RESUMEN

Efficient HTLV-1 viral transmission occurs through cell-to-cell contacts. The Tax viral transcriptional activator protein facilitates this process. Using a comparative transcriptomic analysis, we recently identified a series of genes up-regulated in HTLV-1 Tax expressing T-lymphocytes. We focused our attention towards genes that are important for cytoskeleton dynamic and thus may possibly modulate cell-to-cell contacts. We first demonstrate that Gem, a member of the small GTP-binding proteins within the Ras superfamily, is expressed both at the RNA and protein levels in Tax-expressing cells and in HTLV-1-infected cell lines. Using a series of ChIP assays, we show that Tax recruits CREB and CREB Binding Protein (CBP) onto a c-AMP Responsive Element (CRE) present in the gem promoter. This CRE sequence is required to drive Tax-activated gem transcription. Since Gem is involved in cytoskeleton remodeling, we investigated its role in infected cells motility. We show that Gem co-localizes with F-actin and is involved both in T-cell spontaneous cell migration as well as chemotaxis in the presence of SDF-1/CXCL12. Importantly, gem knock-down in HTLV-1-infected cells decreases cell migration and conjugate formation. Finally, we demonstrate that Gem plays an important role in cell-to-cell viral transmission.

Asunto(s)

Citoesqueleto/metabolismo , Virus Linfotrópico T Tipo 1 Humano , Proteínas de Unión al GTP Monoméricas/metabolismo , Linfocitos T/metabolismo , Linfocitos T/virología , Línea Celular , Quimiotaxis de Leucocito/fisiología , Inmunoprecipitación de Cromatina , Técnica del Anticuerpo Fluorescente , Regulación Viral de la Expresión Génica/fisiología , Productos del Gen tax/metabolismo , Immunoblotting , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/patología , Activación Transcripcional/fisiología , Transducción Genética

RESUMEN

Human T-cell Lymphotropic Viruses type 1 (HTLV-1) is the etiological agent of Adult T-cell Leukemia/Lymphoma. Although associated with lymphocytosis, HTLV-2 infection is not associated with any malignant hematological disease. Similarly, no infection-related symptom has been detected in HTLV-3-infected individuals studied so far. Differences in individual Tax transcriptional activity might account for these distinct physiopathological outcomes. Tax-1 and Tax-3 possess a PDZ binding motif in their sequence. Interestingly, this motif, which is critical for Tax-1 transforming activity, is absent from Tax-2. We used the DNA microarray technology to analyze and compare the global gene expression profiles of different T- and non T-cell types expressing Tax-1, Tax-2 or Tax-3 viral transactivators. In a T-cell line, this analysis allowed us to identify 48 genes whose expression is commonly affected by all Tax proteins and are hence characteristic of the HTLV infection, independently of the virus type. Importantly, we also identified a subset of genes (n = 70) which are specifically up-regulated by Tax-1 and Tax-3, while Tax-1 and Tax-2 shared only 1 gene and Tax-2 and Tax-3 shared 8 genes. These results demonstrate that Tax-3 and Tax-1 are closely related in terms of cellular gene deregulation. Analysis of the molecular interactions existing between those Tax-1/Tax-3 deregulated genes then allowed us to highlight biological networks of genes characteristic of HTLV-1 and HTLV-3 infection. The majority of those up-regulated genes are functionally linked in biological processes characteristic of HTLV-1-infected T-cells expressing Tax such as regulation of transcription and apoptosis, activation of the NF-κB cascade, T-cell mediated immunity and induction of cell proliferation and differentiation. In conclusion, our results demonstrate for the first time that, in T- and non T-cells types, Tax-3 is a functional analogue of Tax-1 in terms of transcriptional activation and suggest that HTLV-3 might share pathogenic features with HTLV-1 in vivo.

Asunto(s)

Transformación Celular Viral , Perfilación de la Expresión Génica , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 3 Humano/genética , Virus Linfotrópico T Tipo 3 Humano/metabolismo , Activación Transcripcional , Línea Celular Tumoral , Análisis por Conglomerados , Expresión Génica , Regulación de la Expresión Génica , Orden Génico , Productos del Gen tax/genética , Redes Reguladoras de Genes , Vectores Genéticos/genética , Células HEK293 , Humanos , Reproducibilidad de los Resultados , Transducción Genética

RESUMEN

Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL), an aggressive and highly chemoresistant malignancy. Rho family GTPases regulate multiple signaling pathways in tumorigenesis: cytoskeletal organization, transcription, cell cycle progression, and cell proliferation. Geranylgeranylation of Rho family GTPases is essential for cell membrane localization and activation of these proteins. It is currently unknown whether HTLV-1-transformed cells are preferentially sensitive to geranylgeranylation inhibitors, such as GGTI-298. In this report, we demonstrate that GGTI-298 decreased cell viability and induced G(2)/M phase accumulation of HTLV-1-transformed cells, independent of p53 reactivation. HTLV-1-LTR transcriptional activity was inhibited and Tax protein levels decreased following treatment with GGTI-298. Furthermore, GGTI-298 decreased activation of NF-κB, a downstream target of Rho family GTPases. These studies suggest that protein geranylgeranylation contributes to dysregulation of cell survival pathways in HTLV-1-transformed cells.

Asunto(s)

Transferasas Alquil y Aril/antagonistas & inhibidores , Benzamidas/farmacología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Prenilación de Proteína/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transferasas Alquil y Aril/metabolismo , Ciclo Celular , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Transformación Celular Viral , Productos del Gen tax/efectos de los fármacos , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Humanos , Proteínas I-kappa B/metabolismo , Leucemia-Linfoma de Células T del Adulto/enzimología , Leucemia-Linfoma de Células T del Adulto/virología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Fosforilación , Transporte de Proteínas/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

Positive transcription elongation factor b (P-TEFb) plays an important role in stimulating RNA polymerase II elongation for viral and cellular gene expression. P-TEFb is found in cells in either an active, low-molecular-weight (LMW) form or an inactive, high-molecular-weight (HMW) form. We report here that human T-lymphotropic virus type 1 (HTLV-1) Tax interacts with the cyclin T1 subunit of P-TEFb, forming a distinct Tax/P-TEFb LMW complex. We demonstrate that Tax can play a role in regulating the amount of HMW complex present in the cell by decreasing the binding of 7SK snRNP/HEXIM1 to P-TEFb. This is seen both in vitro using purified Tax protein and in vivo in cells transduced with Tax expression constructs. Further, we find that a peptide of cyclin T1 spanning the Tax binding domain inhibits the ability of Tax to disrupt HMW P-TEFb complexes. These results suggest that the direct interaction of Tax with cyclin T1 can dissociate P-TEFb from the P-TEFb/7SK snRNP/HEXIM1 complex for activation of the viral long terminal repeat (LTR). We also show that Tax competes with Brd4 for P-TEFb binding. Chromatin immunoprecipitation (ChIP) assays demonstrated that Brd4 and P-TEFb are associated with the basal HTLV-1 LTR, while Tax and P-TEFb are associated with the activated template. Furthermore, the knockdown of Brd4 by small interfering RNA (siRNA) activates the HTLV-1 LTR promoter, which results in an increase in viral expression and production. Our studies have identified Tax as a regulator of P-TEFb that is capable of affecting the balance between its association with the large inactive complex and the small active complex.

Asunto(s)

Productos del Gen tax/metabolismo , Proteínas Nucleares/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Factores de Transcripción/metabolismo , Western Blotting , Proteínas de Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Ciclina T/genética , Ciclina T/metabolismo , Citometría de Flujo , Productos del Gen tax/genética , Infecciones por HTLV-I/genética , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/virología , Células HeLa/metabolismo , Células HeLa/virología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Humanos , Peso Molecular , Proteínas Nucleares/genética , Factor B de Elongación Transcripcional Positiva/genética , Unión Proteica , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas Nucleares Pequeñas/genética , Secuencias Repetidas Terminales/genética , Factores de Transcripción/genética , Transcripción Genética , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo

RESUMEN

Human T-lymphotropic virus type 1 (HTLV-1) encodes the viral protein Tax, which is believed to act as a viral transactivator through its interactions with a variety of transcription factors, including CREB and NF-kappaB. As is the case for all retroviruses, the provirus is inserted into the host DNA, where nucleosomes are deposited to ensure efficient packaging. Nucleosomes act as roadblocks in transcription, making it difficult for RNA polymerase II (Pol II) to proceed toward the 3' end of the genome. Because of this, a variety of chromatin remodelers can act to modify nucleosomes, allowing for efficient transcription. While a number of covalent modifications are known to occur on histone tails in HTLV-1 infection (i.e., histone acetyltransferases [HATs], histone deacetylases [HDACs], and histone methyltransferases [HMTs]), evidence points to the use of chromatin remodelers that use energy from ATP hydrolysis to remodel nucleosomes. Here we confirm that BRG1, which is the core subunit of eight chromatin-remodeling complexes, is essential not only for Tax transactivation but also for viral replication. This is especially evident when wild-type infectious clones of HTLV-1 are used. BRG1 associates with Tax at the HTLV-1 long terminal repeat (LTR), and coexpression of BRG1 and Tax results in increased rates of transcription. The interaction of BRG1 with Tax additionally recruits the basal transcriptional machinery and removes some of the core histones from the nucleosome at the start site (Nuc 1). When using the BRG1-deficient cell lines SW13, C33A, and TSUPR1, we observed little viral transcription and no viral replication. Importantly, while these three cell lines do not express detectable levels of BRG1, much of the SWI/SNF complex remains assembled in the cells. Knockdown of BRG1 and associated SWI/SNF subunits suggests that the BRG1-utilizing SWI/SNF complex PBAF is responsible for HTLV-1 nucleosome remodeling. Finally, HTLV-1 infection of cell lines with a knockdown in BRG1 or the PBAF complex results in a significant reduction in viral production. Overall, we concluded that BRG1 is required for Tax transactivation and HTLV-1 viral production and that the PBAF complex appears to be responsible for nucleosome remodeling.

Asunto(s)

Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Replicación Viral , Línea Celular , ADN Viral/metabolismo , Humanos , Unión Proteica , Mapeo de Interacción de Proteínas , Secuencias Repetidas Terminales

RESUMEN

Nuclear factor (NF)-kappaB is a major survival pathway engaged by the Human T-Lymphotropic Virus type 1 (HTLV-1) Tax protein. Tax1 activation of NF-kappaB occurs predominantly in the cytoplasm, where Tax1 binds NF-kappaB Essential Modulator (NEMO/IKKgamma) and triggers the activation of IkappaB kinases. Several independent studies have shown that Tax1-mediated NF-kappaB activation is dependent on Tax1 ubiquitination. Here, we identify by co-immunoprecipitation assays NEMO-Related Protein (NRP/Optineurin) as a binding partner for Tax1 in HTLV-1 infected and Tax1/NRP co-expressing cells. Immunofluorescence studies reveal that Tax1, NRP and NEMO colocalize in Golgi-associated structures. The interaction between Tax1 and NRP requires the ubiquitin-binding activity of NRP and the ubiquitination sites of Tax1. In addition, we observe that NRP increases the ubiquitination of Tax1 along with Tax1-dependent NF-kappaB signaling. Surprisingly, we find that in addition to Tax1, NRP interacts cooperatively with the Tax1 binding protein TAX1BP1, and that NRP and TAX1BP1 cooperate to modulate Tax1 ubiquitination and NF-kappaB activation. Our data strongly suggest for the first time that NRP is a critical adaptor that regulates the assembly of TAX1BP1 and post-translationally modified forms of Tax1, leading to sustained NF-kappaB activation.

Asunto(s)

Productos del Gen tax/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Factor de Transcripción TFIIIA/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular , Línea Celular Tumoral , Productos del Gen tax/genética , Aparato de Golgi , Células HeLa , Humanos , Inmunoprecipitación , Espacio Intracelular/metabolismo , Proteínas de Transporte de Membrana , Dominios y Motivos de Interacción de Proteínas , Técnicas del Sistema de Dos Híbridos , Ubiquitinación

RESUMEN

Adult T-cell leukemia/lymphoma (ATL) is an aggressive and fatal disease. We have examined 32 patients with smoldering, chronic, lymphoma and acute leukemia using Affymetrix HG-U133A2.0 arrays. Using the BRB array program, we identified genes differentially expressed in leukemia cells compared with normal lymphocytes. Several unique genes were identified that were overexpressed in leukemic cells, including TNFSF11, RGS13, MAFb, CSPG2, C/EBP-alpha, and TCF4; 200 of the most highly overexpressed ATL genes were analyzed by the Pathway Studio, version 4.0 program. ATL leukemia cells were characterized by an increase in genes linked to "central" genes CDC2/cyclin B1, SYK/LYN, proliferating cell nuclear antigen, and BIRC5. Because of its potential therapeutic importance, we focused our studies on the regulation and function of BIRC5, whose expression was increased in 13 of 14 leukemia samples. TCF4 reporter assays and transfection of DN-TCF4 demonstrated that TCF4 regulates BIRC5 gene expression. Functionally, transfection of ATL cells with BIRC5 shRNA decreased BIRC5 expression and cell viability 80%. Clinical treatment of ATL patients with Zenapax or bortezomib decreased BIRC5 expression and cell viability. These experiments represent the first direct experimental evidence that BIRC5 plays an important role in ATL cell viability and provides important insight into ATL genesis and potential targeted therapies.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Leucemia-Linfoma de Células T del Adulto/metabolismo , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Factores de Transcripción/metabolismo , Adulto , Anciano , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Ácidos Borónicos/uso terapéutico , Bortezomib , Linfocitos T CD4-Positivos/metabolismo , Supervivencia Celular , Proteínas de Unión al ADN/genética , Daclizumab , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoglobulina G/uso terapéutico , Proteínas Inhibidoras de la Apoptosis , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Leucemia-Linfoma de Células T del Adulto/genética , Masculino , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Pirazinas/uso terapéutico , Interferencia de ARN , Survivin , Factor de Transcripción 4 , Factores de Transcripción/genética , Células Tumorales Cultivadas

RESUMEN

Kaposi's sarcoma (KS) is an angioproliferative inflammatory disorder induced by endothelial cell infection with the KS-associated herpesvirus (KSHV). ORFK13/vFLIP, one of the KSHV genes expressed in KS, encodes a 188-amino-acid protein which binds to the Ikappab kinase (IKK) complex to activate NF-kappaB. We examined ORFK13/vFLIP contribution to KS phenotype and potential for therapeutic targeting. Retroviral transduction of ORFK13/vFLIP into primary human endothelial cells induces the spindle morphology distinctive of KS cells and promotes the formation of abnormal vascular networks typical of KS vasculature; upregulates the expression of proinflammatory cytokines, chemokines, and interferon-responsive genes; and stimulates the adhesion of inflammatory cells characteristic of KS lesions. Thymidine phosphorylase, a cellular enzyme markedly induced by ORFK13/vFLIP, can metabolize the prodrug 5-fluoro-5-deoxyuridine (5-dFUrd) to 5-fluouridine (5-FU), a potent thymidine synthase inhibitor, which blocks DNA and RNA synthesis. When tested for cytotoxicity, 5-dFUrd (0.1 to 1 microM) selectively killed ORFK13/vFLIP-expressing endothelial cells while sparing control cells. These results demonstrate that ORFK13/vFLIP directly and indirectly contributes to the inflammatory and vascular phenotype of KS and identify 5-dFUrd as a potential new drug that targets KSHV latency for the treatment of KS and other KSHV-associated malignancies.

Asunto(s)

Células Endoteliales/virología , Herpesvirus Humano 8/metabolismo , Sarcoma de Kaposi/genética , Proteínas Virales/metabolismo , Forma de la Célula , Células Cultivadas , Células Endoteliales/citología , Regulación de la Expresión Génica , Herpesvirus Humano 8/genética , Humanos , Sarcoma de Kaposi/virología , Transducción Genética , Proteínas Virales/genética

RESUMEN

Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, the bromodomain protein Brd4 has been shown to interact with the low-molecular-weight, active P-TEFb complex and recruit P-TEFb to the HIV type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions +14 and +36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.

Asunto(s)

Quinasa 9 Dependiente de la Ciclina/metabolismo , VIH/fisiología , Proteínas Nucleares/metabolismo , ARN Viral/biosíntesis , Treonina/metabolismo , Factores de Transcripción/metabolismo , Replicación Viral , Proteínas de Ciclo Celular , Inmunoprecipitación de Cromatina , Células HeLa , Humanos , Fosforilación , Factor B de Elongación Transcripcional Positiva/metabolismo , Unión Proteica , Transcripción Genética

RESUMEN

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of the aggressive and fatal disease adult T-cell leukemia. Previous studies have demonstrated that the HTLV-1-encoded Tax protein inhibits the function of tumor suppressor p53 through a Tax-induced NF-kappaB pathway. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-kappaB and p53. In the present study, we have examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G(1) and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is "druggable" and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-kappaB suggests a promising strategy for the treatment of HTLV-1-transformed cells.

Asunto(s)

Aminacrina/farmacología , Anticarcinógenos/farmacología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Proteína p53 Supresora de Tumor/biosíntesis , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Transformación Celular Viral , Relación Dosis-Respuesta a Droga , Fase G1/efectos de los fármacos , Genes Reporteros , Humanos , Etiquetado Corte-Fin in Situ/métodos , Luciferasas/metabolismo , FN-kappa B/antagonistas & inhibidores , Plásmidos , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Transfección

RESUMEN

We and others have uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3). We have now generated an HTLV-3 proviral clone. We established that gag, env, pol, pro, and tax/rex as well as minus-strand mRNAs are present in cells transfected with the HTLV-3 clone. HTLV-3 p24(gag) protein is detected in the cell culture supernatant. Transfection of 293T-long terminal repeat (LTR)-green fluorescent protein (GFP) cells with the HTLV-3 clone promotes formation of syncytia, a hallmark of Env expression, together with the appearance of fluorescent cells, demonstrating that Tax is expressed. Viral particles are visible by electron microscopy. These particles are infectious, as demonstrated by infection experiments with purified virions.

Asunto(s)

Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 3 de los Primates/genética , ARN Mensajero/metabolismo , Línea Celular , Clonación Molecular , Cartilla de ADN/genética , Células Gigantes/virología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Virus Linfotrópico T Tipo 3 de los Primates/ultraestructura , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Transcription consists of a series of highly regulated steps: assembly of the preinitiation complex (PIC) at the promoter, initiation, elongation, and termination. PIC assembly is nucleated by TFIID, a complex composed of the TATA-binding protein (TBP) and a series of TBP-associated factors (TAFs). One component, TAF7, is incorporated in the PIC through its interaction with TFIID but is released from TFIID upon transcription initiation. We now report that TAF7 interacts with the transcription factors, TFIIH and P-TEFb, resulting in the inhibition of their Pol II CTD kinase activities. Importantly, in in vitro transcription reactions, TAF7 inhibits steps after PIC assembly and formation of the first phosphodiester bonds. Further, in vivo TAF7 coelongates with P-TEFb and Pol II downstream of the promoter. We propose a model in which TAF7 contributes to the regulation of the transition from PIC assembly to initiation and elongation.

Asunto(s)

Regulación de la Expresión Génica , Factor B de Elongación Transcripcional Positiva/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factores Asociados con la Proteína de Unión a TATA/fisiología , Factor de Transcripción TFIID/metabolismo , Factor de Transcripción TFIIH/metabolismo , Línea Celular , Humanos , Complejos Multiproteicos , Unión Proteica , Factor de Transcripción TFIID/fisiología , Transcripción Genética , Transfección

RESUMEN

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia. The HTLV-1-encoded protein Tax transactivates the viral long terminal repeat and plays a critical role in virus replication and transformation. Previous work from our laboratory demonstrated that coactivator-associated arginine methytransferase 1, a protein arginine methytransferase, was important for Tax-mediated transactivation. To further investigate the role of methyltransferases in viral transcription, we utilized adenosine-2,3-dialdehyde (AdOx), an adenosine analog and S-adenosylmethionine-dependent methyltransferase inhibitor. The addition of AdOx decreased Tax transactivation in C81, Hut102, and MT-2 cells. Unexpectedly, we found that AdOx potently inhibited the growth of HTLV-1-transformed cells. Further investigation revealed that AdOx inhibited the Tax-activated NF-kappaB pathway, resulting in reactivation of p53 and induction of p53 target genes. Analysis of the NF-kappaB pathway demonstrated that AdOx treatment resulted in degradation of the IkappaB kinase complex and inhibition of NF-kappaB through stabilization of the NF-kappaB inhibitor IkappaBalpha. Our data further demonstrated that AdOx induced G(2)/M cell cycle arrest and cell death in HTLV-1-transformed but not control lymphocytes. These studies demonstrate that protein methylation plays an important role in NF-kappaB activation and survival of HTLV-1-transformed cells.

Asunto(s)

Apoptosis , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Metiltransferasas/antagonistas & inhibidores , Replicación Viral/fisiología , Línea Celular , Productos del Gen tax/metabolismo , Humanos , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B , Metiltransferasas/fisiología , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis

RESUMEN

The phosphatidylinositol-3-kinase (PI3K) and AKT (protein kinase B) signaling pathways play an important role in regulating cell cycle progression and cell survival. In previous studies, we demonstrated that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to p53 inhibition and cell survival. In the present study, we extend these observations to identify regulatory pathways affected by AKT in HTLV-1-transformed cells. We demonstrate that inhibition of AKT reduces the level of phosphorylated Bad, an important member of the pro-apoptotic family of proteins. Consistent with the decrease of phosphorylated Bad, cytochrome c is released from the mitochondria and caspase-9 is activated. Pretreatment of the cells with caspase-9 specific inhibitor z-LEHD-FMK or pan caspase inhibitor Ac-DEVD-CHO prevented LY294002-induced apoptosis. Of interest, p53 siRNA prevents LY294002-induced apoptosis in HTLV-1-transformed cells, suggesting that p53 reactivation is linked to apoptosis. In conclusion, the AKT pathway is involved in targeting multiple proteins which regulate caspase- and p53-dependent apoptosis in HTLV-1-transformed cells. Since AKT inhibitors simultaneously inhibit NF-kappaB and activate p53, these drugs should be promising candidates for HTLV-1-associated cancer therapy.

Asunto(s)

Apoptosis/fisiología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Caspasa 9/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Línea Celular Transformada , Cromonas/farmacología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/farmacología , Citocromos c/metabolismo , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Modelos Biológicos , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal , Tiofenos/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Proteína Letal Asociada a bcl/metabolismo

RESUMEN

Positive transcription elongation factor (P-TEFb), which is composed of CDK9 and cyclin T1, plays an important role in cellular and viral gene expression. Our lab has recently demonstrated that P-TEFb is required for Tax transactivation of the viral long terminal repeat (LTR). P-TEFb is found in two major complexes: the inactive form, which is associated with inhibitory subunits 7SK snRNA and HEXIM1, and the active form, which is associated with, at least in part, Brd4. In this study, we analyzed the effect of Brd4 on human T-lymphotropic virus type 1 (HTLV-1) transcription. Overexpression of Brd4 repressed Tax transactivation of the HTLV-1 LTR in a dose-dependent manner. In vitro binding studies suggest that Tax and Brd4 compete for binding to P-TEFb through direct interaction with cyclin T1. Tax interacts with cyclin T1 amino acids 426 to 533, which overlaps the region responsible for Brd4 binding. In vivo, overexpression of Tax decreased the amount of 7SK snRNA associated with P-TEFb and stimulates serine 2 phosphorylation of the RNA polymerase II carboxyl-terminal domain, suggesting that Tax regulates the functionality of P-TEFb. Our results suggest the possibility that Tax may compete and functionally substitute for Brd4 in P-TEFb regulation.

Asunto(s)

Regulación Viral de la Expresión Génica , Productos del Gen tax/fisiología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Proteínas Nucleares/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Unión Competitiva , Proteínas de Ciclo Celular , Productos del Gen tax/metabolismo , Humanos , Unión Proteica , Secuencias Repetidas Terminales/genética , Transcripción Genética

RESUMEN

Exenatide (Ex-4) is an antidiabetic drug that acts through the glucagon-like peptide 1 receptor and has recently been approved for the treatment of type 2 diabetes mellitus. Ex-4 also has been shown to affect beta cell gene expression and increase beta cell mass in rodent models of type 1 diabetes mellitus, but the mechanisms are not fully understood. We therefore analyzed the pathways affected by Ex-4 in human islets by using oligonucleotide microarrays and the PathwayStudio software (Ariadne Genomics, Rockville, MD). We identified the JAK1-STAT1 pathway as a novel target of Ex-4 and confirmed the Ex-4-mediated down-regulation of JAK1 and STAT1 by quantitative reverse transcription-polymerase chain reaction in human islets and INS-1 cells. JAK1-STAT1 is the major signaling pathway mediating the interferon gamma effects on beta cell apoptosis in type 1 diabetes mellitus. Thus, these findings suggest that Ex-4 treatment may also be beneficial in type 1 diabetes mellitus, where it may help protect beta cells from cytokine-induced cell death by inhibiting JAK1-STAT1.

Asunto(s)

Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Janus Quinasa 1/antagonistas & inhibidores , Péptidos/farmacología , Factor de Transcripción STAT1/antagonistas & inhibidores , Ponzoñas/farmacología , Células Cultivadas , Exenatida , Perfilación de la Expresión Génica , Humanos , Células Secretoras de Insulina/metabolismo , Interferón gamma/farmacología , Janus Quinasa 1/genética , Factor de Transcripción STAT1/genética , Transducción de Señal/efectos de los fármacos

RESUMEN

In this study, we demonstrate that the coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3 and other proteins such as p300/CBP, is positively involved in the regulation of Tax transactivation. First, transfection studies demonstrated that overexpression of CARM1 wild-type protein resulted in increased Tax transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR). In contrast, transfection of a catalytically inactive CARM1 methyltransferase mutant did not enhance Tax transactivation. CARM1 facilitated Tax transactivation of the CREB-dependent cellular GEM promoter. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro glutathione S-transferase-Tax binding assays, in vivo coimmunoprecipitation, and confocal microscopy experiments. Finally, chromatin immunoprecipitation analysis of the activated HTLV-1 LTR promoter showed the association of CARM1 and methylated histone H3 with the template DNA. In vitro, Tax facilitates the binding of CARM1 to the transcription complex. Together, our data provide evidence that CARM1 enhances Tax transactivation of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of histone H3 (R2, R17, and R26).

Asunto(s)

ADN Viral/metabolismo , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Proteína-Arginina N-Metiltransferasas/metabolismo , Secuencias Repetidas Terminales , Transcripción Genética , Línea Celular , Genes Reporteros , Histonas/metabolismo , Humanos , Inmunoprecipitación , Luciferasas/análisis , Luciferasas/genética , Microscopía Confocal , Unión Proteica

RESUMEN

Human T-lymphotropic virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose function is essential for viral transcription and replication. Tax transactivates the viral long-terminal repeat through a series of protein-protein interactions which facilitate CREB and CBP/p300 binding. In addition, Tax dissociates transcription repressor histone deacetylase 1 interaction with the CREB response element. The subsequent events through which Tax interacts and communicates with RNA polymerase II and cyclin-dependent kinases (CDKs) are not clearly understood. Here we present evidence that Tax recruits positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T1) to the viral promoter. This recruitment likely involves protein-protein interactions since Tax associates with P-TEFb in vitro as demonstrated by glutathione S-transferase fusion protein pull-down assays and in vivo as shown by co-immunoprecipitation assays. Functionally, small interfering RNA directed toward CDK9 inhibited Tax transactivation in transient assays. Consistent with these findings, the depletion of CDK9 from nuclear extracts inhibited Tax transactivation in vitro. Reconstitution of the reaction with wild-type P-TEFb, but not a kinase-dead mutant, recovered HTLV-1 transcription. Moreover, the addition of the CDK9 inhibitor flavopiridol blocked Tax transactivation in vitro and in vivo. Interestingly, we found that Tax regulates CDK9 kinase activity through a novel autophosphorylation pathway.

Asunto(s)

Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Activación Transcripcional/fisiología , Línea Celular , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/fisiología , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Fosforilación , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/fisiología , ARN Viral/biosíntesis , Treonina/metabolismo

RESUMEN

Drug-induced liver disease (DILD) continues to cause significant morbidity and mortality and impair new drug development. Mounting evidence suggests that DILD is a complex, multifactorial disease in which no one factor is likely to be an absolute indicator of susceptibility. As an approach to better understand the multifactorial basis of DILD, we recently compared the hepatic proteomes of mice that were resistant (SJL) and susceptible (C57Bl/6) to APAP-induced liver disease (AILD) wherein we identified potential risk factors and mechanistic pathways responsible for DILD. In this study, we have uncovered additional potential risk factors by comparing hepatic mRNA expression profiles of the same two strains of mice with that of SJLxB6-F1 hybrid (F1) mice, which were found to be of intermediate susceptibility to AILD. Global hepatic gene expression profiling over a 24 h period following APAP treatment revealed elevated patterns in the mRNA expression of cytoprotective genes in resistant SJL mice as compared to susceptible B6 mice, while F1 mice had intermediate mRNA expression levels of these genes. One of these genes encoded for heat shock protein (HSP) 70 whose relative protein expression among the three strains of mice was found to parallel that of their mRNA levels, suggesting that this protein had a protective role against AILD. However, there was no difference in the susceptibility of HSP70 knockout (KO) mice to AILD as compared to wild-type (WT) mice. There were also protoxicant genes, such as osteopontin (OPN), with elevated mRNA expression levels in the B6 mice as compared to the SJL mice and with intermediate levels in the F1 mice, suggesting that they may play a role in exacerbating liver injury after APAP treatment. In support of this hypothesis, OPN KO mice were found to be more resistant to AILD than WT mice. Additionally, the results from both the proteomic and the genomic studies were compared. The two approaches were found to be complementary to each other and not simply overlapping. Our findings suggest that comparative gene expression analysis of susceptible and resistant mouse strains may lead to the identification of factors that could have a role in determining the susceptibility of individuals to DILD.

Asunto(s)

Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas , Predisposición Genética a la Enfermedad , Hepatopatías/genética , Acetaminofén/química , Animales , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/deficiencia , Inyecciones Intraperitoneales , Hígado/metabolismo , Hígado/patología , Hepatopatías/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Osteopontina , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factores de Riesgo , Sialoglicoproteínas/biosíntesis , Sialoglicoproteínas/genética , Factores de Tiempo

RESUMEN

The human hair follicle bulge is an important niche for keratinocyte stem cells (KSCs). Elucidation of human bulge cell biology could be facilitated by analysis of global gene expression profiles and identification of unique cell-surface markers. The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells and KSCs. In this study, we determined the distribution of label-retaining cells to define the human anagen bulge. Using navigated laser capture microdissection, bulge cells and outer root sheath cells from other follicle regions were obtained and analyzed with cDNA microarrays. Gene transcripts encoding inhibitors of WNT and activin/bone morphogenic protein signaling were overrepresented in the bulge, while genes responsible for cell proliferation were underrepresented, consistent with the existence of quiescent noncycling KSCs in anagen follicles. Positive markers for bulge cells included CD200, PHLDA1, follistatin, and frizzled homolog 1, while CD24, CD34, CD71, and CD146 were preferentially expressed by non-bulge keratinocytes. Importantly, CD200+ cells (CD200hiCD24loCD34loCD71loCD146lo) obtained from hair follicle suspensions demonstrated high colony-forming efficiency in clonogenic assays, indicating successful enrichment of living human bulge stem cells. The stem cell behavior of enriched bulge cells and their utility for gene therapy and hair regeneration will need to be assessed in in vivo assays.

Asunto(s)

Folículo Piloso/citología , Folículo Piloso/fisiología , Células Madre/citología , Células Madre/fisiología , Antígenos CD/análisis , División Celular , Ensayo de Unidades Formadoras de Colonias , Folículo Piloso/inmunología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Cuero Cabelludo , Células Madre/inmunología