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1.
Apoptosis ; 17(8): 762-76, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22484480

RESUMEN

Apoptin, a protein derived from the chicken anaemia virus, induces cell death in various cancer cells but shows little or no cytotoxicity in normal cells. The mechanism of apoptin-induced cell death is currently unknown but it appears to induce apoptosis independent of p53 status. Here we show that p73, a p53 family member, is important in apoptin-induced apoptosis. In p53 deficient and/or mutated cells, apoptin induced the expression of TAp73 leading to the induction of apoptosis. Knockdown of p73 using siRNA resulted in a significant reduction in apoptin-induced cytotoxicity. The p53 and p73 pro-apoptotic target PUMA plays an important role in apoptin-induced cell death as knockdown of PUMA significantly reduced cell sensitivity to apoptin. Importantly, apoptin expression resulted in a marked increase in TAp73 protein stability. Investigation into the mechanisms of TAp73 stability showed that apoptin induced the expression of the ring finger domain ubiquitin ligase PIR2 which is involved in the degradation of the anti-apoptotic ∆Np73 isoform. Collectively, our results suggest a novel mechanism of apoptin-induced apoptosis through increased TAp73 stability and induction of PIR2 resulting in the degradation of ∆Np73 and activation of pro-apoptotic targets such as PUMA causing cancer cell death.


Asunto(s)
Apoptosis , Proteínas de la Cápside/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de la Cápside/biosíntesis , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Puntos de Control de la Fase G2 del Ciclo Celular , Semivida , Humanos , Proteínas Nucleares/genética , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteolisis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitinación
2.
J Cell Biol ; 155(5): 719-24, 2001 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-11714728

RESUMEN

Apoptosis or programmed cell death plays a pivotal role in embryonic development and maintenance of homeostasis. It is also involved in the etiology of pathophysiological conditions such as cancer, neurodegenerative, autoimmune, infectious, and heart diseases. Consequently, the study of apoptosis is now at center of both basic and clinical research applications. Therefore, sensitive and simple apoptosis detection techniques are required. Here we describe a monoclonal antibody-defined novel antigen, namely NAPO (negative in apoptosis), which is specifically lost during apoptosis. The anti-NAPO antibody recognizes two nuclear polypeptides of 60 and 70 kD. The antigen is maintained in quiescent and senescent cells, as well as in different phases of the cell cycle, including mitosis. Thus, immunodetection of NAPO antigen provides a specific, sensitive, and easy method for differential identification of apoptotic and nonapoptotic cells.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos/análisis , Apoptosis , Proteínas Nucleares/análisis , Animales , Antígenos/inmunología , Ciclo Celular/fisiología , Línea Celular , Núcleo Celular/inmunología , Senescencia Celular/fisiología , Medio de Cultivo Libre de Suero , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Microscopía Fluorescente , Proteínas Nucleares/inmunología , Pruebas de Precipitina , Células Tumorales Cultivadas
3.
Oncogene ; 20(37): 5111-7, 2001 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-11526499

RESUMEN

p53 and p73 proteins activate similar target genes and induce apoptosis and cell cycle arrest. However, p53, but not p73 is considered a tumour-suppressor gene. Unlike p53, p73 deficiency in mice does not lead to a cancer-prone phenotype, and p73 gene is not mutated in human cancers, including hepatocellular carcinoma. Here we report that normal liver cells express only DeltaN-p73 transcript forms giving rise to the synthesis of N-terminally truncated, transcriptionally inactive and dominant negative p73 proteins. In contrast, most hepatocellular carcinoma cells express TA-p73 transcript forms encoding full-length and transcriptionally active p73 proteins, in addition to DeltaN-p73. We also show that together with the acquired expression of TA-p73, the 'retinoblastoma pathway' is inactivated, and E2F1-target genes including cyclin E and p14(ARF) are activated in hepatocellular carcinoma. However, there was no full correlation between 'retinoblastoma pathway' inactivation and TA-p73 expression. Most TA-p73-expressing hepatocellular carcinoma cells have also lost p53 function either by lack of expression or missense mutations. The p73 gene, encoding only DeltaN-p73 protein, may function as a tumour promoter rather than a tumour suppressor in liver tissue. This may be one reason why p73 is not a mutation target in hepatocellular carcinoma.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Células COS , Carcinoma Hepatocelular/genética , ADN Complementario/metabolismo , Genes Dominantes , Genes Supresores de Tumor , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/genética , Ratones , Ratones Endogámicos BALB C , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Fenotipo , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteína Tumoral p73 , Proteínas Supresoras de Tumor
4.
Oncogene ; 20(11): 1398-401, 2001 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-11313883

RESUMEN

Three monoclonal antibodies (Mabs) were generated against p53 DNA-binding core domain. When tested by immunoprecipitation, Western blot and immunofluorescence techniques, Mab 9E4, as well as 7D3 and 6B10 reacted with both wild-type and various mutant p53 proteins. The epitopes recognized by Mabs 7D3, 9E4 and 6B10 were located respectively within the amino acid residues 211-220, 281-290 and 291-300 of human p53 protein. The epitope recognized by 9E4 Mab coincides with helix 2, also called p53 DNA binding helix, which allows the direct contact of the protein with its target DNA sequences. This antibody may be useful to study transcription-dependent and transcription-independent activities of wild-type and mutant p53 proteins.


Asunto(s)
Anticuerpos Monoclonales , Proteína p53 Supresora de Tumor/inmunología , Especificidad de Anticuerpos , Sitios de Unión/inmunología , Epítopos , Humanos , Hibridomas , Fragmentos de Péptidos/inmunología , Estructura Secundaria de Proteína , Proteína p53 Supresora de Tumor/química
5.
Int J Biochem Cell Biol ; 60: 130-8, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25541373

RESUMEN

Urocortin (Ucn 1), a 40 amino acid long peptide related to corticotropin releasing factor (CRF) was discovered 19 years ago, based on its sequence homology to the parent molecule. Its existence was inferred in the CNS because of anatomical and pharmacological discrepancies between CRF and its two receptor subtypes. Although originally found in the brain, where it has opposing actions to CRF and therefore confers stress-coping mechanisms, Ucn 1 has subsequently been found throughout the periphery including heart, lung, skin, and immune cells. It is now well established that this small peptide is involved in a multitude of physiological and pathophysiological processes, due to its receptor subtype distribution and promiscuity in second messenger signalling pathways. As a result of extensive studies in this field, there are now well over one thousand peer reviewed publications involving Ucn 1. In this review, we intend to highlight some of the less well known actions of Ucn 1 and in particular its role in neuronal cell protection and maintenance of the skeletal system, both by conventional methods of reviewing the literature and using bioinformatics, to highlight further associations between Ucn 1 and disease conditions. Understanding how Ucn 1 works in these tissues, will help to unravel its role in normal and pathophysiological processes. This would ultimately allow the generation of putative medical interventions for the alleviation of important diseases such as Parkinson's disease, arthritis, and osteoporosis.


Asunto(s)
Enfermedad de Parkinson/metabolismo , Urocortinas/metabolismo , Animales , Artritis/genética , Artritis/metabolismo , Humanos , Osteoporosis/genética , Osteoporosis/metabolismo , Enfermedad de Parkinson/genética , Urocortinas/genética
6.
Oncogene ; 33(42): 5039-46, 2014 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-24186203

RESUMEN

Activation of serine biosynthesis supports growth and proliferation of cancer cells. Human cancers often exhibit overexpression of phosphoglycerate dehydrogenase (PHGDH), the metabolic enzyme that catalyses the reaction that diverts serine biosynthesis from the glycolytic pathway. By refueling serine biosynthetic pathways, cancer cells sustain their metabolic requirements, promoting macromolecule synthesis, anaplerotic flux and ATP. Serine biosynthesis intersects glutaminolysis and together with this pathway provides substrates for production of antioxidant GSH. In human lung adenocarcinomas we identified a correlation between serine biosynthetic pathway and p73 expression. Metabolic profiling of human cancer cell line revealed that TAp73 activates serine biosynthesis, resulting in increased intracellular levels of serine and glycine, associated to accumulation of glutamate, tricarboxylic acid (TCA) anaplerotic intermediates and GSH. However, at molecular level p73 does not directly regulate serine metabolic enzymes, but transcriptionally controls a key enzyme of glutaminolysis, glutaminase-2 (GLS-2). p73, through GLS-2, favors conversion of glutamine in glutamate, which in turn drives the serine biosynthetic pathway. Serine and glutamate can be then employed for GSH synthesis, thus the p73-dependent metabolic switch enables potential response against oxidative stress. In knockdown experiment, indeed, TAp73 depletion completely abrogates cancer cell proliferation capacity in serine/glycine-deprivation, supporting the role of p73 to help cancer cells under metabolic stress. These findings implicate p73 in regulation of cancer metabolism and suggest that TAp73 influences glutamine and serine metabolism, affecting GSH synthesis and determining cancer pathogenesis.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/fisiología , Serina/biosíntesis , Proteínas Supresoras de Tumor/fisiología , Línea Celular Tumoral , Proliferación Celular , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glutaminasa/genética , Glutaminasa/metabolismo , Humanos , Fosfoglicerato-Deshidrogenasa/genética , Fosfoglicerato-Deshidrogenasa/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Isoformas de Proteínas/fisiología , Transaminasas/genética , Transaminasas/metabolismo , Transcripción Genética , Proteína Tumoral p73
7.
Oncogene ; 32(40): 4758-65, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23128396

RESUMEN

ΔNp63 is a transcription factor that is critical for the development of stratified epithelia and is overexpressed or amplified in >80% of squamous cell carcinomas (SCCs). We identified the RING finger E3 ubiquitin ligase PIR2/Rnf144b as a direct transcriptional target of ΔNp63α and showed that its expression parallels that of ΔNp63α in keratinocytes, SCC cell lines and SCCs. We used primary keratinocytes as a model system to investigate the function of PIR2/Rnf144b in stratified epithelia. Depletion of PIR2/Rnf144b severely impaired keratinocyte proliferation and differentiation, associated with accumulation of p21(WAF1/CIP1); a known target of PIR2/Rnf144b. More importantly, we found that PIR2/Rnf144b binds and mediates proteasomal degradation of ΔNp63α, generating a hitherto unknown auto-regulatory feedback loop. These findings substantiate PIR2/Rnf144b as a potentially critical component of epithelial homeostasis, acting downstream of ΔNp63α to regulate cellular levels of p21(WAF1/CIP1) and ΔNp63α.


Asunto(s)
Proteínas Portadoras/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Epitelio/metabolismo , Homeostasis/fisiología , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Empalme Alternativo , Diferenciación Celular , Línea Celular , Proliferación Celular , Humanos , Queratinocitos/citología , Proteolisis , Activación Transcripcional , Ubiquitina-Proteína Ligasas/genética
8.
Cell Death Dis ; 3: e285, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22419114

RESUMEN

The transcription factor p73 is a member of the p53 family that can be expressed as at least 24 different isoforms with pro- or anti-apoptotic attributes. The TAp73 isoforms are expressed from an upstream promoter and are regarded as bona fide tumor suppressors; they can induce cell cycle arrest/apoptosis and protect against genomic instability. On the other hand, ΔNp73 isoforms lack the N-terminus transactivation domain; hence, cannot induce the expression of pro-apoptotic genes, but still can oligomerize with TAp73 or p53 to block their transcriptional activities. Therefore, the ratio of TAp73 isoforms to ΔNp73 isoforms is critical for the quality of the response to a genomic insult and needs to be delicately regulated at both transcriptional and post-translational level. In this review, we will summarize the current knowledge on the post-translational regulatory pathways involved to keep p73 protein under control. A comprehensive understanding of p73 post-translational modifications will be extremely useful for the development of new strategies for treating and preventing cancer.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Acetilación , Empalme Alternativo , Animales , Apoptosis , Proteínas de Unión al ADN/genética , Humanos , Ratones , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Fosforilación , Regiones Promotoras Genéticas , Isoformas de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Transducción de Señal , Activación Transcripcional , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética
9.
Oncogene ; 30(41): 4219-30, 2011 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-21516125

RESUMEN

P73 is important in drug-induced apoptosis in some cancer cells, yet its role in the regulation of chemosensitivity in ovarian cancer (OVCA) is poorly understood. Furthermore, if and how the deregulation of p73-mediated apoptosis confers resistance to cisplatin (CDDP) treatment is unclear. Here we demonstrate that TAp73α over-expression enhanced CDDP-induced PARP cleavage and apoptosis in both chemosensitive (OV2008 and A2780s) and their resistant counterparts (C13* and A2780cp) and another chemoresistant OVCA cells (Hey); in contrast, the effect of ΔNp73α over-expression was variable. P73α downregulation attenuated CDDP-induced PUMA and NOXA upregulation and apoptosis in OV2008 cells. CDDP decreased p73α steady-state protein levels in OV2008, but not in C13*, although the mRNA expression was identical. CDDP-induced p73α downregulation was mediated by a calpain-dependent pathway. CDDP induced calpain activation and enhanced its cytoplasmic interaction and co-localization with p73α in OV2008, but not C13* cells. CDDP increased the intracellular calcium concentration ([Ca(2+)](i)) in OV2008 but not C13* whereas cyclopiazonic acid (CPA), a Ca(2+)-ATPase inhibitor, caused this response and calpain activation, p73α processing and apoptosis in both cell types. CDDP-induced [Ca(2+)](i) increase in OV2008 cells was not effected by the elimination of extracellular Ca(2+), but this was attenuated by the depletion of internal Ca(2+) store, indicating that mobilization of intracellular Ca(2+]) stores was potentially involved. These findings demonstrate that p73α and its regulation by the Ca(2+)-mediated calpain pathway are involved in CDDP-induced apoptosis in OVCA cells and that dysregulation of Ca(2+)/calpain/p73 signaling may in part be the pathophysiology of CDDP resistance. Understanding the cellular and molecular mechanisms of chemoresistance will direct the development of effective strategies for the treatment of chemoresistant OVCA.


Asunto(s)
Apoptosis/efectos de los fármacos , Calcio/metabolismo , Calpaína/metabolismo , Cisplatino/farmacología , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Western Blotting , Calpaína/antagonistas & inhibidores , Calpaína/genética , Línea Celular Tumoral , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Unión al ADN/genética , Dipéptidos/farmacología , Resistencia a Antineoplásicos , Femenino , Humanos , Inmunoprecipitación , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Proteínas Nucleares/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/genética
10.
Oncogene ; 27(31): 4363-72, 2008 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-18362891

RESUMEN

The p73 protein, a member of the p53 family, has both developmental and tumorigenic functions. Here we show that p73 is cleaved by caspase-3 and -8 both in vitro and in vivo during apoptosis elicited by DNA-damaging drugs and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor ligation. TAp73 and some of its cleavage products are localized to mitochondria. siRNA-mediated downregulation of p73 expression induced a small but significant change in the susceptibility of HCT116 cells to TRAIL-induced apoptosis. A transcription-deficient mutant of TAp73 enhanced TRAIL-induced apoptosis suggesting that p73 protein has transcription-independent functions during death receptor-mediated apoptosis. Additionally, recombinant p73 protein induced cytochrome c release from isolated mitochondria providing evidence that nonnuclear p73 may have additional functions in the progression of apoptosis.


Asunto(s)
Apoptosis , Caspasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Mutación , Proteínas Nucleares/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular Tumoral , Células HeLa , Humanos , Masculino , Mitocondrias/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteína Tumoral p73
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