as important determinants of chromatin wiring.

The compounds reducing tumor cell viability and disrupting DNA topoisomerase reactions have been widely used in anticancer drug development. Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is a potent intercalating agent that interferes with nucleic acid processing through interaction with DNA topoisomerase II. Although ellipticine is a well-characterized compound, it is not a widely-accepted drug due to the adverse effects detected upon administration. We have previously reported two novel ellipticine derivatives, N-methyl-5-demethyl ellipticine (ET-1) and 2-methyl-N-methyl-5-demethyl ellipticinium iodide (ET-2) as potent compounds targeting DNA topoisomerase II. This study covers an extended synthesis, characterization, and activity data for five new salts of N-methyl 5-demetyl ellipticine (Z-1, Z-2, Z-4, Z-5 and Z-6) having several organic halides and their effects on human topoisomerase II enzymes. Moreover, combined in silico studies were conducted for better understanding of modes of action of studied molecules at the binding pocket of target. Our results showed that three of the derivatives (Z-1, Z-2, and Z-6) have considerable effect on the catalytic activity of human topoisomerase II implying the influence of alkyl groups added to the parental structure of ellipticine. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.

DNA topoisomerase and telomerase enzymes are popular targets of several anti-tumor drugs. Smooth proceeding of telomeric recombination requires Topoisomerase II (Top2), which is involved in telomere-telomere recombination through functioning in relaxation of positive supercoils among the cells adopting telomerase-independent Alternative lengthening of telomere (ALT) pathway. Most of the inhibitors reported so far have been designed to targetsolely telomerase-positive cells, which can potentially lead to therapeutic failure because tumor cells treated with telomerase inhibitors can activate the ALT pathway for telomere maintenance. Knowing that ALT cells are more sensitive against a Top2 inhibitor, ICRF-93 agent, compared to telomerase-positive cells, we analyzed two selected ellipticine derivatives that we recently reported as TopII-targeting compounds, to assess their effects on the formation of DNA breaks and suppression of ALT pathway.Cell viability, Comet, C-Circle assays, dot blot, immunofluorescence staining, and telomere fluorescence in situ hybridization (FISH) staining were used for determining the effect of the compounds on ALT status of tumor cells.Treatment of ALT cells with ellipticine derivatives resulted in the formation of DNA breaks and suppression of ALT-associated phenotypes in vitro. Our results will contribute to the development of therapeutic strategies combining telomerase and ALT pathway inhibitors.

Aims: Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoma. This study was designed to compare epigenetic alterations observed in Enhancer of Zeste Homolog 2 (EZH2)-target genes between plasma-derived exosomes and primary tumors in DLBCL patients. Main methods: Exosomes were isolated from plasma of 21 DLBCL patients and 21 controls. We analyzed the methylation status of the target genes using methylation-specific PCR. We also examined whether the exosomes and the tumor samples contained transcripts of the target genes. Key findings: We found that CDKN2A and CDKN2B were methylated in both plasma exosomes and primary tumor tissue samples. None of the transcripts were found in the exosomes except CDKN1B which was expressed in 8 (38%) of the exosome samples. Significance: This study showed that plasma exosomes might preferably package certain target molecules from primary tumors and the exosomes containing dual methylated DNAs of CDKN2A and CDKN2B, or CDKN1B transcript may contribute to DLBCL pathogenesis. © 2020, Federación de Sociedades Españolas de Oncología (FESEO).

Background DNA topoisomerase and telomerase enzymes are popular targets of several anti-tumor drugs. Smooth proceeding of telomeric recombination requires Topoisomerase II (Top2), which is involved in telomere-telomere recombination through functioning in relaxation of positive supercoils among the cells adopting telomerase-independent Alternative lengthening of telomere (ALT) pathway. Most of the inhibitors reported so far have been designed to targetsolely telomerase-positive cells, which can potentially lead to therapeutic failure because tumor cells treated with telomerase inhibitors can activate the ALT pathway for telomere maintenance. Knowing that ALT cells are more sensitive against a Top2 inhibitor, ICRF-93 agent, compared to telomerase-positive cells, we analyzed two selected ellipticine derivatives that we recently reported as TopII-targeting compounds, to assess their effects on the formation of DNA breaks and suppression of ALT pathway. Methods Cell viability, Comet, C-Circle assays, dot blot, immunofluorescence staining, and telomere fluorescence in situ hybridization (FISH) staining were used for determining the effect of the compounds on ALT status of tumor cells. Results and conclusions Treatment of ALT cells with ellipticine derivatives resulted in the formation of DNA breaks and suppression of ALT-associated phenotypes in vitro. Our results will contribute to the development of therapeutic strategies combining telomerase and ALT pathway inhibitors., Turkish Scientific and Technical Research AssemblyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [115Z349], This research was supported by Grant 115Z349 (Z.T. and S.Z.) from the Turkish Scientific and Technical Research Assembly. the cell lines used in this study were provided by Drs. Shu-Chun Teng and Tsai-Kun Li (College of Medicine, National Taiwan University, Taipei, Taiwan).

Pioneer transcription factors (pTFs) bind to target sites within compact chromatin initiating chromatin remodeling and controlling the recruitment of downstream factors. The mechanisms by which pTFs overcome the chromatin barrier are not well understood. Here we reveal, using single-molecule fluorescence approaches, how the yeast transcription factor Rap1 invades and remodels chromatin. Using a reconstituted chromatin system replicating yeast promoter architecture we demonstrate that Rap1 can bind nucleosomal DNA within a chromatin fiber, but with shortened dwell times compared to naked DNA. Moreover, we show that Rap1 binding opens chromatin fiber structure by inhibiting nucleosome-nucleosome contacts. Finally, we reveal that Rap1 collaborates with the chromatin remodeler RSC to destabilize promoter nucleosomes, paving the way to form long-lived bound states on now exposed DNA. Together, our results provide a mechanistic view of how Rap1 gains access and opens chromatin, thereby establishing an active promoter architecture and controlling gene expression.

The FATC domain of Tel1 is studied via introduction of substitution and truncation mutations. It is found to be required for localization to sites of DNA damage and is essential for phosphorylation of exogenous substrates but dispensable for the intrinsic kinase activity., Two large phosphatidylinositol 3-kinase–related protein kinases (PIKKs), ATM and ATR, play a central role in the DNA damage response pathway. PIKKs contain a highly conserved extreme C-terminus called the FRAP-ATM-TRRAP-C-terminal (FATC) domain. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. In this study, we characterized functions of the FATC domain of Tel1 by introducing substitution or truncation mutations. One substitution mutation, termed tel1-21, and a truncation mutation, called tel1-ΔC, did not significantly affect the expression level. The tel1-21 mutation impaired the cellular response to DNA damage and conferred moderate telomere maintenance defect. In contrast, the tel1-ΔC mutation behaved like a null mutation, conferring defects in both DNA damage response and telomere maintenance. Tel1-21 protein localized to DNA ends as effectively as wild-type Tel1 protein, whereas Tel1-ΔC protein failed. Introduction of a hyperactive TEL1-hy mutation suppressed the tel1-21 mutation but not the tel1-ΔC mutation. In vitro analyses revealed that both Tel1-21 and Tel1-ΔC proteins undergo efficient autophosphorylation but exhibit decreased kinase activities toward the exogenous substrate protein, Rad53. Our results show that the FATC domain of Tel1 mediates localization to DNA ends and contributes to phosphorylation of target proteins.

WOS: 000417698200025, PubMed ID: 29232376, General Control Non-derepressible 5 (GCN5) and Alteration/Deficiency in Activation 2 and 3 proteins (ADA2 and ADA3, respectively) are subunits of the Histone AcetylTransferase (HAT) module of SAGA-and ATAC-type co-activators. We previously reported four new interacting partners of human ADA3 identified by screening a human fetal brain cDNA library using yeast two hybrid technology. One of these partners was Apoptosis-Antagonizing Transcription Factor (AATF), also known as Che-1, an RNA polymerase II-binding protein with a number of roles in different cellular processes including regulation of transcription, cell proliferation, cell cycle control, DNA damage responses and apoptosis. Che-1/AATF is a potential therapeutic target for cancer treatments. In this study, we aimed to identify whether besides ADA3, other components of the HAT modules of SAGA and ATAC complexes, human ADA2 and GCN5 also interact with Che-1/AATF. Co-immunoprecipitation and co-localization experiments were used to demonstrate association of AATF both with two ADA2 isoforms, ADA2A and ADA2B and with GCN5 proteins in human cells and yeast two-hybrid assays to delineate domains in the ADA2 and GCN5 proteins required for these interactions. These findings provide new insights into the pathways regulated by ADA-containing protein complexes., Turkish Scientific and Technological Research Assembly [112T429]; Hungarian NRDIO [GINOP-2.3.2-15-2016-00020], This work was supported by the Turkish Scientific and Technological Research Assembly (112T429) (Dr. Sevil Zencir), and Hungarian NRDIO-GINOP-2.3.2-15-2016-00020 (Prof Imre M. Boros). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Summary What is known and Objective: Imatinib is a specific BCR/ABL inhibitor, commonly used for the treatment of chronic myeloid leukaemia (CML), a hematological malignancy resulting from a chromosomal translocation that generates the BCR/ABL fusion protein. Recent studies showed that the imatinib has cytotoxic and apoptotic effects on many BCR/ABL-negative cancers. Numerous compounds with cytotoxic potential exert their functions by interfering with the DNA topoisomerase. In this study, we examined the effects of imatinib on tumour cell-killing in relation to DNA topoisomerase enzyme inhibition. Methods: We determined the cytotoxicity by cell proliferation assay (XTT; tetrazolium hydroxide), using the human K562 CML cells, and loss of mitochondrial membrane potential by monitoring the changes in caspase-3 enzyme activity. Type I and II topoisomerase activities were measured by supercoiled plasmid relaxation and minicircle DNA decatenation assays respectively. Results and Discussion: Imatinib-induced apoptosis and inhibited cell proliferation in a dose-dependent manner. We also found that the imatinib was effective in both type I and type II topoisomerase reactions to a varying degree between 94% and 7% for the concentration range of 1 mm–0·02 mm in a dose-dependent manner. What is new and Conclusion: Our results suggest that the inhibition of topoisomerases may be a significant factor in imatinib-induced apoptosis in CML.

Benzimidazoles are important compounds because of their antibacterial, antifungal, antimicrobial, antiprotozoal and antihelmintic activities. Some benzimidazole derivatives also interfere with the reactions of DNA topoisomerases, enzymes functioning at almost all stages of the cell cycle. In this study, nine 1H-benzimidazole derivatives with substituents at positions 2 and 5 were synthesized and the structure of the compounds was elucidated by instrumental methods. The characterized compounds were screened to identify if they interfered with mammalian type I DNA topoisomerase activity via in vitro supercoil relaxation assays. Selected compounds were subjected to cytostatic assays using HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells. Our results showed that 5-chloro-2-(2-hydroxyphenyl)-1H-benzimidazole exerted the most profound topoisomerase I inhibition and cytotoxicity. © 2008.

WOS: 000372677900019, PubMed ID: 26894873, Two metabolites from the ascomycete fungus Septofusidium berolinense were recently identified as having antineoplastic activity [Ekiz et al. (2015) J. Antibiot., DOI: 10.1038/ja.2015.84]. However, the basis for this activity is not known. One of the compounds [3,6-dihydroxy-2-propylbenzaldehyde (GE-1)] is a hydroquinone, and the other [2-hydroxymethyl-3-propylcyclohexa-2,5-diene-1,4-dione (GE-2)] is a quinone. Because some hydroquinones and quinones act as topoisomerase II poisons, the effects of GE-1 and GE-2 on DNA cleavage mediated by human topoisomerase IIa were assessed. GE-2 enhanced DNA cleavage similar to 4-fold and induced scission with a site specificity similar to that of the anticancer drug etoposide. Similar to other quinone-based topoisomerase II poisons, GE-2 displayed several hallmark characteristics of covalent topoisomerase II poisons, including (1) the inability to poison a topoisomerase IIa construct that lacks the N-terminal domain, (2) the inhibition of DNA cleavage when the compound was incubated with the enzyme prior to the addition of plasmid, and (3) the loss of poisoning activity in the presence of a reducing agent. In contrast to GE-2, GE-1 did not enhance DNA cleavage mediated by topoisomerase IIa except at very high concentrations. However, the activity and potency of the metabolite were dramatically enhanced under oxidizing conditions. These results suggest that topoisomerase IIa may play a role in mediating the cytotoxic effects of these fungal metabolites., National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM033944]; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [109S361]; EBILTEMEge University [2012/BIL/028]; Turkish Scientific and Technical Research AssemblyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [115Z349]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R25 GM062459, T32 GM008320], This research was supported by grant GM033944 (N.O.) from the National Institutes of Health, grants from TUBITAK (109S361) and EBILTEM (2012/BIL/028) (E.B.), and grant 115Z349 (Z.T.) from the Turkish Scientific and Technical Research Assembly. K.R.V. was a trainee under NIH grants R25 GM062459 and T32 GM008320 and was supported in part by a Research Supplement to Grant GM033944 to Promote Diversity in Health-Related Research.

WOS: 000371344600033, PubMed ID: 26906637, Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is an antineoplastic agent that intercalates into DNA and alters topoisomerase II activity. Unfortunately, this compound displays a number of adverse properties. Therefore, to investigate new ellipticine-based compounds for their potential as topoisomerase II-targeted drugs, we synthesized two novel derivatives, N-methyl-5-demethyl ellipticine (ET1) and 2-methyl-N-methyl-5-demethyl ellipticinium iodide (ET-2). As determined by DNA decatenation and cleavage assays, ET-1 and ET-2 act as catalytic inhibitors of human topoisomerase II alpha and are both more potent than the parent compound. Neither compound impairs the ability of the type II enzyme to bind its DNA substrate. Finally, the potency of ET-1 and ET-2 as catalytic inhibitors of topoisomerase II alpha appears to be related to their ability to intercalate into the double helix. (C) 2016 Elsevier Ltd. All rights reserved., National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM033944, R25-GM062459, T32-GM08320]; Turkish Scientific and Technical Research AssemblyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [115Z349], The authors declare no competing financial interests. This research was supported by grant GM033944 (N.O.) from the National Institutes of Health and grant 115Z349 (Z.T.) from the Turkish Scientific and Technical Research Assembly. K.R.V. was a trainee under National Institutes of Health grants R25-GM062459 and T32-GM08320 and was supported in part by a Research Supplement to Grant GM033944 to Promote Diversity in Health-Related Research. We are grateful to Rachel E. Ashley, Lorena Infante Lara, and Elizabeth G. Gibson for critical reading of the manuscript.

Background: Myeloid differentiation primary response 88 (MYD88) is a common adaptor protein that is responsible for signaling from several receptors; mutations in this gene may play a role in the pathogenesis of lymphoma. Aim: We aimed to determine the MYD88 L265P mutation frequency, the level of MYD88 expression, and their associations with clinicopathological parameters in mature B-cell non-Hodgkin lymphomas (NHLs). Methods: A total of 68 patients were included in the study. The presence of the MYD88 L265P mutation was analyzed by real-time polymerase chain reaction and direct sequencing. MYD88 protein expression was evaluated by immunohistochemistry (IHC) using two different scoring systems. Results: MYD88 L265P mutation was present in eight (18.6%) diffuse large B-cell lymphoma (DLBCL) patients. We also observed a significant association between the loss of MYD88 expression and advanced stage in both mature B-cell NHL and DLBCL according to the first IHC scoring systems (p=0.015 and p=0.024, respectively). An association was also seen between MYD88 overexpression and low clinical risk in both mature B-cell NHL and DLBCL according to the second IHC scoring system (p=0.027 and p=0.024, respectively). Conclusions: The L265P mutation may be helpful for understanding the pathogenesis of immune-privileged site-associated DLBCLs. The presence of the mutation, together with its protein overexpression, could also be used as a prognostic marker in advanced stage DLBCLs. © Copyright 2015, Mary Ann Liebert, Inc.

WOS: 000316038500003, PubMed ID: 23395680, Regulation of gene expression in cells is mediated by protein-protein, DNA-protein and receptor-ligand interactions. PDZ (PSD-95/Discs-large/ZO-1) domains are protein-protein interaction modules. PDZ-containing proteins function in the organization of multi-protein complexes controlling spatial and temporal fidelity of intracellular signaling pathways. In general, PDZ proteins possess multiple domains facilitating distinct interactions. The human glutaminase interacting protein (hGIP) is an unusual PDZ protein comprising entirely of a single PDZ domain and plays pivotal roles in many cellular processes through its interaction with the C-terminus of partner proteins. Here, we report the identification by yeast two-hybrid screening of two new hGIP-interacting partners, DTX1 and STAU1. Both proteins lack the typical C-terminal PDZ recognition motif but contain a novel internal hGIP recognition motif recently identified in a phage display library screen. Fluorescence resonance energy transfer and confocal microscopy analysis confirmed the in vivo association of hGIP with DTX1 and STAU1 in mammalian cells validating the previous discovery of S/T-X-V/L-D as a consensus internal motif for hGIP recognition. Similar to hGIP, DTX1 and STAU1 have been implicated in neuronal function. Identification of these new interacting partners furthers our understanding of GIP-regulated signaling cascades and these interactions may represent potential new drug targets in humans. (C) 2013 Elsevier Inc. All rights reserved., United States Department of AgricultureUnited States Department of Agriculture (USDA) [2003-35302-12930, 2011-65503-20030]; National Science FoundationNational Science Foundation (NSF) [IOS-0628064]; National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [DK082397]; NSERCNatural Sciences and Engineering Research Council of Canada [155268]; [TUBITAK 108T945], This research was financially supported by United States Department of Agriculture PECASE award 2003-35302-12930, and AFRI award 2011-65503-20030, National Science Foundation Grant IOS-0628064, and National Institutes of Health Grant DK082397 to Smita Mohanty, NSERC Grant 155268 to Melanie Dobson, and the Grant TUBITAK 108T945 to Zeki Topcu.

A considerable number of agents with chemotherapeutic potentials reported over the past years were shown to interfere with the reactions of DNA topoisomerases, the essential enzymes that regulate conformational changes in DNA topology. Gossypol, a naturally occurring bioactive phytochemical is a chemopreventive agent against various types of cancer cell growth with a reported activity on mammalian topoisomerase II. The compounds targeting topoisomerases vary in their mode of action; class I compounds act by stabilizing covalent topoisomerase-DNA complexes resulting in DNA strand breaks while class II compounds interfere with the catalytic function of topoisomerases without generating strand breaks. In this study, we report Gossypol as the interfering agent with type I topoisomerases as well. We also carried out an extensive set of assays to analyze the type of interference manifested by Gossypol on DNA topoisomerases. Our results strongly suggest that Gossypol is a potential class II inhibitor as it blocked DNA topoisomerase reactions with no consequently formed strand breaks., Scientific and Technological Research Council of Turkey (TBAG-108T548)

ADA (alteration/deficiency in activation) 3 is a conserved component of several transcriptional adaptor and HAT (histone acetyltransferase) complexes that regulate RNA polymerase IImediated gene expression. Within the HAT complexes ADA3 is associated with ADA2 and the HAT GCN5 (general control non-repressed 5). ADA3 plays roles in diverse cellular processes and also in malignancies by modulating GCN5 catalytic activity and/or by interactions with other regulators. To gain a better understanding of ADA3 function, we used a yeast two-hybrid approach to screen a human fetal cDNA library for proteins that interacted with hADA3 (human ADA3). We identified three novel hADA3-interacting partners, a transcriptional regulator, AATF (apoptosis-antagonizing transcription factor), and regulatory subunits of the PP1 (protein phosphatase 1) and PP2A (protein phosphatase 2A) [PPP1R7 (PP1 regulatory subunit 7) and PPP2R5D (PP2A 56 kDa regulatory subunit δ isoform) respectively]. Analysis of truncated versions of hADA3 indicated that the C-terminal ADA2-interacting domain was not required for these interactions. Fluorescent microscopy analysis and co-immunoprecipitation provided support for the co-localization and interaction of hADA3 with these proteins in human cells. Expression of the interacting proteins altered expression of an hADA3-regulated reporter gene, suggesting functional consequences for the interactions. The detected interactions of hADA3 might extend the spectrum of mechanisms by which ADA3 can contribute to the regulation of gene expression and shed light on processes mediated by these newly identified ADA3 partners. © 2013 Biochemical Society.

Despite inherent limitations, the ease and rapidity of their use make transiently expressed reporter gene assays the most frequently used techniques for analyzing promoters and transcriptional regulators. The results of transient reporter gene assays are generally accepted to reflect transcriptional processes correctly, though these assays study regulatory sequences outside of the chromosomal environment and draw conclusions on transcription based on enzyme activity determination. For transient reporter gene assays, often more than one promoter is introduced into one cell. In addition to the one driving the primary reporter gene expression, a further one might serve to ensure the production of an internal control second reporter or/and a trans-acting factor. We demonstrate here by various examples that interference between physically unlinked promoters can profoundly affect reporter expression. Results of reporter gene assays performed by combinations of the cytomegalovirus promoter and various other promoter constructs (human immunodeficiency virus [HIV], Human T-cell Leukemia Virus Type I (HTLV-I), NF-?B-responsive, and p53-responsive) and trans-activator factors (HIV-Tat and p53) in different host cell lines (U2OS, HeLa, and L929) prove that interference between active transcription units can modify transcription responses dramatically. Since the interference depends on the promoters used, on the amount of transfected DNA, on the host cells, and on other factors, extra caution is required in interpreting results of transient reporter gene assays. © Copyright 2012, Mary Ann Liebert, Inc..

WOS: 000294317300024, PubMed ID: 21787750, The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-I, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, beta-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kit 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP. (C) 2011 Elsevier Inc. All rights reserved., USDA PECASE [2003-35302-12930]; NSFNational Science Foundation (NSF) [IBN-0628064]; USDAUnited States Department of Agriculture (USDA) [2011-65503-20030]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [DK082397]; NSERCNatural Sciences and Engineering Research Council of Canada [155268]; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108T945], This research was financially supported by USDA PECASE Presidential Early Career Award for Scientists and Engineers award 2003-35302-12930, NSF Grant IBN-0628064, USDA Grant 2011-65503-20030 and NIH Grant DK082397 to Smita Mohanty, NSERC Grant 155268 to Melanie Dobson, and the Grant TUBITAK 108T945 to Zeki Topcu. We thank Dr. David L. Zoetewey for critical reading of this manuscript.

PubMedID: 20824388 We investigated the effect of extremely low-frequency electromagnetic field (ELF-EMF) with pulse trains exposure on lipid peroxidation, and, hence, oxidative stress in the rat liver tissue. The parameters that we measured were the levels of plasma alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase as well as plasma albumin, bilirubin, and total protein levels in 30 adult male Wistar rats exposed to ELF. We also determined the percentage of apoptotic and necrotic cells of the kidney extracts from the animals by flow cytometry method. Apoptotic cell death was further characterized by monitoring DNA degradation using gel electrophoresis. The results showed an increase in the levels of oxidative stress indicators, and the flow cytometric data suggested a possible relationship between the exposure to magnetic field and the cell death. We showed significantly lower necrotic cell percentages in experimental animals compared to either unexposed or sham control groups. However, DNA ladder analyses did not differentiate between the groups. Our results were discussed in relation to the response of biological systems to EMF. © 2010 Springer Science+Business Media, LLC. Acknowledgment The authors acknowledge with thanks the support provided by the Biochemistry department of Cukurova Medical School by way of valuable contributions in the determination of oxidative stress parameters.

Human Cytochrome P450 (CYP) comprises a multigene family of microsomal enzymes that metabolize a wide variety of xenobiotics, including drugs and carcinogens. Although the a number of CYP enzymes were also detected in epithelial cells along the gastrointestinal tract, little is known about the expression of CYP genes in gastric tissue.In this study, the expression patterns of CYP isoforms was investigated in a total of 14 antral biopsy tissues obtained from the patients with either chronic gastritis (n = 6) or cancer (n = 8) by gene-specific real-time reverse transcriptase -PCR analyses. We employed primer sets specific for CYPs -1A1, -1A2, -2A6, -2B6, -2C, -2D6, -2E1, and -3A5.Among the isoforms CYP1A1, CYP2C and CYP2D6 gave rise to detectable mRNAs in all 14 gastric tissues while the mRNAs for the other CYPs were detected in some of the tissues. The expression patterns were compared to clinical parameters. There were no significant differences in the parameters between the two groups; however the mRNA expression of CYP2A6 was significantly higher in women than man (p0.05).Our data suggests that the CYP isoforms were independently expressed with respect to the pathological status in human gastric tissue.

Chalcones (1,3-diaryl-2-propen-1-ones) are ?, ß-unsaturated ketones with cytotoxic and anticancer properties. Several reports have shown that compounds with cytotoxic properties may also interfere with DNA topoisomerase functions. Five derivatives of 4'-hydroxychalcones were examined for cytotoxicity against transformed human T (Jurkat) cells as well as plasmid supercoil relaxation experiments using mammalian DNA topoisomerase I. The compounds were 3-phenyl-1-(4'-hydroxyphenyl)-2-propen-1-one (I), 3-(p-methylphenyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (II), 3-(p-methoxyphenyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (III), 3-(p-chlorophenyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (IV), and 3-(2- thienyl)-1-(4'-hydroxyphenyl)-2-propen-1-one (V). The order of the cytotoxicity of the compounds was; IV > III > II > I > V. Compound IV, had the highest Hammett and log P values (0.23 and 4.21, respectively) and exerted both highest cytotoxicity and strongest DNA topoisomerase I inhibition. Compounds I and II gave moderate interference with the DNA topoisomerase I while III & V did not interfere with the enzyme. © 2009 Informa UK Ltd.

The generation of urothelial carcinoma is caused by the accumulation of various molecular changes, as in most malignancies. There are conflicting data about the status of HER-2/neu oncogene in urothelial carcinomas. The aim of this study was to determine the status of HER-2/neu oncogene in high-grade invasive urothelial carcinoma of urinary bladder both in protein and DNA level. We evaluated HER-2/neu protein overexpression by immunohistochemistry (IHC) and gene amplification by fluorescent in situ hybridization (FISH) and real-time quantitative PCR in paraffin-embedded samples of high-grade invasive urothelial carcinoma obtained from 36 patients. Polysomy 17 was also assessed by FISH. Immunohistochemically, HER-2/neu protein overexpression was observed in 22 (61.1%) tumors (ten tumors with score 3+ and 12 with score 2+). Fourteen of 36 tumors (38.9%) were evaluated as negative (score 0 or 1+). Complete concordance between FISH and the PCR was seen in all of the samples scored as 0 and 1+ by IHC. HER-2/neu gene amplification was observed in three of 27 (11.1%) tumors by FISH (nine samples were non-informative) and in eight of 36 (22.2%) tumors by the PCR. The complete concordance between HER2-2/neu protein overexpression and gene amplification was seen only in three of 27 tumors. Polysomy 17 was seen in nine tumors (33.3%). The results indicated that, in contrast to breast cancer, there was no strong association between HER-2/neu overexpression and gene amplification in invasive urothelial carcinomas, and polysomy 17 was higher in tumors showing HER-2/neu overexpression. © 2008 Arányi Lajos Foundation.

PubMed ID: 19040121, Cytochrome P450 (CYP) is a heme-containing enzyme superfamily metabolizing a wide variety of xenobiotics, including drugs and carcinogens. The majority of CYP genes are expressed in the liver, however, some CYP isoforms are also reported for a number of extra hepatic tissues. We analyzed Cytochrome P450-2A6, -3A5 and -4B1 mRNAs using real-time reverse-transcriptase polymerase chain reaction (RT-PCR) in a total of 21 homogenized prostate tissues with or without malignancy. We detected a consistent expression of CYP2A6 and CYP3A5 in all, and of CYP4B1 in some (11/21) of the samples at mRNA level. Neither the histopathological status nor the smoking habit of the individuals affected CYP4B1 expression. Our results reflect possible roles for these particular CYPs in therapy and protection of prostate tissue. © 2008 Verlag der Zeitschrift für Naturforschung.

Reelin is an extracellular matrix-associated protein important in the regulation of neuronal migration during cerebral cortical development. Point mutations in the RELN gene have been shown to cause an autosomal recessive human brain malformation termed lissencephaly with cerebellar hypoplasia (LCH). Recent work has raised the possibility that reelin may also play a pathogenic role in other neuropsychiatric disorders. We sought, therefore, to define more precisely the phenotype of RELN gene disruption. To do this, we performed a clinical, radiological, and molecular study of a family in whom multiple individuals carry a chromosomal inversion that disrupts the RELN locus. A 6-year-old girl homozygous for the pericentric inversion 46,XX,inv7(p11.2q22) demonstrated the same clinical features that have been previously described in association with RELN point mutations. The girl's brain magnetic resonance imaging (MRI) findings, including pachygyria and severe cerebellar hypoplasia, were identical to those seen with RELN point mutations. Fluorescence in situ hybridization confirmed that one of the breakpoints of this inversion mapped to within the RELN gene, and Western blotting revealed an absence of detectable serum reelin protein. Several relatives who were heterozygous for this inversion were neurologically normal and had no signs of psychotic illness. Our findings demonstrate the distinctive phenotype of LCH, which is easily distinguishable from other forms of lissencephaly. Although RELN appears to be critical for normal cerebral and cerebellar development, its role, if any, in the pathogenesis of psychiatric disorders remains unclear. © 2006 Wiley-Liss, Inc.

Developing small molecule inhibitors in anti-cancer drug research is an integral part of clinical oncology. Gefitinib (Iressa) is a promising small molecule inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase. It was shown to have the potential to improve the clinical effectiveness of EGFR-targeted therapies and gained a significant impact in treatment of non-small-cell lung cancer (NSCLC). Over the past years, several naturally occurring and synthetic compounds with anti-cancer activities were shown to exert their actions via interfering with normal DNA topoisomerase reactions. DNA topoisomerases are ubiquitous enzymes that regulate DNA topology by making transient breakages at nucleic acid backbone and rejoining of DNA strands. Topoisomerases carry out their reactions by two different mechanisms; Type I topoisomerases make a single-stranded break in a DNA duplex while type II topoisomerases create transient breaks in both strands of a duplex. In this study we aimed to examine the effects of Gefitinib on these enzymes to extend the evaluation of its biological activities by plasmid supercoil relaxation and kinetoplast decatenation assays for type I and type II topoisomerases, respectively. Our results are discussed in relation to the outcomes of Gefitinib reported in clinical trials, which together would contribute in identifying the mechanisms of Gefitinib-induced tumor cell-killing. Key Words; Gefitinib; DNA topoisomerases; anti-cancer drugs 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 1688.

AIM: To characterize and compare genotype profiles of H pylori strains isolated from patients with chronic gastritis and duodenal ulcer in western part of Turkey. METHODS: A total of 46 patients [30 chronic gastritis (CG) and 16 duodenal ulcer (DU)] who had undergone endoscopy because of dyspeptic complaints were studied. The antral biopsy specimens were evaluated for the presence of H pylori by rapid urease test and culture, and the genotype profiles were determined by real-time PCR. RESULTS: The cagA gene was observed in 43 (93.5%) isolates. The vacA s1m2 genotype was the predominant subtype, found in 63.3% and 68.7% of isolates in patients with CG and DU, respectively. Twenty (66.6%) isolates from patients with CG were iceA2 positive while the iceA1 was predominant in those with DU (68.8%). In terms of the association of the iceA alleles to other genes, both alleles were significantly associated with the cagA vacA s1m2 genotype. CONCLUSION: The prevalent circulating genotypes in CG and DU were cagA vacA s1m2 iceA2 and cagA vacA s1m2 iceA1 genotype, respectively. It was found that cagA vacA s1m2 genotype seems to be common virulence factors in both CG and DU while iceA alleles show specificity for gastroduodenal pathologies in this study.

Summary Pioneer transcription factors (pTFs) bind to target sites within compact chromatin, initiating chromatin remodeling and controlling the recruitment of downstream factors. The mechanisms by which pTFs overcome the chromatin barrier are not well understood. Here, we reveal, using single-molecule fluorescence, how the yeast transcription factor Rap1 invades and remodels chromatin. Using a reconstituted chromatin system replicating yeast promoter architecture, we demonstrate that Rap1 can bind nucleosomal DNA within a chromatin fiber but with shortened dwell times compared to naked DNA. Moreover, we show that Rap1 binding opens chromatin fiber structure by inhibiting inter-nucleosome contacts. Finally, we reveal that Rap1 collaborates with the chromatin remodeler RSC to displace promoter nucleosomes, paving the way for long-lived bound states on newly exposed DNA. Together, our results provide a mechanistic view of how Rap1 gains access and opens chromatin, thereby establishing an active promoter architecture and controlling gene expression., Graphical Abstract, Highlights • The yeast transcription factor Rap1 can invade compact chromatin • Rap1 directly opens chromatin structure by preventing nucleosome stacking • Stable Rap1 binding requires collaboration with RSC to shift promoter nucleosomes, Mivelaz et al. use single-molecule fluorescence approaches and highly defined chromatin systems to show that the yeast transcription factor Rap1 can invade compact chromatin fibers and directly open chromatin structure. For stable binding, Rap1 then collaborates with RSC to displace nucleosomes from its binding sites, generating an active promoter state.