RESUMEN
Redo coronary artery bypass grafting (CABG) is still associated with increased morbidity and mortality compared to primary operation. Myocardial protection is one of the key issues in redo on pump CABG and is still a matter of debate. Off pump redo CABG seems to be an attractive alternative as native coronary blood flow remains and cross clamping of the aorta is avoided. The aim of this retrospective study was to compare the outcome of redo CABG with and without CPB. From 1/1998 to 5/2004 redo CABG was performed in 195 patients (pts): 162 male (83.1%) and 33 female (16.9%) pts, age 66 +/- 9 years. In 160 pts, CPB with isolated antegrade myocardial protection was used for redo CABG. Off pump redo CABG was performed in 35 pts (30 male (85.7%) and 5 female (14.3%), age 67 +/- 8 years). Perioperative overall mortality rate was 3.6% (n = 7) and comparable in both groups (on pump 3.8% versus off pump 2.9%; p = 0.90), as well as perioperative myocardial infarction, intraaortic balloon pump implantation rate and secondary morbidity. Complete revascularization was achieved in 139 pts (86.9%) after on pump CABG and in 17 pts (48.6%) of the off pump group (p < 0.01). The average number of grafts was significantly higher in the on pump group (2.8 +/- 0.78 versus 1.6 +/- 0.6; p = 0.04).Furthermore, 20 pts (12.5%) in the on pump group died during follow-up (50 +/- 16 months). Five pts (25.0%) died due to cardiac reasons. In the off pump group 3 pts (8.6%) died during follow-up (44 +/- 13 months), noncardiac related. Overall survival was 83.8% in the on pump group and 88.6% in the off pump group (p = 0.92). On pump redo CABG and off pump redo CABG can be safely performed with low mortality and morbidity. Off pump redo CABG might be limited due to incomplete revascularization.
Asunto(s)
Puente de Arteria Coronaria , Enfermedad Coronaria/cirugía , Anciano , Puente Cardiopulmonar , Puente de Arteria Coronaria Off-Pump , Enfermedad Coronaria/mortalidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reoperación , Resultado del TratamientoRESUMEN
Oncoproteins encoded by the early region 1A (E1A) of adenoviruses (Ads) have been shown to be powerful tools to study gene regulatory mechanisms. As E1A proteins lack a sequence-specific DNA-binding activity, they modulate viral and cellular gene expression by interacting directly with a diverse array of cellular factors, among them sequence-specific transcription factors, proteins of the general transcription machinery, co-activators and chromatin-modifying enzymes. By making use of these factors, E1A affects major cellular events such as cell cycle control, differentiation, apoptosis, and oncogenic transformation. In this review we will focus on the interaction of E1A with cellular components involved in the cAMP/PKA signal transduction pathway and we will discuss the consequences of these interactions in respect to the activation of CREB/CBP-dependent target genes.
Asunto(s)
Proteínas E1A de Adenovirus/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/fisiología , Adenoviridae/química , Proteínas E1A de Adenovirus/metabolismo , Animales , Proteína de Unión a CREB , Humanos , Unión Proteica , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
The usual mechanism for syringe contamination is spread of micro-organisms along the inside of the barrel. Two or more full strokes of the plunger will inevitably transport a contaminant from the inside wall into the sterile compartment. We modified syringes by replacing the plunger with a modified version that has an additional central O-ring seal. Conventional and modified syringes were tested for their susceptibility to contamination using standard microbiological and radioisotope methods, and the flow-rate stability of the modified syringe design was assessed in a model with commercially available infusion pumps. Although conventional syringes consistently became contaminated with the second full excursion of the plunger, no contamination was observed in modified syringes, even after 100 aspiration/injection cycles. With respect to flow-rate stability, the new syringe design complies with DIN (German Institute for Standardization) standards.
Asunto(s)
Infección Hospitalaria/prevención & control , Diseño de Equipo , Jeringas/microbiología , Contaminación de EquiposRESUMEN
Major histocompatibility complex (MHC) class I proteins are an essential component of the immune system allowing the organism to protect from viral infections and neoplastic transformation. Expression of the MHC class I genes is regulated by a variety of cis-regulatory promoter elements among which the enhancer A is of particular importance. This enhancer is synergistically activated through AP-1/ATF and NF-kappa B transcription factors. NF-kappa B recruits the histone acetyltransferase (HAT) p300/CREB-binding protein (CBP) to the multiprotein complex bound to the enhancer A. Here we present evidence that acetylation and deacetylation processes are involved in the activation of the enhancer A. The p300/CBP associated factor PCAF, but not p300/CBP, counteracts the repression of the enhancer A mediated by the histone deacetylase HDAC1. Furthermore, overexpression of PCAF results in an increase in the acetylation of histone H4 bound to the enhancer A and HDAC1 counteracts the PCAF-mediated H4 acetylation. The activation function of PCAF requires the p300/CBP binding motif indicating that PCAF might be recruited to the enhancer A through an association with p300/CBP. Moreover, PCAF and the Brahma/SWI2-related protein BRG-1, which is a key factor of the human ATP-dependent chromatin remodelling complex SWI/SNF, synergistically up-regulate the enhancer A. Synergistic activation requires the HAT domain of PCAF. Taken together our data suggest that members of two different groups of chromatin modifying complexes are involved in the activation of the enhancer A of the MHC class I promoter.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Elementos de Facilitación Genéticos/genética , Genes MHC Clase I/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Acetilación , Acetiltransferasas/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , ADN Helicasas , Regulación de la Expresión Génica , Células HeLa , Histona Acetiltransferasas , Histonas/metabolismo , Humanos , Proteínas Nucleares/genética , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Transfección , Células Tumorales Cultivadas , Factores de Transcripción p300-CBPRESUMEN
Neurodegeneration in Huntington's disease (HD) is associated with an elongated glutamine tract in the widely expressed huntingtin protein. Although the pathogenic mechanisms are still unknown, the distinct physical properties of mutant huntingtin in the brain suggest that other factors including huntingtin-interacting proteins might play a specific role. We have previously identified a DNA-binding motif in the proximal E1A promoter of adenovirus serotype 12 as responsible for E1A autoregulation. Here, we identified the p231HBP protein as a DNA-binding factor, the C-terminal portion of which has recently been characterized as the huntingtin-interacting protein HYPB of unknown function. We have determined the full-length cDNA sequence, identified several domains supporting its gene regulatory functions, and mapped the HBP231 gene to chromosome 3p21.2-p21.3. Our results provide an interesting molecular link between huntingtin and a DNA-binding factor, implicating that this interaction might result in the alteration of cellular gene expression involved in HD pathogenesis.
Asunto(s)
Cromosomas Humanos Par 3 , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas E1A de Adenovirus/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , ADN Complementario , Expresión Génica/fisiología , Células HeLa , Humanos , Proteína Huntingtina , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/fisiología , Conejos , Técnicas del Sistema de Dos HíbridosRESUMEN
The adenovirus type 12 (Ad12) E1A12S oncoprotein utilizes the cAMP/protein kinase A (PKA) signal transduction pathway to activate expression of the viral E2 gene, the products of which are essential for viral replication. A central unsolved question is, however, whether E1A12S interacts directly with PKA in the process of promoter activation. We show here that E1A12S binds to the regulatory subunits (R) of PKA in vitro and in vivo. Interaction depends on the N-terminus and the conserved region 1 (CR1) of E1A12S. Both domains are also essential for the activation of viral E2 gene expression. Infection of cells with Ad12 leads to the cellular redistribution of RIIalpha from the cytoplasm into the nucleus. Furthermore, RIIalpha is also located in the nucleus of cells transformed by E1 of Ad12 and transient expression of E1A12S leads to the redistribution of RIIalpha into the nucleus in a N-terminus- and CR1-dependent manner. Cotransfection of E1A12S with RIIalpha results in strong activation of the E2 promoter. Based on these results we conclude that E1A12S functions as a viral A-kinase anchoring protein redistributing RIIalpha from the cytoplasm into the nucleus where it is involved in E1A12S-mediated activation of the E2 promoter.
Asunto(s)
Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Núcleo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas E2 de Adenovirus/metabolismo , Animales , Sitios de Unión , Células COS , Línea Celular , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico , Citoplasma/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Transducción de SeñalRESUMEN
Activation of the transcription unit early region 2 (E2) promoter of the oncogenic adenovirus serotype 12 (Ad12), which regulates the expression of proteins essential for viral replication, requires the assembly of a ternary complex consisting of cAMP response element-binding protein (CREB)-1/activating transcription factor (ATF)-1, the Ad12 12S oncogene product of early region 1A (E1A(12S)), and the co-activator p300/CBP on the E2(Ad12) cAMP response element (E2-CRE). Here we show that the active E2(Ad12) promoter is associated with acetylated histone H4 whereas an E2-CRE point-mutated promoter which is transcriptionally inactive due to its inability to assemble this ternary complex is not bound by acetylated histone H4. The histone deacetylase 1 as well as Roscovitine, which blocks the activation of the histone acetyltransferase (HAT) activity of CBP by cyclin E-Cdk2, prevents E2(Ad12) promoter activation through E1A(12S). p300/CBP counteracts the repressive function of histone deacetylase 1 in a HAT domain-dependent manner whereas the p300/CBP-associated factor PCAF failed to rescue E2(Ad12) promoter activity. E1A(12S) bound p300/CBP displays strong HAT activity. Most interestingly, E1A(12S)-mediated activation of the E2(Ad12) promoter correlates well with the ability of the viral protein to associate with the HAT activity of p300/CBP in vivo. Taken together these data indicate that the recruitment of the HAT activity of p300/CBP by E1A(12S) plays an important role in E2(Ad12) promoter activation.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas E1A de Adenovirus/fisiología , Proteínas E2 de Adenovirus/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Proteínas E1A de Adenovirus/genética , Animales , Proteína de Unión a CREB , Línea Celular , Histona Acetiltransferasas , HumanosRESUMEN
A crucial function of the BCR-ABL chimeric gene in chronic myeloid leukemia is the prolongation of cell survival by inhibition of apoptosis. BCR-ABL expression confers cross-resistance to multiple genotoxic anticancer drugs by inhibition of the apoptotic response to DNA damage in association with cell cycle arrest at the G2-M restriction point. Previous reports indicated that BCR-ABL exerts its antiapoptotic effect against various apoptotic stimuli upstream to the cleavage and activity of caspase-3. Here we show that the adenovirus E1A protein induces substantial apoptosis in BCR-ABL expressing K562 and LAMA-84 leukemia cells. This apoptotic activity of E1A is accompanied by processing of caspase-3 and cleavage of poly(ADP-ribose) polymerase and can be significantly blocked by z-VAD-fmk Z-Val-Ala-Asp(OCH3)-CH2F and the caspase-3-specific inhibitor Z-DEVD-FMK Z-Asp(OCH3)-Glu-Val-Asp(OCH3)-CH2F. Moreover, E1A renders K562 cells, which are particularly resistant to cell death irrespective of the inducing agent, susceptible to induction of apoptosis by the chemotherapeutic agents etoposide and daunorubicin. Counteracting the DNA damage-induced inactivation of cdc2 kinase, E1A reverses the drug-induced G2-M arrest These results indicate that solitary delivery of E1A significantly antagonizes BCR-ABL-induced antiapoptotic functions and circumvents the inherent resistance to DNA damage-induced apoptosis, supporting the use of E1A in combination with chemotherapeutic agents as a promising therapeutic strategy for successful treatment of Philadelphia chromosome-positive leukemia in vivo.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Apoptosis/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Leucemia/metabolismo , Antibióticos Antineoplásicos/farmacología , Western Blotting , Proteína Quinasa CDC2/metabolismo , Caspasa 3 , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , División Celular , Daño del ADN/efectos de los fármacos , Daunorrubicina/farmacología , Relación Dosis-Respuesta a Droga , Etopósido/farmacología , Citometría de Flujo , Humanos , Células K562 , Leucemia/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Luciferasas/metabolismo , Microscopía de Contraste de Fase , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Péptidos/farmacología , Plásmidos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Adenoviral E1 A proteins exhibit a strong tumor-suppressive activity in human tumor cells. However, E1 A is capable of transforming rodent and human cells in cooperation with other oncoproteins, such as activated RAS. Thus, the therapeutic use of wild-type E1A harbors the principal risk of enhancing tumor malignancy. This prompted us to construct E1A 13S cDNA-derived mutants that were unable to transform baby mouse kidney cells in cooperation with E1B and to test their tumor-suppressive activity in BLM human melanoma cells. Anchorage-independent growth in soft agar was reduced for those cell lines expressing the E1AdelCR2 mutant, which lacks the entire conserved region 2 (CR2) sequences, or for cells expressing the E1AcR3Ex2 mutant, which contains CR3 plus exon 2 sequences. In contrast, cell lines expressing the entire E1A wild-type (E1AWT) or only the exon 2 sequences (E1AEx2) grew like the parental BLM cells. Moreover, inoculation of nude mice with BLM cells or cells expressing E1AEx2 revealed large tumors after 2 weeks. In contrast, tumors derived from E1AdelCR2- or E1ACR3Ex2-expressing cells exhibited a substantial delay in tumor growth accompanied by a loss of E1A expression in the outgrown tumors. Cell lines expressing E1AWT showed an intermediate phenotype. Thus, expression of CR3 plus exon 2 sequences is sufficient to enhance both the antioncogenic properties and the therapeutic safety of E1A in our system.
Asunto(s)
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Transformación Celular Neoplásica/genética , Terapia Genética , Melanoma Experimental/terapia , Proteínas E1A de Adenovirus/metabolismo , Animales , Western Blotting , ADN de Neoplasias/análisis , Virus Defectuosos , Genes Supresores de Tumor , Humanos , Melanoma Experimental/metabolismo , Ratones , Ratones Desnudos , Reacción en Cadena de la Polimerasa , Transfección , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
The transfer of pro-apoptotic genes to tumors is one of the most promising strategies for anticancer gene therapy. However, the use of potentially toxic genes, such as tumor suppressor genes or apoptotic genes, needs controllable transgene activation. To achieve regulation of the transgene at a desired time, we developed an adenovirus (Ad) vector, in which the apoptotic activity of the target gene has been made 4-OHT-dependent by fusion to the ligand binding-domain of the estrogen receptor (ER). For evaluation of the system in human tumor cells, we used the E2F1 gene which encodes a transcription factor that triggers massive apoptosis in several human cancers. AdER-E2F1 expressed high levels of transgene over at least 1 week. Upon activation of E2F1 by the ligand 4-hydroxy-tamoxifen (4-OHT) the ER-E2F1 fusion protein correctly translocated from the cytosol to the nucleus, transactivated E2F-dependent promoters, and rapidly induced substantial E2F1-related toxicity. Finally, experiments in nude mice showed tightly regulated tumor growth suppression in vivo. Taken together, our system represents a powerful approach for tight regulation and rapid induction of cytotoxicity as the major criteria for safe gene delivery.
Asunto(s)
Adenoviridae/genética , Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Neoplasias Experimentales/terapia , Factores de Transcripción/genética , Animales , Apoptosis/genética , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Antagonistas de Estrógenos/uso terapéutico , Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , Proteína 1 de Unión a Retinoblastoma , Tamoxifeno/uso terapéutico , Factor de Transcripción DP1RESUMEN
Expression of the transcription unit early region 2 (E2) is of crucial importance for adenoviruses because this region encodes proteins essential for viral replication. Here, we demonstrate that the E1A(12S) protein of the oncogenic adenovirus serotype 12 activates the E2 promoter in dependence of the N terminus and the conserved region 1. Activation is mediated through a cAMP-response element that is bound by CREB-1 and ATF-1. Moreover, the Ad12 E2 promoter is inducible by protein kinase A and repressed by either a dominant-negative cAMP-response element-binding protein (CREB) mutant or the highly specific protein kinase A inhibitor protein underscoring the participation of CREB-1/ATF-1 in promoter activation. E1A(12S) binds to CREB-1 and ATF-1 in dependence of the N terminus and CR1 and is recruited to the E2 cAMP-response element through both cellular transcription factors. Most interestingly, point mutations revealed that E1A(12S) domains essential for binding to CREB-1/ATF-1 and for activation of the Ad12 E2 promoter are also essential for binding to the CREB-binding protein. Due to these data and results obtained in DNA-dependent protein-protein interaction assays, we propose a model in which the cAMP-independent activation of the Ad12 E2 promoter is mediated through a ternary complex consisting of CREB-1/ATF-1, E1A(12S), and CREB-binding protein, which assembles on the E2 cAMP-response element.
Asunto(s)
Proteínas E2 de Adenovirus/genética , AMP Cíclico/metabolismo , Proteínas de Unión al ADN , Regulación Viral de la Expresión Génica , Regiones Promotoras Genéticas , Elementos de Respuesta , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 1 , Adenoviridae/genética , Adenoviridae/crecimiento & desarrollo , Proteínas E1A de Adenovirus/metabolismo , Secuencia de Bases , Proteína de Unión a CREB , Secuencia Conservada , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Proteínas Nucleares/metabolismo , Virus Oncogénicos/genética , Virus Oncogénicos/crecimiento & desarrollo , Unión Proteica , Transactivadores/metabolismo , Activación TranscripcionalRESUMEN
Induction of apoptosis seems to be a key function in maintaining normal cell growth by exerting negative controls on cell proliferation and suppressing tumorigenesis. The adenovirus E1A oncogene shows both cell cycle progression and apoptotic functions. To understand the mechanism of E1A-induced apoptosis, the apoptotic function of E1A 13S was investigated in p53-null cells. We show here that E1A is sufficient by itself to induce substantial apoptosis independent of p53 and other adenoviral genes. The apoptotic function of E1A is accompanied by processing of caspase-3 and cleavage of poly(ADP-ribose)-polymerase. Cell death is significantly blocked by the caspase inhibitor zVAD-fmk and when coexpressed with E1B19K, Bcl-2 or the retinoblastoma protein (RB). Analyses of E1A mutants indicated that the apoptotic activity of E1A correlates closely with the ability to bind the key regulators of E2F1-induced apoptosis, p300 and RB. Finally, in vivo relevance of down-modulation of p53-independent apoptosis for efficient transformation is demonstrated.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Apoptosis/genética , Regulación Viral de la Expresión Génica , Proteína p53 Supresora de Tumor/genética , Adenoviridae/genética , Animales , Línea Celular , Técnicas de Transferencia de Gen , Genes ViralesRESUMEN
Major histocompatibility complex (MHC) class I genes encode highly polymorphic antigens that play an essential role in a number of immunological processes. Their expression is activated in response to a variety of signals and is mediated through several promoter elements among which the enhancer A is one of the key control regions. It contains binding sites for several transcription factors, for example: (i) a well-characterized binding site for rel/NF-kappaB transcription factors in its 3'-end (the H2TF1 or kappaB1 element), (ii) a second kappaB site (the kappaB2 element), which is located immediately adjacent 5' to the H2TF1 element and which is recognized by p65/relA in the human HLA system, and (iii) an AP-1/ATF recognition sequence in the 5' end (EnA-TRE). Here we demonstrate that latter element is bound by at least two distinct heterodimers of the AP-1/ATF transcription factor family, namely c-Jun/ATF-2 and c-Jun/Fra2. Moreover, our data reveal that the enhancer A is simultaneously bound by AP-1/ATF and rel/NF-kappaB transcription factors and that the cellular coactivator p300, which enhances enhancer A-driven reporter gene expression if cotransfected, is recruited to the enhancer A through this multiprotein complex. In contrast to the complete enhancer A, neither the EnA-TRE nor the H2TF1 element on their own are able to confer activation on a heterologous promoter in response to the phorbol ester tumor promoter TPA or the cytokine TNFalpha. Moreover, deletion of any one of the enhancer A control elements results in a dramatic loss of its inducibility by TNFalpha, and point mutations in either the EnA-TRE or the H2TF1 element lead to the loss of AP-1/ATF or NF-kappaB binding, respectively, and to the loss of enhancer A inducibility. Therefore, we conclude that the enhancer A is synergistically activated through a multiprotein complex containing AP-1/ATF, NF-kappaB transcription factors as well as the cellular coactivator p300.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Elementos de Facilitación Genéticos , Antígenos H-2/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción Activadores , Animales , Proteína de Unión a CREB , Cloranfenicol O-Acetiltransferasa/metabolismo , Regulación de la Expresión Génica , Antígenos H-2/genética , Células HeLa , Humanos , Ratones , Subunidad p52 de NF-kappa B , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Transcriptional control of p53 expression participates in the generation of appropriate levels of active p53 in response to mitogenic stimulation. This prompted us to study the role of a putative AP-1 and a NF-kappaB motif in the human p53 promoter for transcriptional regulation. We show that mutation of the AP-1 or the NF-kappaB motif abolishes transcription from the human p53 promoter in HeLa, HepG2 and adenovirus type 5 E1-transformed 293 cells. In comparison, mutation of the previously characterized Myc/Max/USF binding site in the human p53 promoter reduces the transcription rate fivefold. The AP-1 motif in the human p53 promoter binds c-Fos and c-Jun and the NF-kappaB motif binds p50(NF-kappaB) and p65RelA. The cooperative nature of transcriptional activation by these factors was documented by repression of c-fos or NF-kappaB1 translation: Pretreatment of the cells with a c-fos or p50(NF-kappaB1) antisense oligonucleotide suppresses transcription from the human p53 promoter completely. In addition, we show that (a) the level of endogenous p53 mRNA and (b) transcription from the strictly p53-dependent human mdm2 promoter are reduced in the presence of c-fos, c-jun, p50(NF-kappaB1), p65RelA or c-myc antisense oligonucleotides, underscoring the importance of these transcription factors for the expression of functional p53.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Genes p53 , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Animales , Humanos , Ratones , FN-kappa B/fisiología , Oligonucleótidos Antisentido , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-myc/fisiología , Factor de Transcripción AP-1/fisiología , Células Tumorales CultivadasRESUMEN
Transformation of primary rodent cells by functions of the adenovirus type 12 (Ad12) early region 1 (E1) is reduced severalfold compared with transformation by E1 of Ad2. We analyzed whether the unique spacer region of Ad12 E1A that borders the conserved region (CR) 2 and represents an oncogenic determinant of Ad12 E1A is involved in this impaired transformation property, putatively by modulating transformation-relevant biological E1A functions. We show that a mutant (E1ASpm1) that lacks 12 amino-terminal residues of the spacer binds p105-Rb and p130 as Ad12 E1A wild type (E1Awt), whereas a second spacer mutant (E1ASpm2) that lacks an adjacent stretch of six alanines exhibits highly reduced binding to p105-Rb. The binding of this mutant to the p130 pocket protein is, however, little impaired. E1ASpm1 diminishes the formation of the p105-Rb-E2F complex more efficiently than E1Awt or, least efficient, E1ASpm2. These properties of the spacer mutants to target and to disintegrate the p105-Rb-E2F complex correspond with their ability to transform primary mouse cells in combination with E1B: E1ASpm1 (plus Ad12 E1B)-transfected cells could be easily established as cell lines, comparable to Ad12 E1Awt- or Ad2 E1Awt-transfected cells. In contrast, cells transfected with E1ASpm2 or Ad12 E1AdelCR2 (lacking the entire CR2) died within 6-10 weeks after replating, although foci were formed in all cases. Of note, the E1ASpm1-transformed cells grow as fast as the Ad2 E1Awt-transformed cells, with a doubling rate of 15 h, whereas the doubling of the Ad12 E1Awt-transformed cells takes approximately 120 h. Moreover, in the established cell lines, the affinity of E1ASpm1 to p105-Rb was higher than with that of E1Awt. Our data suggest the presence of a transformation-suppressing domain within the carboxyl-terminal 12 residues of the Ad12 E1A-unique spacer, whereas the hydrophobic stretch of six alanines in the spacer is required for stable transformation.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Transformación Celular Viral , Proteína de Retinoblastoma/metabolismo , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Secuencia de Aminoácidos , Animales , Western Blotting , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Proteína de Retinoblastoma/genética , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Association with the cellular coactivators p300 and CBP is required for the growth-regulatory function of adenoviral (Ad) early region 1A (E1A) proteins. E1A regions necessary for these interactions overlap with domains involved in the induction of tumours in immunocompetent rodents through highly oncogenic Ad12. Differences in the association of cellular factors with the respective E1A domains of Ad12 and nononcogenic Ad2 might therefore be involved in serotype-specific oncogenicity. We analyzed the interaction of the Ad12 E1A 235R protein with p300 and CBP. Here we demonstrate that in the case of Ad12, but not Ad2/5, amino acids (aa) 1-29 of E1A proteins are sufficient to bind the p300-C/H3 domain in vivo and wild-type p300 in vitro. The conserved arginine-2, which is essential for the interaction between Ad2 E1A and p300, was dispensable for the Ad12 E1A 235R-p300 interaction in vitro. In addition to the p300-C/H3 region, we identified a second domain within p300 (aa 1999-2200) binding to the 235R protein. Contrary to p300, the amino-terminus and CR1 are necessary to associate with CBP. The aa 1-29 of the 235R protein but not CR1 are essential for the repression of colTRE-driven gene expression. This repression function is strictly dependent on p300 but not on CBP.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Proteínas E1A de Adenovirus/genética , Adenovirus Humanos/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Proteína de Unión a CREB , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores/genéticaRESUMEN
Early region 1A (E1A) gene products of adenoviruses (Ad) play an essential role in both productive infection and cellular transformation. Besides their function to induce the expression of all other adenoviral genes they modulate the expression of specific cellular genes to ensure an efficient viral reproduction. Gene regulatory functions of E1A proteins are mainly located in the conserved regions 1-3 (CRs) and in the non-conserved amino terminal end and are mediated via protein/protein interactions with cellular factors. We could show recently, that the E1A N-terminus (amino acids [aa] 1-29) of oncogenic Ad12 contains a unique 'trans'-activation domain. Here we demonstrate that this region binds to rap30/TF(II)F and to the TATA-box binding protein TBP in vitro. Mutation analyses suggest that binding to rap30 and 'trans'-activation are two independent functions as a mutant which failed to interact with rap30 was still able to induce gene expression with wildtype efficiency. Moreover loss of transcriptional activity does not correlate with a loss of TBP binding suggesting that this association is not necessary for the N-terminal 'trans'-activating activity. Interestingly, aa 1-29 of Ad2 E1A binds also to rap30 indicating that this interaction might be a common feature of E1A proteins from different serotypes.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Aminoácidos , Sitios de Unión , Humanos , Datos de Secuencia Molecular , Serotipificación , Proteína de Unión a TATA-BoxRESUMEN
The recent cloning of human cytidine deaminase (CDD) revealed two variants with a nonconservative amino acid deviation (Gln<-->Lys) at codon 27 within a region of structural homology to a core domain of bacterial CDDs. We here confirm the occurrence of both CDD sequences by cDNA cloning and show that at cytarabine (ara-C) concentrations of 1x10(-8) to 2x10(-2) M, the recombinant enzyme corresponding to the Lys-carrying natural variant (CDD-2) exerts a 1.3- to 3.3-fold higher in vitro deamination rate of ara-C than the Gln-carrying enzyme (CDD-1). These results suggest that this genetic polymorphism contributes to the different deamination phenotypes of ara-C observed in vivo, and that investigation of CDD allelotype frequencies and their correlation with ara-C resistance in patients with acute leukemia may be warranted. In addition, our data may be relevant to recently considered CDD gene transfer strategies for the detoxification of hematopoietic stem cells during high-dose therapy with cytosine nucleoside analogs.
Asunto(s)
Citidina Desaminasa/química , Citidina Desaminasa/genética , Proteínas Recombinantes/química , Citarabina/química , Citidina/química , ADN Complementario/genética , Desaminación , Resistencia a Antineoplásicos/genética , Variación Genética/fisiología , Humanos , Análisis de Secuencia de ADNRESUMEN
The transformation-defective Vero cell host range mutant CS-1 of the highly oncogenic adenovirus type 12 (Ad12) (Ad12-CS-1) has a 69-bp deletion in the early region 1A (E1A) gene that removes the carboxy-terminal half of conserved region 2 and the amino-terminal half of the Ad12-specific so-called spacer that seems to play a pivotal role in the oncogenicity of the virus. Despite its deficiency in immortalizing and transforming primary rodent cells, we found that the E1A 13S protein of Ad12-CS-1 retains the ability to bind p105-RB, p107, and p130 in nuclear extract binding assays with glutathione S-transferase-E1A fusion proteins and Western blot analysis. Like wild-type E1A, the mutant protein was able to dissociate E2F from retinoblastoma-related protein-containing complexes, as judged from gel shift experiments with purified 12S and 13S proteins from transfection experiments with an E1A expression vector or from infection with the respective virus. Moreover, in transient expression assays, the 12S and 13S products of wild-type Ad12 and Ad12-CS-1 were shown to transactivate the Ad12 E1A promoter containing E2F-1 and E2F-5-motifs, respectively, in a comparable manner. The same results were obtained from transfection assays with the E2F motif-dependent E2 promoter of adenovirus type 5 or the human dihydrofolate reductase promoter. These data suggest that efficient infection by Ad12 and the correlated virus-induced reprogramming of the infected cells, including the induction of cell cycle-relevant mechanisms (e.g. E2F activation), can be uncoupled from the transformation properties of the virus.
Asunto(s)
Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Virus Defectuosos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Proteínas , Proteína de Retinoblastoma/metabolismo , Transactivadores , Factores de Transcripción/metabolismo , Activación Transcripcional , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Secuencia de Aminoácidos , Transformación Celular Viral , Virus Defectuosos/genética , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F5 , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína 1 de Unión a Retinoblastoma , Proteína p107 Similar a la del Retinoblastoma , Proteína p130 Similar a la del Retinoblastoma , Eliminación de Secuencia , Factor de Transcripción DP1 , Células Tumorales CultivadasRESUMEN
We have analysed the different regulation of cell-cycle-relevant proteins by interferon alpha (IFN alpha) in IFN alpha-sensitive and resistant U937 leukemic cell lines. In contrast to the INF alpha-sensitive U937 variant cell line U937V, the IFN alpha-resistant derivative (U937VR) is insensitive to the antiproliferative activity of IFN alpha. As we found no differences between these cell lines concerning the induction by IFN alpha of the pathway involving tyrosine-protein kinases and the signal transducer and activator of transcription (Jak-Stat), we examined whether cell-cycle-regulating proteins are differently affected by IFN alpha in U937VR and U937VR cells. In U937V cells IFN alpha induced the formation of the complex between early-region-2 transcription factor 1 (E2F-1) and retinoblastoma protein (RB) which is known to repress transcription of E2F-1-inducible genes, necessary for cell cycle progression. Formation of this complex was not inducible by IFN alpha in U937VR cells, although the suitable binding partners (E2F-1 and under-phosphorylated RB) were present. Interestingly, treatment of nuclear extracts from logarithmically growing U937V and U937VR cells with an antiserum against cyclin A that disrupts cyclin-A-containing complexes, led to the formation of the E2F-1-RB complex, suggesting the presence of under-phosphorylated (active) RB, trapped in nuclear complexes that contain cyclin A. This suggestion was supported by combined immunoprecipitation/western blot experiments that revealed a physical interaction between phosphorylated as well as under-phosphorylated forms of RB and cyclin A complex(es) in U937V and U937VR cells. RB, especially the under-phosphorylated form, was released by treatment with IFN alpha from this complex(es) in the case of U937V cells but not U937VR cells. We conclude that the missing induction of the E2F-1-RB transrepressor by IFN alpha and the failure to release RB from cyclin-A-containing complexes might contribute to the resistance of U937VR cells to the antiproliferative effects of IFN alpha.