RESUMEN
The biological role of interleukin 6 (IL-6) molecules in human B cell tumorigenesis was studied by using an episomal expression vector, pHEBoSV-IL6, to introduce stably the human IL-6 gene into human Epstein Barr virus (EBV)-transformed B lymphoblasts. The gene was present in the IL-6-transfected cells in a high copy number and was efficiently expressed, resulting in the secretion of consistent levels of IL-6 molecules. The constitutive expression of the IL-6 gene led to an altered pattern of growth and to a malignant phenotype, as shown by clonogenicity in to an altered pattern of growth and to a malignant phenotype, as shown by clonogenicity in soft agar cultures and tumorigenicity in nude mice. These data suggest that the combined action of EBV, which exerts an immortalizing function, and of the growth-promoting activity of IL-6 molecules, can give rise to fully transformed B cell tumors in immunodeficient subjects.
Asunto(s)
Linfocitos B/citología , Transformación Celular Neoplásica/genética , Herpesvirus Humano 4 , Interleucina-6/genética , Animales , Linfocitos B/microbiología , Northern Blotting , Línea Celular Transformada , Transformación Celular Viral , Femenino , Expresión Génica , Herpesvirus Humano 4/genética , Interleucina-6/biosíntesis , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Plásmidos , Transcripción Genética , TransfecciónRESUMEN
Human immunodeficiency virus 1 (HIV1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin 6 (IL-6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL-6 secretion of HIV1-infected cells may include transactivation of the IL-6 gene by HIV1. To test this hypothesis, we used the pIL6Pr-chloramphenicol acetyltransferase (CAT) plasmid, an IL-6 promoter-CAT construct, as a target of the transactivating function of the HIV1 TAT protein. By cotransfecting the pIL6Pr-CAT and the tat-expressing pSVT8 plasmid in MC3 B-lymphoblastoid or in HeLa epithelial cells, we observed that TAT transactivates the human IL-6 promoter. These results were confirmed when pIL6Pr-CAT was transfected in MC3 or HeLa cells that constitutively expressed the tat gene in a sense (pSVT8 cells) or antisense (pSVT10 cells) orientation. 5' deletion plasmids of pIL6Pr-CAT, in which regions at -658, -287, and -172 were inserted 5' to the cat gene, were transiently transfected in pSVT10 and pSVT8 cells and showed that TAT-induced activation of the IL-6 promoter required a minimal region located between -287 and -54 bp. Moreover, experiments with plasmids carrying the -658, -287, and -172 bp regions of the IL-6 promoter inserted downstream to a TAR-deleted HIV1-LTR identified the sequence of -172 to -54 as the minimal region of the IL-6 promoter required for TAT to transactivate the TAR-deleted HIV1-LTR. By DNA-protein binding experiments, tat-transfected cells expressed a consistent increase in kappa B and nuclear factor (NF)-IL-6 binding activity. Accordingly, the pDRCAT and IL-1REK9CAT, carrying tandem repeats of NF-kappa B or NF-IL6 binding motifs, respectively, were activated in TAT-expressing cells. The biological relevance of the TAT-induced IL-6 secretion was addressed by generating 7TD1 cells, an IL-6-dependent mouse cell line, stably expressing the tat gene. These tat-positive cells expressed the endogenous IL-6 gene, secreted high amounts of murine IL-6, and grew efficiently in the absence of exogenous IL-6. Moreover, the tat-positive 7TD1 cells sustained the growth of parental 7TD1 cells and showed a dramatic increase in their tumorigenic potency. These results suggest that TAT protein may play a role in the pathogenesis of some HIV1-associated diseases by modulating the expression of host cellular genes.
Asunto(s)
Expresión Génica , Productos del Gen tat/metabolismo , VIH-1/genética , Interleucina-6/biosíntesis , Animales , Linfocitos B , Secuencia de Bases , Línea Celular , Línea Celular Transformada , Transformación Celular Neoplásica , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/metabolismo , Cartilla de ADN , Femenino , Productos del Gen tat/biosíntesis , Genes tat , VIH-1/metabolismo , Células HeLa , Humanos , Interleucina-6/genética , Cinética , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Activación Transcripcional , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Vaccinia virus (vv) early transcription can be reconstituted in vitro from purified virions; in this assay mRNAs are made inside the viral core and subsequently extruded. Although the in vitro process has been extensively characterized, relatively little is known about vv early transcription in vivo. In the present study the fate of vv early mRNAs in infected HeLa cells was followed by BrUTP transfection and confocal and electron microscopy. The extruded vv early mRNAs were found to be organized into unique granular cytoplasmic structures that reached a size up to 1 microm. By EM these structures appeared as amorphous electron-dense cytoplasmic aggregates that were surrounded by ribosomes. Confocal images showed that the RNA structures were located some distance away from intracellular cores and that both structures appeared to be aligned on microtubules (MTs), implying that MT tracks connected mRNAs and cores. Accordingly, intact MTs were found to be required for the typical punctate organization of viral mRNAs. Biochemical evidence supported the notion that vv mRNAs were MT associated and that MT depletion severely affected viral (but not cellular) mRNA synthesis and stability. By confocal microscopy the viral mRNA structures appeared to be surrounded by molecules of the translation machinery, showing that they were active in protein synthesis. Finally, our data suggest a role for a MT and RNA-binding viral protein of 25 kDa (gene L4R), in mRNA targeting away from intracellular cores to their sites of cytoplasmic accumulation.
Asunto(s)
Citoplasma/virología , Microtúbulos/metabolismo , ARN Viral/metabolismo , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Ensamble de Virus , Western Blotting , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/ultraestructura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Células HeLa , Humanos , Microscopía Electrónica , Microscopía Fluorescente , Microtúbulos/ultraestructura , Microtúbulos/virología , Mitocondrias/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Transfección , Virus Vaccinia/química , Virus Vaccinia/ultraestructuraRESUMEN
Several studies associate foetal human exposure to bisphenol A (BPA) to metabolic/endocrine diseases, mainly diabesity. They describe the role of BPA in the disruption of pancreatic beta cell, adipocyte and hepatocyte functions. Indeed, the complexity of the diabesity phenotype is due to the involvement of different endoderm-derived organs, all targets of BPA. Here, we analyse this point delineating a picture of different mechanisms of BPA toxicity in endoderm-derived organs leading to diabesity. Moving from epidemiological data, we summarize the in vivo experimental data of the BPA effects on endoderm-derived organs (thyroid, pancreas, liver, gut, prostate and lung) after prenatal exposure. Mainly, we gather molecular data evidencing harmful effects at low-dose exposure, pointing to the risk to human health. Although the fragmentation of molecular data does not allow a clear conclusion to be drawn, the present work indicates that the developmental exposure to BPA represents a risk for endoderm-derived organs development as it deregulates the gene expression from the earliest developmental stages. A more systematic analysis of BPA impact on the transcriptomes of endoderm-derived organs is still missing. Here, we suggest in vitro toxicogenomics approaches as a tool for the identification of common mechanisms of BPA toxicity leading to the diabesity in organs having the same developmental origin.
Asunto(s)
Compuestos de Bencidrilo/toxicidad , Diabetes Mellitus Tipo 2/fisiopatología , Endodermo/efectos de los fármacos , Enfermedades Metabólicas/fisiopatología , Obesidad/fisiopatología , Fenoles/toxicidad , Animales , Diabetes Mellitus Tipo 2/inducido químicamente , Modelos Animales de Enfermedad , Tracto Gastrointestinal/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Pulmón/efectos de los fármacos , Masculino , Enfermedades Metabólicas/inducido químicamente , Obesidad/inducido químicamente , Páncreas/efectos de los fármacos , Próstata/efectos de los fármacos , Glándula Tiroides/efectos de los fármacosRESUMEN
Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 × 10(-9 )M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-κB pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-κB activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions.
Asunto(s)
Compuestos de Bencidrilo/toxicidad , Glucemia/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Fenoles/toxicidad , Animales , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Homeostasis/efectos de los fármacos , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Toxicogenética/métodosRESUMEN
Antihypertensive efficacy, effects on left ventricular mass index (LVMI) and tolerability of telmisartan, an angiotensin II receptor blocker, were compared with those of hydrochlorothiazide (HCTZ). Adult patients with mild-to-moderate hypertension and an optimal acoustic window by two-dimensional echocardiography were randomised at baseline to 12 months' double-blind, once-daily treatment with telmisartan 80 mg or HCTZ 25 mg. Two-dimensional echocardiography and freehand precordial three-dimensional echocardiography and 24-h ambulatory blood pressure monitoring were performed at baseline and after treatment. Of the 41 telmisartan group patients and 28 HCTZ group patients, 40 and 25, respectively, completed the study. Following treatment, 24-h mean SBP (telmisartan 157 +/- 11 vs 133 +/- 7 mmHg, P<0.001; HCTZ 154 +/- 10 vs 144 +/- 11 mmHg, P<0.003) and DBP (telmisartan 96 +/- 6 vs 83 +/- 5 mmHg, P<0.001; HCTZ 95 +/- 7 vs 87 +/- 8 mmHg, P<0.003) were significantly reduced. Telmisartan produced significantly greater 24-h mean SBP and DBP reductions than HCTZ (P<0.001). LVMI was significantly reduced by telmisartan (141 +/- 16 vs 125 +/- 19 g/m2, P<0.001), but not by HCTZ (139 +/- 20 vs 135 +/- 22 g/m(2)). Incidences of adverse events in both the treatment groups were low; two cases of hypokalaemia occurred with HCTZ. In conclusion, telmisartan 80 mg was well tolerated and significantly reduced SBP, DBP and LVMI after 12 months' treatment compared with HCTZ.
Asunto(s)
Antihipertensivos/uso terapéutico , Bencimidazoles/uso terapéutico , Benzoatos/uso terapéutico , Hidroclorotiazida/uso terapéutico , Hipertensión/tratamiento farmacológico , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Adulto , Anciano , Antagonistas de Receptores de Angiotensina , Presión Sanguínea/efectos de los fármacos , Monitoreo Ambulatorio de la Presión Arterial , Método Doble Ciego , Ecocardiografía Tridimensional , Femenino , Humanos , Hipertensión/diagnóstico por imagen , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Telmisartán , Resultado del TratamientoRESUMEN
DNOC, Ferbam and Imidan were tested in (C3H X C57BL/6) F1 mice to assess their potential testicular toxicity. Chemicals were administered i.p. and per os at different doses for 5 consecutive days. After 35 days the testicular was toxicity was evaluated by measuring the testicular weights, the sperm counts and the percentage of abnormal sperm. DNOC and Imidan failed to induce teratospermia in mice treated by both routes of administration. Conversely Ferbam induced a statistically significant increase in teratospermia only following per os administration to mice at a dose of 1000 mg/kg b.w./day. These data indicate that per os administration of Ferbam succeeded in producing active metabolites able to interfere with the differentiation process of spermatogenic cells.
Asunto(s)
Cresoles/toxicidad , Dimetilditiocarbamato/toxicidad , Dinitrocresoles/toxicidad , Insecticidas/toxicidad , Plaguicidas/toxicidad , Fosmet/toxicidad , Espermatozoides/efectos de los fármacos , Tiocarbamatos/toxicidad , Administración Oral , Animales , Relación Dosis-Respuesta a Droga , Inyecciones Intraperitoneales , Masculino , Ratones , Mutágenos , Tamaño de los Órganos/efectos de los fármacos , Recuento de Espermatozoides/efectos de los fármacos , Espermatozoides/citología , Testículo/anatomía & histologíaAsunto(s)
Herpesvirus Humano 4/genética , Interleucina-6/genética , Linfoma de Células B/genética , Plásmidos/genética , Receptores Inmunológicos/genética , Transcripción Genética/genética , Transfección , Infecciones Tumorales por Virus/genética , Animales , Linfocitos B/inmunología , Diferenciación Celular , Línea Celular , Inmunoglobulinas/análisis , Interleucina-6/biosíntesis , Linfoma de Células B/metabolismo , Ratones , Ratones Desnudos , Fenotipo , Receptores Inmunológicos/biosíntesis , Receptores de Interleucina-6 , Infecciones Tumorales por Virus/metabolismoAsunto(s)
Modelos Animales de Enfermedad , Leucemia/patología , Mutación/genética , Proteínas Nucleares/fisiología , Secuencias Repetidas en Tándem/genética , Tirosina Quinasa 3 Similar a fms/genética , Enfermedad Aguda , Animales , Femenino , Humanos , Leucemia/genética , Leucemia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Nucleofosmina , Tasa de SupervivenciaRESUMEN
The activation of the human immunodeficiency virus, type 1 (HIV-1) by the DNA alkylating agents ethyl methanesulfonate, methyl methanesulfonate, and mitomycin C was observed in human B lymphocytes transiently transfected with plasmids in which the HIV-1 long terminal repeat (LTR) directed the expression of the bacterial chloramphenicol acetyltransferase gene. Deletion of the two NF-kappa B-binding sites of LTR abolished the HIV-1 activation induced by the three mutagens, while deletion of the three Sp1-binding sites slightly reduced it. Electrophoretic mobility shift assays revealed an increased binding to the kappa B sites of HIV-1 LTR in the nuclear extracts of human B lymphocytes upon mutagen treatment, while binding to Sp1 sites was unaffected. The TAR region was also involved in the mutagen-mediated activation of HIV-1 LTR inasmuch as a small deletion in the TAR sequence (nucleotides +34 to +37) greatly decreased the induction of HIV-1 expression. Moreover, an enhanced binding activity to the TAR DNA sequence (nucleotides +24 to +47) was observed in nuclear extracts of mutagen-treated lymphocytes. Thus, both the enhancer and the 5'-untranslated region of HIV-1 functionally cooperate in the mutagen-mediated induction of HIV-1 expression.
Asunto(s)
Alquilantes/farmacología , Duplicado del Terminal Largo de VIH/efectos de los fármacos , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Linfocitos/microbiología , Mutágenos/farmacología , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Metanosulfonato de Etilo/farmacología , VIH-1/efectos de los fármacos , Humanos , Metilmetanosulfonato/farmacología , Mitomicina/farmacología , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Factor de Transcripción Sp1/metabolismo , Activación Viral/efectos de los fármacos , Activación Viral/genéticaRESUMEN
A promoterless neo gene was stably transfected in rodent fibroblasts to act as a reporter gene for rearrangements resulting in its expression at the different genomic integration sites. Nine clones were isolated which had integrated a varying copy number of neo at one or more genomic sites but still displayed a Neo- phenotype (G418-sensitive). These clones were analyzed for their ability to become Neo+ (G418-resistant) either spontaneously or after mutagen treatment. They were all able to generate G418-resistant subclones spontaneously at frequencies ranging from 2 x 10(-8) to 6 x 10(-5). The acquired G418-resistance was always associated with amplification and enhanced transcription of neo. No correlation was observed between the frequency of occurrence of G418-resistance and the number of copies or integration sites of neo. When treated with the mutagens mitomycin C or methylmethane sulfonate, only one clone, RH15, produced G418-resistant subclones in a dose-related fashion. In this mutagen-inducible clone, DNA lesions of a different nature (monoadducts or cross-links) were equally efficient in the induction of G418-resistance. Amplification and enhanced transcription of the neo gene were observed in both the spontaneous and mutagen-induced G418-resistant subclones of RH15 cell line. These findings indicate that the exogenous neo gene integrated at different genomic sites was acting as a reporter gene for amplification. Interestingly, while all nine integration sites were observed to amplify spontaneously, only one could be induced to amplify by mutagens. This suggests that different genomic regions display differing susceptibilities to mutagen-mediated amplification. This may be important in view of the major role played by mutagen-mediated gene amplification in carcinogenesis.
Asunto(s)
Amplificación de Genes/genética , Regulación Neoplásica de la Expresión Génica/genética , Animales , Southern Blotting , Resistencia a Medicamentos/genética , Fibroblastos , Amplificación de Genes/efectos de los fármacos , Gentamicinas/farmacología , Metilmetanosulfonato/farmacología , Mitomicina/toxicidad , Ratas , TransfecciónRESUMEN
The 5'-untranslated leader region of human immunodeficiency virus, type 1 (HIV-1), includes a complex array of putative regulatory elements whose role in the viral expression is not completely understood. Here we demonstrate the presence of an NF-kappaB-responsive element in the trans-activation response (TAR) region of HIV-1 that confers the full induction of HIV-1 long terminal repeat (LTR) in response to NF-kappaB-activating stimuli, such as DNA alkylating agents, phorbol 12-myristate 13-acetate, and tumor necrosis factor-alpha. The TAR NF-kappaB site GGGAGCTCTC spans from positions +31 to +40 and cooperates with the NF-kappaB enhancer upstream of the TATA box in the NF-kappaB-mediated induction of HIV-1 LTR. The conclusion stems from the following observations: (i) deletion of the two NF-kappaB sites upstream of the TATA box reduces, but does not abolish, the HIV-1 LTR activation by NF-kappaB inducers; (ii) deletion or base pair substitutions of the TAR NF-kappaB site significantly reduce the HIV-1 LTR activation by NF-kappaB inducers; (iii) deletions of both the NF-kappaB sites upstream of the TATA box and the TAR NF-kappaB site abolish the activation of HIV-1 LTR in response to NF-kappaB inducers. Moreover, the p50 p65 NF-kappaB complex binds to the TAR NF-kappaB sequence and trans-activates the TAR NF-kappaB-directed expression. The identification of an additional NF-kappaB site in the HIV-1 LTR points to the relevance of NF-kappaB factors in the HIV-1 life cycle.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación Viral de la Expresión Génica , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , FN-kappa B/metabolismo , Secuencia de Bases , Línea Celular , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Unión Proteica , ARN Mensajero/genética , Eliminación de Secuencia , Transcripción Genética , Activación TranscripcionalRESUMEN
In this study, we addressed the question of whether carcinogens affected the expression of interleukin-1 alpha (IL1 alpha), interleukin-1 beta (IL1 beta), and interleukin-6 (IL6) genes and the production of the relative proteins. Primary cultures of human monocytes were exposed to the alkylating agents mitomycin C (Mit C), methylmethane sulfonate (MMS), and ethylmethane sulfonate (EMS) and tested for the production of IL1 alpha, IL1 beta, and IL6 proteins, as well as for the expression of IL1 alpha, IL1 beta, and IL6 transcripts. The production of IL1 beta and IL6 was significantly augmented by all the three chemicals after 24-48 h of treatment. IL1 alpha was also increased by Mit C and MMS. By Northern blotting analysis, the increased expression of IL1 alpha, IL1 beta, and IL6 genes was shown to occur at 30 min of Mit C and MMS treatment and to decline after 8 h. Similarly, EMS up-regulated the expression of IL1 beta and IL6 genes. The mutagen-mediated increase in interleukin transcripts did not require de novo protein synthesis, and it was due to the enhanced half-life of IL1 alpha, IL1 beta, and IL6 mRNAs rather than to the increased rate of gene transcription. These results suggest that carcinogens, in addition to causing DNA mutations and rearrangements, may also affect cell growth and differentiation by enhancing the expression of cytokine genes.
Asunto(s)
Carcinógenos/farmacología , Daño del ADN/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/genética , Interleucina-6/genética , ARN Mensajero/metabolismo , Células Cultivadas , Metanosulfonato de Etilo/farmacología , Humanos , Interleucina-1/biosíntesis , Interleucina-6/metabolismo , Metilmetanosulfonato/farmacología , Mitomicina/farmacología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , ARN Mensajero/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Transcription factors of the NF-kappa B/Rel family are retained in the cytoplasm as inactive complexes through association with I kappa B inhibitory proteins. Several NF-kappa B activators induce the proteolysis of I kappa B proteins, which results in the nuclear translocation and DNA binding of NF-kappa B complexes. Here, we report a novel mechanism of NF-kappa B regulation mediated by p105 (NF-kappa B1) precursor of p50 directly at the nuclear level. In Epstein-Barr virus-immortalized B cells, p105 was found in the nucleus, where it was complexed with p65. In concomitance with NF-kappa B activation, mitomycin C induced the processing of p105 to p50 in the nucleus, while it did not affect the steady-state protein levels of I kappa B alpha and p105 in the cytoplasm. Differently, phorbol 12-myristate 13-acetate induced a significant proteolysis of both I kappa B alpha and p105 in the cytoplasm, while it did not affect the protein level of p105 in the nucleus. These results suggest that in Epstein-Barr virus-positive B cell lines the nuclear processing of p105 can contribute to NF-kappa B activation in response to specific signaling molecules, such as DNA-damaging agents.
Asunto(s)
Linfocitos B/citología , Proteínas Bacterianas/metabolismo , Núcleo Celular/metabolismo , Transformación Celular Viral , Herpesvirus Humano 4/fisiología , FN-kappa B/metabolismo , Secuencia de Bases , Línea Celular , ADN , Mitomicina/farmacología , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Live-attenuated human immunodeficiency viruses (HIVs) are candidates for Acquired Immunodeficiency Syndrome (AIDS) vaccine. Based on the simian immunodeficiency virus (SIV) model for AIDS, loss-of-function (e.g. deletion of accessory genes such as nef) has been forwarded as a primary approach for creating enfeebled, but replication-competent, HIV-1/SIV. Regrettably, recent evidence suggests that loss-of-function alone is not always sufficient to prevent the emergence of virulent mutants. New strategies that attenuate via mechanisms distinct from loss-of-function are needed for enhancing the safety phenotype of viral genome. Here, we propose gain-of-function to be used simultaneously with loss-of-function as a novel approach for attenuating HIV-1. We have constructed an HIV-1 genome carrying the cDNA of a proteolysis-resistant nuclear factor-kappaB inhibitor (IkappaB-alphaS32/36A) in the nef region. HIV-1 expressing IkappaB-alphaS32/36A down-regulates viral expression and is highly attenuated in both Jurkat and peripheral blood mononuclear cells. We provide formal proof that the phenotypic and attenuating characteristics of IkappaB-alphaS32/36A permit its stable maintenance in a live, replicating HIV-1 despite 180 days of forced ex vivo passaging in tissue culture. As compared with other open-reading frames embedded into HIV/SIV genome, this degree of stability is unprecedented. Thus, IkappaB-alphaS32/36A offers proof-of-principle that artifactually gained functions, when used to attenuate the replication of live HIV-1, can be stable. These findings illustrate gain-of-function as a feasible strategy for developing safer live-attenuated HIVs to be tested as candidates for AIDS vaccine.
Asunto(s)
Vacunas contra el SIDA/inmunología , Proteínas de Unión al ADN/genética , VIH-1/genética , Proteínas I-kappa B , FN-kappa B/antagonistas & inhibidores , Línea Celular , Regulación hacia Abajo , Regulación Viral de la Expresión Génica/inmunología , Genes Virales/inmunología , Genes nef , Duplicado del Terminal Largo de VIH , VIH-1/inmunología , Humanos , Células Jurkat/virología , Monocitos/virología , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Fenotipo , ARN Viral/genética , Pase Seriado , Virus de la Inmunodeficiencia de los Simios/genética , Transfección , Vacunas Atenuadas/genética , Replicación ViralRESUMEN
Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line, which suggests that EBV and HIV-1 infection of non-B cells may result in HIV-1 promoter activation. Therefore, a specific gene product of EBV, EBNA2, can transactivate HIV-1 and possibly contribute to the clinical progression of AIDS.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/genética , Antígenos Virales/genética , Proteínas de Unión al ADN/genética , Regulación Viral de la Expresión Génica/genética , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Antígenos Virales/biosíntesis , Antígenos Virales/farmacología , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/farmacología , Antígenos Nucleares del Virus de Epstein-Barr , Regulación Viral de la Expresión Génica/efectos de los fármacos , Productos del Gen tat/biosíntesis , Productos del Gen tat/genética , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/inmunología , Inmunohistoquímica , Datos de Secuencia Molecular , FN-kappa B/biosíntesis , FN-kappa B/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp1/genética , Transactivadores/genética , Transactivadores/farmacología , Activación Transcripcional , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin-6 (IL6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL6 secretion of HIV-1-infected cells may include transactivation of the IL6 gene by HIV-1. Here we report the molecular mechanisms of Tat activity on the expression of the IL6 gene. By using 5' deletion mutants of pIL6Pr-CAT and using IL6:HIV-1-LTR hybrid constructs where discrete regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we identified a short sequence of the 5'-untranslated region of the IL6 mRNA that is required for Tat to trans-activate the IL6 promoter. This sequence acquires a stem-loop structure and includes a UCU sequence that binds to Tat and is necessary for full trans-activation. In addition, we provide the evidence that Tat can function by enhancing the CAAT enhancer-binding protein (C/EBP) DNA binding activity and is able to complex with in vitro translated C/EBPbeta, which is a major mediator of IL6 promoter function. By using the yeast two-hybrid system and immunoprecipitation, we observed that the interaction of Tat with C/EBP proteins also occurred in vivo. The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes. In addition, Tat may function by protein-protein interactions, leading to the generation of heterodimers with specific transcription factors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Productos del Gen tat/metabolismo , VIH-1/fisiología , Interleucina-6/biosíntesis , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Potenciadoras de Unión a CCAAT , Cloranfenicol O-Acetiltransferasa/biosíntesis , Clonación Molecular , Cartilla de ADN , Productos del Gen tat/biosíntesis , Genes tat , VIH-1/genética , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Sondas de Oligonucleótidos , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Eliminación de Secuencia , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
We report the characterization of a CAAT enhancer-binding protein (C/EBP) (NF-IL6) element encompassing the region from -174 to -166 of the U3 long terminal repeat (LTR) region of HIV-1. This C/EBP cis sequence was found to bind to C/EBPbeta and C/EBPdelta factors in DNA band shift assay. Transfection of NTera-2 cells with a HIV-1-LTR CAT construct (pC15CAT), together with C/EBPbeta or C/EBPdelta expression plasmids showed that C/EBP proteins strongly activated the HIV-1 promoter. Deletions encompassing the C/EBP-binding site resulted in the enhancement of the LTR activation mediated by C/EBP proteins, suggesting that other sequences located 3' to -170 were indeed the target for C/EBP factors. This possibility was confirmed by using the pCD54E9CAT plasmid, in which the NF-kappaB enhancer was inserted 5' to the HIV-1 LTR TATA box. A NF-kappaB1(p50) expression plasmid was also utilized to test for functional co-operation between NF-kappaB and C/EBP factors. We observed that p50 middle dotC/EBPbeta and p50 middle dotC/EBPdelta complexes were generated in tested cells and strongly activated the HIV-1 LTR by binding to the NF-kappaB sequences. The physical association of NF-kappaB1(p50) with C/EBP factors was assayed by direct interaction of in vitro translated p50 proteins with C/EBPbeta or C/EBPdelta produced as glutathione S-transferase fusion proteins. Moreover, p50 middle dotC/EBPbeta complexes were observed in vivo by using DNA affinity studies with biotinylated NF-kappaB oligonucleotides. By using mutant forms of p50 or C/EBPbeta proteins we found that the transactivation of HIV-1 LTR by p50 middle dotC/EBPbeta complexes required the DNA-binding domain of p50 and the transcription activation domain of C/EBPbeta.