RESUMEN
The persistence and spreading of HTLV-I infected cells relies upon their clonal expansion through cellular replication. The development of adult T cell leukemia (ATLL) occurs decades following primary infection by HTLV-I. Moreover, identical provirus integration sites have been found in samples recovered several years apart from infected individuals. These observations suggest that infected cells persist in the host for an extended period of time. To endure long term proliferation, HTLV-I pre-leukemic cells must acquire critical oncogenic events, two of which are the bypassing of apoptosis and replicative senescence. In the early stages of disease, interleukin-2 (IL-2)/IL-2R signaling likely plays a major role in combination with activation of anti-apoptotic pathways. Avoidance of replicative senescence in HTLV-I infected cells is achieved through reactivation of human telomerase (hTERT). We have previously shown that HTLV-I viral Tax transcriptionally activates the hTERT promoter. In this study we demonstrate that Tax can stimulate hTERT enzymatic activity independently of its transcriptional effects. We further show that this occurs through Tax-mediated NF-KB activating functions. Our results suggest that in ATLL cells acquire Tax-transcriptional and post-transcriptional events to elevate telomerase activity.
RESUMEN
The rate of failure or recurrence after ulnar nerve release at the elbow is up to 25%. Various biomaterials have been developed to protect nerves from postoperative adhesions. The aim of this study was to review a case series of 40 surgical revision procedures of the ulnar nerve at the elbow, protected by a collagen membrane (Cova™ ORTHO). Forty patients who had this revision surgery between January 2013 and December 2017 were reviewed: 34 were evaluated in person, 6 were evaluated over the phone. The operation consisted in release of the ulnar nerve, anterior subcutaneous transposition and nerve protection using a collagen membrane. We assessed the following parameters with an average follow-up of 4 years and 3 months: paresthesia, night awakening, quality of life (QuickDASH score) and neuropathic pain (DN4 questionnaire). The outcome was determined with the Gabel & Amadio score. The patients' satisfaction was evaluated. A significant decrease in paresthesia and night awakening was found (p < 0.05). The average Gabel & Amadio score improved from 4.4 to 6.7 with 5 excellent, 19 good, 9 fair, and 1 poor result. The average DN4 was 5/10 and the QuickDASH score was 40.1. Eighty percent of patients were satisfied or very satisfied with the outcome. Surgical revision of the ulnar nerve at the elbow remains a delicate operation without a gold standard. This case series found good or excellent results in 70% of patients. Surgical revision of the ulnar nerve with a collagen membrane is a reliable alternative among other possibilities for ulnar nerve release at the elbow.
Asunto(s)
Articulación del Codo , Síndromes de Compresión del Nervio Cubital , Colágeno , Codo , Humanos , Calidad de VidaRESUMEN
Our hypothesis was that immediate repetition of a microsurgery-suturing task will improve its execution and outcome. This was an experimental animal study. Ten surgeons were divided into two groups of five surgeons. Each performed two end-to-end carotid anastomoses on the same rat, one after the other. The anastomosis was evaluated by the surgeon and an instructor. The primary endpoint was permeability. The outcome was evaluated using an objective and subjective assessment grid yielding 1 to 3 points per item. The total scores for each of the 10 surgeons were used to compare the anastomosis of carotid 1 versus 2, using the ratings given by the surgeon and the instructor. Twenty anastomoses were performed, but 1 rat died intraoperatively, leaving 18 anastomoses for evaluation. No significant differences were found on the main endpoint of permeability, with all anastomoses being permeable. The surgeon's self-assessment was significantly better for the second carotid artery (P=0.05), but this was not confirmed by the proxy assessment (instructor). The analysis by subgroups-morning versus afternoon-found the second carotid anastomosis was significant better in the self-assessment and proxy assessment for the morning group (P<0.001, P=0.024). There was no significant difference in clamping times. The immediate repetition of a microsurgical procedure seems to favor its execution, which leads us to propose that the more difficult or important anastomosis should be done after an easier or less important one during complex surgeries. LEVEL OF EVIDENCE: 2B.
Asunto(s)
Anastomosis Quirúrgica/educación , Competencia Clínica , Educación Médica Continua/métodos , Microcirugia/educación , Cirujanos , Suturas , Animales , Arterias Carótidas/cirugía , Humanos , Ratas Sprague-Dawley , Grado de Desobstrucción VascularRESUMEN
BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia/lymphoma (ATLL), a lymphoproliferative malignancy with a dismal prognosis and limited therapeutic options. Recent evidence shows that HTLV-1-transformed cells present defects in both DNA replication and DNA repair, suggesting that these cells might be particularly sensitive to treatment with a small helicase inhibitor. Because the "Werner syndrome ATP-dependent helicase" encoded by the WRN gene plays important roles in both cellular proliferation and DNA repair, we hypothesized that inhibition of WRN activity could be used as a new strategy to target ATLL cells. METHODS: Our analysis demonstrates an apoptotic effect induced by the WRN helicase inhibitor in HTLV-1-transformed cells in vitro and ATL-derived cell lines. Inhibition of cellular proliferation and induction of apoptosis were demonstrated with cell cycle analysis, XTT proliferation assay, clonogenic assay, annexin V staining, and measurement of mitochondrial transmembrane potential. RESULTS: Targeted inhibition of the WRN helicase induced cell cycle arrest and apoptosis in HTLV-1-transformed leukemia cells. Treatment with NSC 19630 (WRN inhibitor) induces S-phase cell cycle arrest, disruption of the mitochondrial membrane potential, and decreased expression of anti-apoptotic factor Bcl-2. These events were associated with activation of caspase-3-dependent apoptosis in ATL cells. We identified some ATL cells, ATL-55T and LMY1, less sensitive to NSC 19630 but sensitive to another WRN inhibitor, NSC 617145. CONCLUSIONS: WRN is essential for survival of ATL cells. Our studies suggest that targeting the WRN helicase with small inhibitors is a novel promising strategy to target HTLV-1-transformed ATL cells.
Asunto(s)
Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Helicasa del Síndrome de Werner/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Leucemia-Linfoma de Células T del Adulto/patología , Maleimidas/farmacología , Células Tumorales Cultivadas , Helicasa del Síndrome de Werner/fisiologíaRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus associated with the lymphoproliferative disease adult T-cell leukemia/lymphoma (ATL) and the neurodegenerative disorder tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). Replication of HTLV-1 is under the control of two major trans-acting proteins, Tax and Rex. Previous studies suggested that Tax activates transcription from the viral long terminal repeat (LTR) through recruitment of cellular CREB and transcriptional coactivators. Other studies reported that Rex acts posttranscriptionally and allows the cytoplasmic export of unspliced or incompletely spliced viral mRNAs carrying gag/pol and env only. As opposed to HIV's Rev-responsive element (RRE), the Rex-responsive element (RxRE) is present in all viral mRNAs in HTLV-1. However, based on indirect observations, it is believed that nuclear export and expression of the doubly spliced tax/rex RNA are Rex independent. In this study, we demonstrate that Rex does stimulate Tax expression, through nuclear-cytoplasmic export of the tax/rex RNA, even though a Rex-independent basal export mechanism exists. This effect was dependent upon the RxRE element and the RNA-binding activity of Rex. In addition, Rex-mediated export of tax/rex RNA was CRM1 dependent and inhibited by leptomycin B treatment. RNA immunoprecipitation (RNA-IP) experiments confirmed Rex binding to the tax/rex RNA in both transfected cells with HTLV-1 molecular clones and HTLV-1-infected T cells. Since both Rex and p30 interact with the tax/rex RNA and with one another, this may offer a temporal and dynamic regulation of HTLV-1 replication. Our results shed light on HTLV-1 replication and reveal a more complex regulatory network than previously anticipated.
Asunto(s)
Productos del Gen rex/genética , Productos del Gen tax/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Nucléolo Celular/metabolismo , Regulación Viral de la Expresión Génica , Orden Génico , Productos del Gen rex/metabolismo , Productos del Gen tax/metabolismo , Humanos , Datos de Secuencia MolecularRESUMEN
Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma, and it encodes a number of nonstructural proteins that are involved in virus replication and immune evasion. The viral protein p12 previously has been characterized to interfere with major histocompatibility complex class, ICAM-1, and ICAM-2 expression, and it activates STAT5. Using a previously established T-cell line immortalized with an HTLV-1 molecular clone deleted for p12, we assessed the role of p12 in regulating cellular growth and virus transmission. These cells were complemented for p12 expression by the transduction of a lentivirus vector expressing p12. We report that p12 conferred a selective growth advantage in vitro and increased the colony formation of human T cells in soft-agar assays. Consistently with previous studies, p12- and p12+ cell lines produced similar amounts of virus particles released into the supernatant of cultured cells, although we found that p12 expression greatly enhanced virus transmission. Moreover, we found that interleukin-2 (IL-2) stimulation also increased HTLV-1 transmission whether p12 was expressed or not, and inversely, that the inhibition of Jak signaling significantly reduced HTLV-1 transmission. Intriguingly, IL-2/Jak signaling was not associated with changes in viral gene expression, viral RNA encapsidation, the maturation of the virus particle, cell-cell adherence, or Gag polarization and virological synapse formation. We do demonstrate, however, that IL-2 stimulation and p12 expression significantly increased the rate of syncytium formation, revealing a novel role for IL-2 signaling and Jak activation in HTLV-1 virus transmission.
Asunto(s)
Virus Linfotrópico T Tipo 1 Humano/fisiología , Quinasas Janus/fisiología , Receptores de Interleucina-2/fisiología , Proteínas Reguladoras y Accesorias Virales/fisiología , División Celular/fisiología , Línea Celular Tumoral , Transformación Celular Viral/fisiología , Humanos , Interleucina-2/fisiología , Proteínas Oncogénicas de Retroviridae/fisiología , Factores de Transcripción STAT/fisiología , Transducción de Señal/fisiología , Replicación Viral/fisiologíaRESUMEN
Persistent inhibition of telomerase induces a severe telomere shortening in human T-cell leukemia virus type-1-infected cells which signals a DNA double-strand break damage response, formation of telomere dysfunction-induced foci and activates the ATM pathway. In turn, activation of ATM and its downstream effectors led to an increased phosphorylation and acetylation on specific residues of p53 known to be involved in transcriptional activation. Disruption of Mdm2-p53 complexes coupled with increased proteasomal degradation of MDMX further enhanced reactivation of p53 transcription, ultimately leading to senescence of tumor cells. Induction of senescence in these T-cells was associated with an increased expression of p21, p16 and activation of GSK3beta. Our results support the cancer-aging model and demonstrate that the halt of aging in cancer cells can be reversed through reactivation of p53.
Asunto(s)
Roturas del ADN de Doble Cadena , Virus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T del Adulto/virología , Transducción de Señal/genética , Telómero/patología , Proteína p53 Supresora de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/fisiología , Línea Celular Transformada , Línea Celular Tumoral , Senescencia Celular/genética , Daño del ADN/genética , Proteínas de Unión al ADN/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Células Jurkat , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteína p53 Supresora de Tumor/biosíntesis , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Treatment of patients with adult T-cell leukemia-lymphoma (ATLL) using conventional chemotherapy has limited benefit because human T-cell leukemia virus type 1 (HTLV-1) cells are resistant to most apoptosis-inducing agents. The recent report that arsenic trioxide induces apoptosis in HTLV-1-transformed cells prompted investigation of the mechanism of action of this drug in HTLV-1 and HTLV-2 interleukin-2-independent T cells and in HTLV-1-immortalized cells or in ex vivo ATLL samples. Fluorescence-activated cell sorter analysis, fluorescence microscopy, and measures of mitochondrial membrane potential (Delta Psi m) demonstrated that arsenic trioxide alone was sufficient to induce programmed cell death in all HTLV-1 and -2 cells tested and in ATLL patient samples. I kappa B-alpha phosphorylation strongly decreased, and NF-kappa B translocation to the nucleus was abrogated. Expression of the antiapoptotic protein Bcl-X(L), whose promoter is NF-kappa B dependent, was down-regulated. The collapse of Delta Psi m and the release of cytochrome c to the cytosol resulted in the activation of caspase-3, as demonstrated by the cleavage of PARP. A specific caspase-3 inhibitor (Ac-DEVD-CHO) could reverse this phenotype. The antiapoptotic factor Bcl-2 was then cleaved, converting it to a Bax-like death effector. These results demonstrated that arsenic trioxide induces apoptosis in HTLV-1- and -2-infected cells through activation of the caspase pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Caspasas/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Virus Linfotrópico T Tipo 2 Humano/fisiología , Proteínas I-kappa B , Leucemia-Linfoma de Células T del Adulto/patología , Óxidos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Trióxido de Arsénico , Caspasa 3 , Línea Celular Transformada , Núcleo Celular/metabolismo , Grupo Citocromo c/metabolismo , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Humanos , Interferón-alfa/farmacología , Leucemia-Linfoma de Células T del Adulto/virología , Potenciales de la Membrana , Microscopía Fluorescente , Mitocondrias/ultraestructura , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteína p53 Supresora de Tumor/análisis , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
The proto-oncogene c-myb is essential for a controlled balance between cell growth and differentiation. Aberrant c-Myb activity has been reported for numerous human cancers, and enforced c-Myb transcription can transform cells of lymphoid origin by stimulating cellular proliferation and inhibiting apoptotic pathways. Here we demonstrate that activation of the NF-kappaB pathway by the HTLV-1 Tax protein leads to transcriptional inactivation of c-Myb. This conclusion was supported by the fact that Tax mutants unable to stimulate the NF-kappaB pathway could not inhibit c-Myb transactivating functions. In addition, inhibition of Tax-mediated NF-kappaB activation by coexpression of IkappaBalpha restored c-Myb transcription, and Tax was unable to block c-Myb transcription in a NEMO knockout cell line. Importantly, physiological stimuli, such as signaling with the cellular cytokines tumor necrosis factor alpha, interleukin 1 beta (IL-1beta), and lipopolysaccharide, also inhibited c-Myb transcription. These results uncover a new link between extracellular signaling and c-Myb-dependent transcription. The mechanism underlying NF-kappaB-mediated repression was identified as sequestration of the coactivators CBP/p300 by RelA. Interestingly, an amino-terminal deletion form of p300 lacking the C/H1 and KIX domains and unable to bind RelA retained the ability to stimulate c-Myb transcription and prevented NF-kappaB-mediated repression.
Asunto(s)
Productos del Gen tax/metabolismo , Proteínas I-kappa B , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Transcripción Genética , Animales , Diferenciación Celular , División Celular , Línea Celular , Proteínas de Unión al ADN/metabolismo , Proteína p300 Asociada a E1A , Activación Enzimática , Eliminación de Gen , Immunoblotting , Interleucina-1/metabolismo , Ligasas/metabolismo , Luciferasas/metabolismo , Ratones , Ratones Noqueados , Microscopía Fluorescente , Mutación , Inhibidor NF-kappaB alfa , Proteínas Nucleares/metabolismo , Fenotipo , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proto-Oncogenes Mas , Conejos , Reticulocitos/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
The p12(I) protein, encoded by the pX open reading frame I of the human T-lymphotropic virus type 1 (HTLV-1), is a hydrophobic protein that localizes to the endoplasmic reticulum and the Golgi. Although p12(I) contains 4 minimal proline-rich, src homology 3-binding motifs (PXXP), a characteristic commonly found in proteins involved in signaling pathways, it has not been known whether p12(I) has a role in modulating intracellular signaling pathways. This study demonstrated that p12(I) binds to the cytoplasmic domain of the interleukin-2 receptor (IL-2R) beta chain that is involved in the recruitment of the Jak1 and Jak3 kinases. As a result of this interaction, p12(I) increases signal transducers and activators of transcription 5 (STAT5) DNA binding and transcriptional activity and this effect depends on the presence of both IL-2R beta and gamma(c) chains and Jak3. Transduction of primary human peripheral blood mononuclear cells (PBMCs) with a human immunodeficiency virus type 1-based retroviral vector expressing p12(I) also resulted in increased STAT5 phosphorylation and DNA binding. However, p12(I) could increase proliferation of human PBMCs only after stimulation of T-cell receptors by treatment of cells with low concentrations of alphaCD3 and alphaCD28 antibodies. In addition, the proliferative advantage of p12(I)-transduced PBMCs was evident mainly at low concentrations of IL-2. Together, these data indicate that p12(I) may confer a proliferative advantage on HTLV-1-infected cells in the presence of suboptimal antigen stimulation and that this event may account for the clonal proliferation of infected T cells in vivo. (Blood. 2001;98:823-829)
Asunto(s)
Proteínas de Unión al ADN/efectos de los fármacos , Interleucina-2/farmacología , Proteínas de la Leche , Proteínas Oncogénicas Virales/farmacología , Linfocitos T/virología , Transactivadores/efectos de los fármacos , Factores de Transcripción , Técnicas de Cultivo de Célula , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Sinergismo Farmacológico , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/patología , Humanos , Unión Proteica , Receptores de Interleucina-2/metabolismo , Factor de Transcripción STAT5 , Transducción de Señal/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Transactivadores/genética , Transactivadores/metabolismo , Activación Transcripcional/efectos de los fármacos , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Human T-cell leukemia virus type 1 (HTLV-1) establishes a persistent infection in the host despite a vigorous virus-specific immune response. Here we demonstrate that an HTLV-1-encoded protein, p12(I), resides in the endoplasmic reticulum (ER) and Golgi and physically binds to the free human major histocompatibility complex class I heavy chains (MHC-I-Hc) encoded by the HLA-A2, -B7, and -Cw4 alleles. As a result of this interaction, the newly synthesized MHC-I-Hc fails to associate with beta(2)-microglobulin and is retrotranslocated to the cytosol, where it is degraded by the proteasome complex. Targeting of the free MHC-I-Hc, and not the MHC-I-Hc-beta(2)-microglobulin complex, by p12(I) represents a novel mechanism of viral interference and disrupts the intracellular trafficking of MHC-I, which results in a significant decrease in surface levels of MHC-I on human T-cells. These findings suggest that the interaction of p12(I) with MHC-1-Hc may interfere with antigen presentation in vivo and facilitate escape of HTLV-1-infected cells from immune recognition.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas Oncogénicas Virales/fisiología , Factores de Transcripción , Transporte Biológico , Cisteína Endopeptidasas/fisiología , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Antígeno HLA-A2/metabolismo , Antígeno HLA-B7/metabolismo , Antígenos HLA-C/metabolismo , Células HeLa , Humanos , Complejos Multienzimáticos/fisiología , Complejo de la Endopetidasa Proteasomal , Proteínas Reguladoras y Accesorias Virales , Microglobulina beta-2/metabolismoRESUMEN
The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of fatty acids. The genes for the two isoforms of CPTI-liver (L-CPTI) and muscle (M-CPTI) have been cloned and expressed, and the genes encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Pig L-CPTI encodes for a 772 amino acid protein that shares 86 and 62% identity, respectively, with rat L- and M-CPTI. When expressed in Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics (carnitine, K(m) = 126 microM; palmitoyl-CoA, K(m) = 35 microM) similar to human or rat L-CPTI. However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition (IC(50) = 141 nM) that is characteristic of human or rat M-CPTI enzymes. Therefore, pig L-CPTI behaves like a natural chimera of the L- and M-CPTI isotypes, which makes it a useful model to study the structure--function relationships of the CPTI enzymes.
Asunto(s)
Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Inhibidores Enzimáticos/metabolismo , Malonil Coenzima A/metabolismo , Mitocondrias Hepáticas/enzimología , Mitocondrias Musculares/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carnitina O-Palmitoiltransferasa/biosíntesis , Carnitina O-Palmitoiltransferasa/genética , Clonación Molecular , ADN Complementario/aislamiento & purificación , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Pichia/genética , Ratas , Alineación de Secuencia , PorcinosRESUMEN
The B-myb gene was identified on the basis of its homology with the protooncogene c-myb, homolog of the avian myeloblastosis virus (AMV) and avian leukemia virus (E26) transforming genes. Several studies using antisense constructs or antisense oligonucleotides as well as overexpression experiments suggest that B-Myb plays an important role in the transition from G(1) to S phase of the cell cycle and that B-Myb expression is cell cycle regulated. We have previously demonstrated that the human T cell lymphotropic virus type 1 (HTLV1) trans-activator Tax is able to repress transcription from c-myb promoter reporter constructs as well as from the endogenous c-myb promoter in human T cells and that this effect is mediated through inhibition of the c-Myb trans-activating functions. Here we report that both HTLV-1 as well as HTLV-2 Tax proteins inhibit c-Myb trans-activation in mouse embryo fibroblasts (MEFs). In addition to c-Myb, B-Myb expression is also markedly downregulated in HTLV-1-transformed cells at both RNA and protein levels. Furthermore, by using a Jurkat T cell line stably transfected with a tax gene driven by a cadmium-inducible promoter (JPX9), we were able to demonstrate that Tax directly represses the endogenous B-myb promoter in T cells. Because c-Myb and B-Myb have been involved in cell cycle progression, our results suggest that Tax, by repressing both c-Myb and B-Myb endogenous promoters, may bypass their requirement for cell cycle progression in HTLV-1-transformed T cells.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Productos del Gen tax/farmacología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Virus Linfotrópico T Tipo 2 Humano/fisiología , Linfocitos T/virología , Transactivadores/genética , Activación Transcripcional , Animales , Línea Celular Transformada , Transformación Celular Viral , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Fibroblastos , Humanos , Células Jurkat , Ratones , Regiones Promotoras Genéticas , Transactivadores/metabolismoRESUMEN
Human T cell lymphotropic virus type II (HTLV-2) was originally isolated from a patient with a hairy T cell leukemia. It has been associated with rare cases of CD8(+) T lymphoproliferative disorders, and has a controversial role as a pathogen. The loss of p53 function, as a consequence of mutation or inactivation, increases the chances of genetic damage. Indeed, the importance of p53 as a tumor suppressor is evident from the fact that over 60% of all human cancers have a mutant or inactive p53. p53 status has been extensively studied in HTLV-1-infected cell lines. Interestingly, despite the fact that p53 mutations have been found in only a minority of cells, the p53 functions were found to be impaired. We have analyzed the functional activity of the p53 tumor suppressor in cells transformed with HTLV-2 subtypes A and B. As with HTLV-1-infected cells, abundant levels of the p53 protein are detected in HTLV-2 virus-infected cell lines. Using p53 reporter plasmid or induction of p53-responsive genes in response to gamma-irradiation, the p53 was found to be transcriptionally inhibited in HTLV-2-infected cells. Interestingly, although Tax-2A and-2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B in T cells, but not in fibroblasts.
Asunto(s)
Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Virus Linfotrópico T Tipo 2 Humano/fisiología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Transformada , Infecciones por HTLV-I/virología , Infecciones por HTLV-II/virología , Humanos , Ratones , Linfocitos T/virología , Transcripción Genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Human T cell leukemia/lymphotropic virus types 1 and 2 are two immunologically and phylogenetically related retroviruses that differ in their pathogenicity in vivo. The overall genetic structure of HTLV-1 and -2 is similar. Each contains a unique region at the 3' end of the genome, designated the pX region. p12(I) is a membrane-associated protein encoded by the open reading frame I (ORF I) region of HTLV-1, which lies within the pX region. A corresponding protein, p10(I) is encoded by the ORF I region of HTLV-2 and an additional protein, p11(V), is encoded by ORF V, which overlaps the HTLV-2 ORF I region. As with HTLV-1, the small proteins encoded by the pX region of HTLV-2 appear to be dispensable for viral replication and cellular transformation in vitro. However, the small open reading frames of both viruses are important for viral replication in vivo, which suggests they may play an important role during the viral life cycle. This study was undertaken to investigate and compare the cellular targets of the p10(I), p11(V), and p12(I) putative proteins.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Virus Linfotrópico T Tipo 2 Humano/metabolismo , Proteínas de los Retroviridae/genética , Proteínas de los Retroviridae/metabolismo , Regiones no Traducidas 3'/genética , Línea Celular Transformada , Infecciones por HTLV-I/virología , Infecciones por HTLV-II/virología , Células HeLa , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Virus Linfotrópico T Tipo 2 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/patogenicidad , Humanos , ATPasas de Translocación de Protón/metabolismo , Receptores de Interleucina-2/metabolismo , Linfocitos T/virologíaRESUMEN
The dysregulation of cellular apoptosis pathways has emerged as a critical early event associated with the development of many types of human cancers. Numerous viral and cellular oncogenes, aside from their inherent transforming properties, are known to induce programmed cell death, consistent with the hypothesis that genetic defects are required to support tumor survival. Here, we report that nuclear expression of the CREB-binding protein (CBP)/p300-binding domain of the human T-cell lymphotropic virus type 1 (HTLV-1) transactivator, Tax, triggers an apoptotic death-inducing signal during short-term clonal analyses, as well as in transient cell death assays. Coexpression of the antiapoptotic factor Bcl-2 increased serum stimulation; incubation with the chemical caspase inhibitor z-Val-Ala-DL-Asp fluoromethylketone antagonized Tax-induced cell death. The CBP/p300-binding defective Tax mutants K88A and V89A exhibited markedly reduced cytotoxic effects compared to the wild-type Tax protein. Importantly, nuclear expression of the minimal CBP/p300-binding peptide of Tax induced apoptosis in the absence of Tax-dependent transcriptional activities, while its K88A counterpart did not cause cell death. Further, Tax-mediated apoptosis was effectively prevented by ectopic expression of the p300 coactivator. We also report that activation of the NF-kappaB transcription pathway by Tax, under growth arrest conditions, results in apoptosis that occurs independent of direct Tax coactivator effects. Our results allude to a novel pivotal role for the transcriptional coactivator p300 in determining cell fate and raise the possibility that dysregulated coactivator usage may pose an early barrier to transformation that must be selectively overcome as a prerequisite for the initiation of neoplasia.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Productos del Gen tax/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Secuencia de Bases , Sitios de Unión , Inhibidores de Caspasas , Núcleo Celular/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Productos del Gen tax/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transactivadores/genéticaRESUMEN
We have analyzed the functional activity of the p53 tumor suppressor in human T-cell lymphotropic virus type 2 (HTLV-2)-transformed cells. Abundant levels of the p53 protein were detected in both HTLV-2A and -2B virus-infected cell lines. The p53 was functionally inactive, however, both in transient-transfection assays using a p53 reporter plasmid and in induction of p53-responsive genes in response to gamma irradiation. We further investigated HTLV-2A Tax and HTLV-2B Tax effects on p53 activity. Interestingly, although Tax-2A and -2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B. In transient-cotransfection assays, Tax-1 and Tax-2B inactivated p53 by 80%, while Tax2A reduced p53 activity by 20%. In addition, Tax-2A does not increase the steady-state level of cellular p53 as well as Tax-1 or -2B does in the same assays. Cotransfection assays demonstrated that Tax-2A could efficiently transactivate CREB-responsive promoters to the same level as Tax-1 and Tax-2B, indicating that the protein was functional. This report provides evidence of the first functional difference between the HTLV-2A and -2B subtypes. This comparison of the action of HTLV-1 and HTLV-2 Tax proteins on p53 function will provide important insights into the mechanism of HTLV transformation.
Asunto(s)
Transformación Celular Viral , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Virus Linfotrópico T Tipo 2 Humano/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Transformada , Rayos gamma , Regulación de la Expresión Génica , Productos del Gen tax/genética , Infecciones por HTLV-II/virología , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/genética , Humanos , Células Jurkat , Fosforilación , Linfocitos T/virología , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/efectos de la radiaciónRESUMEN
Human T lymphotropic virus type I (HTLV-I) is the etiological agent of adult T-cell lymphocytic leukemia (ATLL), whereas HTLV-II has not been associated with hematopoietic malignancies. The control of apoptotic pathways has emerged as a critical step in the development of many cancer types. As a result, the underlying mechanism of long-term survival of HTLV-I and HTLV-II was studied in infected T cells in vitro and in ex vivo ATLL samples. Results indicate that HTLV-I- and HTLV-II-infected T cells in vitro express high levels of the antiapoptotic protein Bcl compared with other human leukemic T cell lines or uninfected peripheral blood mononuclear cells. The levels of proapoptotic proteins Bax, BAD, and Bak were not significantly altered. HTLV-I and HTLV-II viral transactivators, Tax1 and Tax2, are known to increase expression of cellular genes. These proteins were tested for increased transcription from the human Bcl2 and Bcl-X(L) promoters. Whereas no effect was observed on the Bcl2 promoter, both Tax1 and Tax2 increased transcription of the Bcl-X(L) promoter in T cells, although Tax1 appeared to be more efficient than Tax2. The biological significance of these observations was validated by the finding of an increased expression of Bcl-X(L) in ex vivo ATLL cells, especially from patients unresponsive to various chemotherapy regimens. Altogether, these data suggest that overexpression of Bcl-X(L )in vivo( )may be in part responsible for the resistance of ATLL cells to chemotherapy. In addition, inefficient activation of the Bcl-X(L) promoter by Tax2 may result in a shorter survival time of HTLV-II-infected cells in vivo and a diminished risk of leukemia development.
Asunto(s)
Virus Linfotrópico T Tipo 1 Humano/fisiología , Virus Linfotrópico T Tipo 2 Humano/fisiología , Leucemia-Linfoma de Células T del Adulto/sangre , Proteínas Proto-Oncogénicas c-bcl-2/genética , Linfocitos T/fisiología , Linfocitos T/virología , Adulto , Línea Celular Transformada , Productos del Gen tax/genética , Genes bcl-2 , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 2 Humano/genética , Humanos , Células Jurkat , Leucemia-Linfoma de Células T del Adulto/inmunología , Recuento de Leucocitos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-bcl-2/sangre , Proteínas Recombinantes/biosíntesis , Valores de Referencia , Linfocitos T/inmunología , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , Proteína bcl-XRESUMEN
Human T-cell lymphotropic virus type I (HTLV-I) transforms T cells in vitro, and the viral transactivator Tax functionally impairs the tumor suppressor p53 protein, which is also stabilized in HTLV-I-infected T cells. Thus, the functional impairment of p53 is essential to maintain the viral-induced proliferation of CD4+ mature T cells. However, in the CD4+ leukemic cells of patients with adult T-cell leukemia/lymphoma (ATLL), the viral transactivator does not appear to be expressed, and p53 mutations have been found only in a fraction of patients. We sought to investigate whether p53 function is impaired, in ex vivo samples from patients with ATLL, in the absence of genetic mutations. Here we demonstrate that the p53 protein is stabilized also in ex vivo ATLL samples (10 of 10 studied) and that at least in 2 patients p53 stabilization was not associated with genetic mutation. Furthermore, the assessment of p53 function after ionizing radiation of ATLL cells indicated an abnormal induction of the p53-responsive genes GADD45 and p21(WAF1) in 7 of 7 patients. In 2 of 2 patients, p53 regulation of cell-cycle progression appeared to be impaired as well. Because p53 is part of a regulatory loop that also involves MDM2 and p14(ARF), the status of the latter proteins was also assessed in cultured or fresh ATLL cells. The p97 MDM2 protein was not detected by Western blot analysis in established HTLV-I-infected T-cell lines or ex vivo ATLL cell lysates. However, the MDM2 protein could be easily detected after treatment of cells with the specific proteasome inhibitor lactacystin, suggesting a normal regulation of the p53-MDM2 regulating loop. Similarly, p14(ARF) did not appear to be aberrantly expressed in ex vivo ATLL cells nor in any of the established HTLV-I-infected T-cell lines studied. Thus, p53 stabilization in HTLV-I infection occurs in the absence of genetic mutation and alteration of the physiologic degradation pathway of p53. (Blood. 2000;95:3939-3944)
Asunto(s)
Genes p53 , Leucemia-Linfoma de Células T del Adulto/genética , Linfocitos/inmunología , Proteínas Nucleares , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Línea Celular , Células Cultivadas , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Linfocitos/efectos de los fármacos , Proteínas de Neoplasias/genética , Proteínas/análisis , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-mdm2 , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Transfección , Proteína p14ARF Supresora de Tumor , Proteína p53 Supresora de Tumor/análisisRESUMEN
The c-Myb proto-oncogene is preferentially expressed in hematopoietic lineages, and highly expressed in several leukemia types. The Human T-cell Leukemia Virus Type I (HTLV-I) is the etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL). A previous report suggested that Tax, the viral transactivator, is able to suppress the transactivation potential of c-Myb protein by competing for recruitment of CBP. We tested whether such a competition could affect transcription from the c-Myb promoter in Tax expressing T-cells. Using several c-Myb promoter reporter constructs carrying mutations in various regions, we demonstrate that Tax suppression of c-Myb transactivation results in transrepression of the c-Myb promoter through the Myb responsive elements in Jurkat T-cells. The ability of Tax mutants M22, M47 and V89A to interact with the full-length CBP and p300 proteins in vitro, and their ability to repress the c-Myb promoter, was then evaluated. Although both M47 and M22 bind to CBP and p300 to a similar extent, only M47 was able to repress the c-Myb promoter, suggesting that competition for CBP/p300 binding was not the mechanism underlying Tax's effect. This concept was further supported by the fact that the Tax mutant V89A transrepresses the c-Myb promoter efficiently in spite of an impaired binding to CBP and p300. Therefore, Tax-mediated repression of the c-Myb promoter appears to be independent from a direct competition between c-Myb and Tax for recruitment of CBP/p300. Interestingly, a decreased transcription from the endogenous c-Myb promoter was observed in several HTLV-I transformed T-cell lines. Finally, the ability of Tax to directly repress the endogenous c-Myb promoter was demonstrated in a Jurkat cell line stably transfected with a tax gene driven by a cadmium-inducible promoter.