RESUMEN
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.
Asunto(s)
Cromatina/metabolismo , Nucleosomas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/metabolismo , Cromatina/genética , Ensamble y Desensamble de Cromatina , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/genética , Nucleosomas/metabolismo , Nucleosomas/fisiología , Regiones Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Factores de Transcripción/genéticaRESUMEN
While expression of ribosomal protein genes (RPGs) in the budding yeast has been extensively studied, a longstanding enigma persists regarding their co-regulation under fluctuating growth conditions. Most RPG promoters display one of two distinct arrangements of a core set of transcription factors (TFs) and are further differentiated by the presence or absence of the HMGB protein Hmo1. However, a third group of promoters appears not to be bound by any of these proteins, raising the question of how the whole suite of genes is co-regulated. We demonstrate here that all RPGs are regulated by two distinct, but complementary mechanisms driven by the TFs Ifh1 and Sfp1, both of which are required for maximal expression in optimal conditions and coordinated downregulation upon stress. At the majority of RPG promoters, Ifh1-dependent regulation predominates, whereas Sfp1 plays the major role at all other genes. We also uncovered an unexpected protein homeostasis-dependent binding property of Hmo1 at RPG promoters. Finally, we show that the Ifh1 paralog Crf1, previously described as a transcriptional repressor, can act as a constitutive RPG activator. Our study provides a more complete picture of RPG regulation and may serve as a paradigm for unravelling RPG regulation in multicellular eukaryotes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Estrés Fisiológico/genética , Transactivadores/metabolismo , Transcripción Genética , Secuenciación de Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas del Grupo de Alta Movilidad/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas Ribosómicas/biosíntesis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sirolimus/farmacología , Estrés Fisiológico/efectos de los fármacos , Transactivadores/genéticaRESUMEN
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.
Asunto(s)
Estrés Oxidativo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Transcripción Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.
Asunto(s)
Cromosomas Fúngicos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/metabolismo , Roturas del ADN de Doble Cadena , Replicación del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Homeostasis del TelómeroRESUMEN
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 IIα were assessed. GE-2 enhanced DNA cleavage â¼4-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 IIα 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 IIα except at very high concentrations. However, the activity and potency of the metabolite were dramatically enhanced under oxidizing conditions. These results suggest that topoisomerase IIα may play a role in mediating the cytotoxic effects of these fungal metabolites.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Benzaldehídos/farmacología , Ciclohexanonas/farmacología , División del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Hongos/química , Hongos/metabolismo , Metabolismo Secundario , Benzaldehídos/química , Benzaldehídos/metabolismo , Ciclohexanonas/química , Ciclohexanonas/metabolismo , Humanos , Estructura MolecularRESUMEN
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 (ET-1) 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 IIα 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 IIα appears to be related to their ability to intercalate into the double helix.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Elipticinas/química , Elipticinas/farmacología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , Antígenos de Neoplasias/metabolismo , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , MetilaciónRESUMEN
ADA (alteration/deficiency in activation) 3 is a conserved component of several transcriptional adaptor and HAT (histone acetyltransferase) complexes that regulate RNA polymerase II-mediated 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.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Histona Acetiltransferasas/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , ADN Complementario/metabolismo , Proteínas de Unión al ADN , Genes Reporteros , Células HeLa , Histona Acetiltransferasas/genética , Humanos , Microscopía Fluorescente , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 2/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
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.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Encéfalo , Proteínas del Citoesqueleto/genética , Feto , Transferencia Resonante de Energía de Fluorescencia , Biblioteca de Genes , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Microscopía Confocal , Datos de Secuencia Molecular , Dominios PDZ , Mapeo de Interacción de Proteínas , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
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-1, 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, ß-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 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.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Humanos , Proteínas del Tejido Nervioso/genética , Péptidos/genética , Péptidos/metabolismo , Espectrometría de Fluorescencia , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND/AIMS: 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. METHODOLOGY: 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. RESULTS: 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 (p < 0.05). CONCLUSIONS: Our data suggests that the CYP isoforms were independently expressed with respect to the pathological status in human gastric tissue.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Gastritis/metabolismo , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Neoplasias Gástricas/metabolismo , Adulto , Anciano , Biopsia , Distribución de Chi-Cuadrado , Cartilla de ADN , Electroforesis en Gel de Agar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Elipticinas/farmacología , Telomerasa/genética , Homeostasis del Telómero/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Antineoplásicos/química , Línea Celular , Elipticinas/química , Técnica del Anticuerpo Fluorescente , Humanos , Hibridación Fluorescente in SituRESUMEN
Benzimidazoles of both natural and synthetic sources are the key components of many bio-active compounds. Several reports have shown antifungal, antiviral, H(2) receptor blocker and antitumor activities for benzimidazoles and their derivatives. In this study, we synthesized twelve bis-benzimidazole derivatives by selecting di(1H-benzo[d]imidazol-2-yl)methane as the main compound. The numbers of carbons at 2 positions of bis-benzimidazole derivatives were changed from 1 to 4, and derivatives were synthesized with methyl substitutions at 5- and/or 6- positions. The compounds were screened via in vitro plasmid superciol relaxation assays using mammalian DNA topoisomerase I and cytostatic assays were carried out against HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells for selected derivatives. Our results suggest that the malonic acid derivatives of bis-benzimidazoles, namely, bis(5-methyl-1H-benzo[d]imidazol-2-yl)methane and bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methane, were remarkably active compounds in interfering with DNA topoisomerase I and the former compound was also found to be cytotoxic against MCF7 and A431 cells. The inhibitory effects obtained with these derivatives are significant as these compounds can be potential sources of anticancer agents.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Bencimidazoles/síntesis química , Bencimidazoles/farmacología , Inhibidores de Topoisomerasa I , Animales , Antineoplásicos/química , Bencimidazoles/química , Neoplasias de la Mama/patología , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo I/metabolismo , Femenino , Células HeLa , Humanos , Análisis Espectral , Relación Estructura-Actividad , Neoplasias del Cuello Uterino/patologíaRESUMEN
Chalcones (1,3-diaryl-2-propen-1-ones) are alpha, beta-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.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Chalconas/síntesis química , Chalconas/farmacología , Inhibidores de Topoisomerasa I , Animales , Antineoplásicos/química , Bovinos , Línea Celular , Chalconas/química , ADN-Topoisomerasas de Tipo I/metabolismo , Humanos , Concentración 50 Inhibidora , Células Jurkat , Plásmidos/metabolismoRESUMEN
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.
Asunto(s)
Amplificación de Genes , Regulación Neoplásica de la Expresión Génica/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Cromosomas Humanos Par 17/genética , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Vejiga Urinaria/patología , Urotelio/metabolismo , Urotelio/patologíaRESUMEN
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.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP3A/genética , Próstata/enzimología , ARN Mensajero/genética , Biopsia , Citocromo P-450 CYP2A6 , Cartilla de ADN , ADN Complementario/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Masculino , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodosRESUMEN
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.
Asunto(s)
Úlcera Duodenal/microbiología , Gastritis/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Factores de Virulencia/genética , Alelos , Enfermedad Crónica , Femenino , Helicobacter pylori/patogenicidad , Humanos , Masculino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Acetiltransferasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Unión al ADN , Humanos , Unión Proteica , Factores de Transcripción/metabolismo , Activación Transcripcional , Factores de Transcripción p300-CBP/metabolismoRESUMEN
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.
Asunto(s)
ADN de Hongos/genética , ADN de Hongos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/genética , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telómero/metabolismoRESUMEN
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.