RESUMEN

TNF/iNOS-producing dendritic cells (Tip-DCs) have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8(+) T cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip-DCs that express high levels of TNF-α and iNOS. Tip-DCs exhibited T-cell priming ability, expressed high levels of MHC class II, upregulated co-stimulatory molecules CD40, CD80, CD86, toll-like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip-DCs was partially dependent on IFN-γ as blocking the IFN-γ receptor on monocytes resulted in a significant decrease in CD40 and CD83 expression and in TNF-α production. Importantly, these Tip-DCs were capable of further driving Th1 responses by priming naive CD4(+) T cells for proliferation and IFN-γ production and this was partially dependent on Tip-DC production of TNF-α and NO. Our study hence identifies a role for CD8(+) T cells in orchestrating Th1-mediating signals through the differentiation of monocytes into Th1-inducing Tip-DCs.

Asunto(s)

Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/metabolismo , Interferón gamma/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos Bloqueadores/farmacología , Antígenos CD/biosíntesis , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Receptor 1 de Quimiocinas CX3C , Comunicación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/patología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Interferón gamma/inmunología , Activación de Linfocitos/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/inmunología , Receptores CCR1/biosíntesis , Receptores de Quimiocina/biosíntesis , Células TH1/efectos de los fármacos , Células TH1/inmunología , Receptores Toll-Like/biosíntesis , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

We recently reported that diadenosine tetraphosphate hydrolase (Ap(4)A hydrolase) plays a critical role in gene expression via regulation of intracellular Ap(4)A levels. This enzyme serves as a component of our newly described lysyl tRNA synthetase (LysRS)-Ap(4)A biochemical pathway that is triggered upon immunological challenge. Here we explored the mechanism of this enzyme's translocation into the nucleus and found its immunologically dependent association with importin beta. Silencing of importin beta prevented Ap(4)A hydrolase nuclear translocation and affected the local concentration of Ap(4)A, which led to an increase in microphthalmia transcription factor (MITF) transcriptional activity. Furthermore, immunological activation of mast cells resulted in dephosphorylation of Ap(4)A hydrolase, which changed the hydrolytic activity of the enzyme.

Asunto(s)

Ácido Anhídrido Hidrolasas/metabolismo , Núcleo Celular/metabolismo , Lisina-ARNt Ligasa/metabolismo , Mastocitos/inmunología , beta Carioferinas/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Fosfatos de Dinucleósidos/análisis , Citometría de Flujo , Expresión Génica , Inmunoglobulina E/inmunología , Inmunoprecipitación , Lisina-ARNt Ligasa/genética , Mastocitos/metabolismo , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Reacción en Cadena de la Polimerasa , Procesamiento Proteico-Postraduccional , Interferencia de ARN , ARN Interferente Pequeño , Ratas , beta Carioferinas/genética

RESUMEN

Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap(4)A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap(4)A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap(4)A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap(4)A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap(4)A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.

Asunto(s)

Regulación de la Expresión Génica , Inmunidad Celular/genética , Lisina-ARNt Ligasa/fisiología , Transducción de Señal , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Fosfatos de Dinucleósidos/biosíntesis , Humanos , Lisina-ARNt Ligasa/metabolismo , Sistema de Señalización de MAP Quinasas , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Fosforilación , Ratas , Serina/metabolismo

RESUMEN

TRIP-Br1 and TRIP-Br2 are potent cell growth promoting factors that function as components of the E2F1/DP1 transcription complex to integrate positive growth signals provided by PHD zinc finger- and/or bromodomain-containing transcription factors. TRIP-Br1 has been demonstrated to be an oncogene. We recently reported that antagonism of the TRIP-Br integrator function by synthetic decoy peptides that compete with TRIP-Br for binding to PHD zinc finger- and/or bromodomain-containing proteins elicit an anti-proliferative effect and induces caspase-3-independent sub-diploidization in cancer cells in vitro. We now demonstrate the chemotherapeutic potential of TRIP-Br decoy peptides for the treatment of cutaneous and intracavitary lesions in vitro as well as in vivo in representative human nasopharyngeal cancer (CNE2), cervical cancer (Ca Ski) and melanoma (MeWo) cancer cell lines. In vitro, BrdU incorporation, colony formation assays and cell cycle analysis confirmed that TRIP-Br decoy peptides possess strong anti-proliferative effects and induce nuclear sub-diploidization in cancer cells. In vivo, CNE2, Ca Ski and MeWo-derived chick embryo chorioallantoic membrane (CAM) tumor xenografts were used to evaluate the effect of topically applied TRIP-Br peptides. Confocal microscopy and flow cytometric analysis demonstrated that cells comprising the tumor xenografts efficiently internalized topically applied FITC-labeled peptides. Fifty muM of TRIP-Br1 decoy peptide significantly suppressed the growth of NPC2-derived human nasopharyngeal tumors, while 50 muM of TRIP-Br2 decoy peptide significantly inhibited tumor growth in all three CAM tumor xenograft models. Two hundred muM of TRIP-Br1 decoy peptide significantly inhibited MeWo-derived tumors. These results suggest that the TRIP-Br integrator function may represent a novel chemotherapeutic target for the treatment of human cutaneous and intracavitary proliferative lesions.

Asunto(s)

Proteínas de Homeodominio/farmacología , Proteínas Nucleares/genética , Fragmentos de Péptidos/farmacología , Transactivadores/genética , Factores de Transcripción/farmacología , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular , Proliferación Celular/efectos de los fármacos , Embrión de Pollo , Femenino , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , ARN Interferente Pequeño/farmacología , Proteínas Represoras , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The TRIP-Br family of transcriptional regulators (TRIP-Br1 and TRIP-Br2) has been proposed to function at E2F-responsive promoters to integrate regulatory signals provided by PHD zinc finger- and/or bromodomain-containing transcription factors. To characterize the TRIP-Br "integrator" function(s), we have employed decoy peptides (*Br1 and *Br2) to antagonize the interaction between TRIP-Br1 or TRIP-Br2 and the PHD zinc finger and/or bromodomain of other transcription factors. Antagonism of the TRIP-Br integrator function elicits anti-proliferative effects through the transcriptional downregulation of a subset of E2F-responsive genes in vivo, and induces aberrant cyclin E accumulation, leading to Geminin deregulation and caspase-3-independent cellular sub-diploidization. The observed cyclin E deregulation is attributed to the downregulation of Fbxw7, which encodes the Fbw7 receptor subunit of the SCF(FBW7) ubiquitin ligase (E3) responsible for targeting cyclin E for proteolysis. Fbxw7 is identified herein as an E2F-responsive and TRIP-Br coregulated gene. Our results demonstrate a physiologic role for TRIP-Br in coupling E2F to novel functions in the regulation of cyclin E expression during cell cycle progression to ensure the proper execution of DNA replication and the maintenance of genomic stability.

Asunto(s)

Ciclina E/genética , Factores de Transcripción E2F/metabolismo , Regulación de la Expresión Génica , Inestabilidad Genómica/genética , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Caspasa 3 , Caspasas/metabolismo , Recuento de Células , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/genética , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Citometría de Flujo , Fase G1 , Humanos , Modelos Biológicos , Proteínas Nucleares/antagonistas & inhibidores , Péptidos/farmacología , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Transactivadores/antagonistas & inhibidores , Factores de Transcripción , Transcripción Genética , Ubiquitina-Proteína Ligasas/genética

RESUMEN

The genital human papillomaviruses (HPVs) are a taxonomic group including HPV types that preferentially cause genital and laryngeal warts ("low-risk types"), such as HPV-6 and HPV-11, or cancer of the cervix and its precursor lesions ("high-risk types"), such as HPV-16. The transforming processes induced by these viruses depend on the proteins E5, E6, and E7. Among these oncoproteins, the E6 protein stands out because it supports a particularly large number of functions and interactions with cellular proteins, some of which are specific for the carcinogenic HPVs, while others are shared among low- and high-risk HPVs. Here we report yeast two-hybrid screens with HPV-6 and -11 E6 proteins that identified TRIP-Br1 as a novel cellular target. TRIP-Br1 was recently detected by two research groups, which described two separate functions, namely that of a transcriptional integrator of the E2F1/DP1/RB cell-cycle regulatory pathway (and then named TRIP-Br1), and that of an antagonist of the cyclin-dependent kinase suppression of p16INK4a (and then named p34SEI-1). We observed that TRIP-Br1 interacts with low- and high-risk HPV E6 proteins in yeast, in vitro and in mammalian cell cultures. Transcription activation of a complex consisting of E2F1, DP1, and TRIP-Br1 was efficiently stimulated by both E6 proteins. TRIP-Br1 has an LLG E6 interaction motif, which contributed to the binding of E6 proteins. Apparently, E6 does not promote degradation of TRIP-Br1. Our observations imply that the cell-cycle promoting transcription factor E2F1/DP1 is dually targeted by HPV oncoproteins, namely (i) by interference of the E7 protein with repression by RB, and (ii) by the transcriptional cofactor function of the E6 protein. Our data reveal the natural context of the transcription activator function of E6, which has been predicted without knowledge of the E2F1/DP1/TRIP-Br/E6 complex by studying chimeric constructs, and add a function to the limited number of transforming properties shared by low- and high-risk HPVs.

Asunto(s)

Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Proteínas Represoras , Transactivadores/metabolismo , Secuencias de Aminoácidos , Animales , Células COS , Línea Celular , Humanos , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Transactivadores/química , Transactivadores/genética , Factores de Transcripción , Técnicas del Sistema de Dos Híbridos