RESUMEN
DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.
Asunto(s)
ADN Primasa/genética , Replicación del ADN/genética , ADN Polimerasa Dirigida por ADN/genética , ADN/genética , Enzimas Multifuncionales/genética , Animales , ADN Helicasas/genética , Reparación del ADN/genética , Femenino , Humanos , Masculino , Mamíferos/genética , RatonesRESUMEN
Nucleolar disruption has recently emerged as a relevant means to activate p53 through inhibition of HDM2 by ribosome-free RPL11. Most drugs that induce nucleolar disruption also possess important genotoxic activity, which can have lasting mutagenic effects. Therefore, it is of interest to identify compounds that selectively produce nucleolar disruption in the absence of DNA damage. Here, we have performed a high-throughput screening to search for nucleolar disruptors. We have identified an acridine derivative (PubChem CID-765471) previously known for its capacity to activate p53 independently of DNA damage, although the molecular mechanism underlying p53 activation had remained uncharacterized. We report that CID-765471 produces nucleolar disruption by inhibiting ribosomal DNA transcription in a process that includes the selective degradation of the RPA194 subunit of RNA polymerase I. Following nucleolar disruption, CID-765471 activates p53 through the RPL11/HDM2 pathway in the absence of detectable DNA damage. In a secondary screening of compounds approved for medical use, we identify two additional acridine derivatives, aminacrine and ethacridine, that operate in a similar manner as CID-765471. These findings provide the basis for non-genotoxic chemotherapeutic approaches that selectively target the nucleolus.
Asunto(s)
Acridinas/farmacología , Neoplasias Óseas/metabolismo , Neoplasias del Colon/metabolismo , Naftiridinas/farmacología , Osteosarcoma/metabolismo , Preparaciones Farmacéuticas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Ribosómicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acridinas/administración & dosificación , Acridinas/química , Northern Blotting , Western Blotting , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Daño del ADN/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunoprecipitación , Naftiridinas/administración & dosificación , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Osteosarcoma/patología , Preparaciones Farmacéuticas/administración & dosificación , Proteínas Proto-Oncogénicas c-mdm2/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Chemotherapy often generates intratumoral senescent cancer cells that strongly modify the tumor microenvironment, favoring immunosuppression and tumor growth. We discovered, through an unbiased proteomics screen, that the immune checkpoint inhibitor programmed cell death 1 ligand 2 (PD-L2) is highly upregulated upon induction of senescence in different types of cancer cells. PD-L2 is not required for cells to undergo senescence, but it is critical for senescent cells to evade the immune system and persist intratumorally. Indeed, after chemotherapy, PD-L2-deficient senescent cancer cells are rapidly eliminated and tumors do not produce the senescence-associated chemokines CXCL1 and CXCL2. Accordingly, PD-L2-deficient pancreatic tumors fail to recruit myeloid-derived suppressor cells and undergo regression driven by CD8 T cells after chemotherapy. Finally, antibody-mediated blockade of PD-L2 strongly synergizes with chemotherapy causing remission of mammary tumors in mice. The combination of chemotherapy with anti-PD-L2 provides a therapeutic strategy that exploits vulnerabilities arising from therapy-induced senescence.
Asunto(s)
Neoplasias Pancreáticas , Animales , Ratones , Neoplasias Pancreáticas/metabolismo , Linfocitos T CD8-positivos/patología , Tolerancia Inmunológica , Terapia de Inmunosupresión , Senescencia Celular , Microambiente TumoralRESUMEN
Collapsin response mediator protein-2 (CRMP-2) is the first described and most studied member of a family of proteins that mediate the addition of tubulin dimers to the growing microtubule. CRMPs have mainly been studied in the nervous system, but recently, they have been described in other tissues where they participate in vesicle transport, migration and mitosis. In this work, we aimed at studying the role of CRMP-2 in lung cancer cell division. We first explored the expression of CRMP-2 and phosphorylated (Thr 514) CRMP-2 in 91 samples obtained from patients with localized nonsmall cell lung cancer. We observed a significant correlation between high levels of nuclear phosphorylated CRMP-2 and poor prognosis in those patients. Interestingly, this association was only positive for untreated patients. To provide a mechanistic explanation to these findings, we used in vitro models to analyze the role of CRMP-2 and its phosphorylated forms in cell division. Thus, we observed by confocal microscopy and immunoprecipitation assays that CRMP-2 differentially colocalizes with the mitotic spindle during cell division. The use of phosphodefective or phosphomimetic mutants of CRMP-2 allowed us to prove that anomalies in the phosphorylation status of CRMP-2 result in changes in the mitotic tempo, and increments in the number of multinucleated cells. Finally, here we demonstrate that CRMP-2 phosphorylation impairment, or silencing induces p53 expression and promotes apoptosis through caspase 3 activation. These results pointed to CRMP-2 phosphorylation as a prognostic marker and potential new target to be explored in cancer therapy.
Asunto(s)
Adenocarcinoma/metabolismo , Biomarcadores de Tumor/sangre , Carcinoma Adenoescamoso/metabolismo , Carcinoma de Células Grandes/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Apoptosis , Western Blotting , Carcinoma Adenoescamoso/mortalidad , Carcinoma Adenoescamoso/patología , Carcinoma de Células Grandes/mortalidad , Carcinoma de Células Grandes/patología , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Ciclo Celular , Proliferación Celular , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/genética , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Microtúbulos/metabolismo , Persona de Mediana Edad , Mutagénesis Sitio-Dirigida , Mutación/genética , Estadificación de Neoplasias , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Fosforilación , Pronóstico , ARN Interferente Pequeño/genética , Huso Acromático , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Although kinase mutations have been identified in various human diseases, much less is known about protein phosphatases. Here, we show that all apoptosis-stimulating proteins of p53 (ASPP) family members can bind protein phosphatase 1 (PP1) via two distinct interacting motifs. ASPP2 interacts with PP1 through an RVXF PP1 binding motif, whereas the inhibitory member of the ASPP family (iASPP) interacts with PP1 via a noncanonical motif (RNYF) that is located within its Src homology 3 domain (SH3). Phe-815 is crucial in mediating iASPP/PP1 interaction, and iASPP(F815A) fails to inhibit the transcriptional and apoptotic function of p53. This study identifies iASPP as a new binding partner of PP1, interacting through a noncanonical PP1 binding motif.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Represoras/metabolismo , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Citometría de Flujo , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/genética , Unión Proteica , Proteína Fosfatasa 1/genética , Proteínas Represoras/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Palbociclib is a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6) approved for the treatment of some cancers. The main mechanism of action of palbociclib is to induce cell cycle arrest and senescence on responsive cells. Here, we report that palbociclib concentrates in intracellular acidic vesicles, where it can be readily observed due to its intrinsic fluorescence, and it is released from these vesicles upon dilution or washing out of the extracellular medium. This reversible storage of drugs into acidic vesicles is generally known as lysosomal trapping and, based on this, we uncover novel properties of palbociclib. In particular, a short exposure of cells to palbociclib is sufficient to produce a stable cell-cycle arrest and long-term senescence. Moreover, after washing out the drug, palbociclib-treated cells release the drug to the medium and this conditioned medium is active on susceptible cells. Interestingly, cancer cells resistant to palbociclib also accumulate and release the drug producing paracrine senescence on susceptible cells. Finally, other lysosomotropic drugs, such as chloroquine, interfere with the accumulation of palbociclib into lysosomes, thereby reducing the minimal dose of palbociclib required for cell-cycle arrest and senescence. In summary, lysosomal trapping explains the prolonged temporal activity of palbociclib, the paracrine activity of exposed cells, and the cooperation with lysosomotropic drugs. These are important features that may help to improve the therapeutic dosing and efficacy of palbociclib. Finally, two other clinically approved CDK4/6 inhibitors, ribociclib and abemaciclib, present a similar behavior as palbociclib, suggesting that lysosomal trapping is a property common to all three clinically-approved CDK4/6 inhibitors.
Asunto(s)
Antineoplásicos/farmacocinética , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Piperazinas/farmacocinética , Piridinas/farmacocinética , Naranja de Acridina/química , Aminopiridinas/farmacocinética , Bencimidazoles/farmacocinética , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Cloroquina/farmacología , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Citocinas/metabolismo , Colorantes Fluorescentes/química , Humanos , Proteínas de Membrana de los Lisosomas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Purinas/farmacocinéticaRESUMEN
Compounds with specific cytotoxic activity in senescent cells, or senolytics, support the causal involvement of senescence in aging and offer therapeutic interventions. Here we report the identification of Cardiac Glycosides (CGs) as a family of compounds with senolytic activity. CGs, by targeting the Na+/K+ATPase pump, cause a disbalanced electrochemical gradient within the cell causing depolarization and acidification. Senescent cells present a slightly depolarized plasma membrane and higher concentrations of H+, making them more susceptible to the action of CGs. These vulnerabilities can be exploited for therapeutic purposes as evidenced by the in vivo eradication of tumors xenografted in mice after treatment with the combination of a senogenic and a senolytic drug. The senolytic effect of CGs is also effective in the elimination of senescence-induced lung fibrosis. This experimental approach allows the identification of compounds with senolytic activity that could potentially be used to develop effective treatments against age-related diseases.
Asunto(s)
Apoptosis/efectos de los fármacos , Glicósidos Cardíacos/farmacología , Senescencia Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Células A549 , Animales , Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Neoplasias de la Mama , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Digoxina/farmacología , Femenino , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Ratones , Osteoartritis , Ouabaína/farmacología , Proscilaridina/farmacología , Fibrosis Pulmonar , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Senescent cells accumulate in multiple aging-associated diseases, and eliminating these cells has recently emerged as a promising therapeutic approach. Here, we take advantage of the high lysosomal ß-galactosidase activity of senescent cells to design a drug delivery system based on the encapsulation of drugs with galacto-oligosaccharides. We show that gal-encapsulated fluorophores are preferentially released within senescent cells in mice. In a model of chemotherapy-induced senescence, gal-encapsulated cytotoxic drugs target senescent tumor cells and improve tumor xenograft regression in combination with palbociclib. Moreover, in a model of pulmonary fibrosis in mice, gal-encapsulated cytotoxics target senescent cells, reducing collagen deposition and restoring pulmonary function. Finally, gal-encapsulation reduces the toxic side effects of the cytotoxic drugs. Drug delivery into senescent cells opens new diagnostic and therapeutic applications for senescence-associated disorders.
Asunto(s)
Senescencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Galactosa/metabolismo , Lisosomas/enzimología , Oligosacáridos/metabolismo , beta-Galactosidasa/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Citotoxinas/administración & dosificación , Citotoxinas/farmacología , Modelos Animales de Enfermedad , Composición de Medicamentos , Colorantes Fluorescentes/metabolismo , Xenoinjertos , Ratones , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Piperazinas/administración & dosificación , Piperazinas/farmacología , Piridinas/administración & dosificación , Piridinas/farmacología , Coloración y EtiquetadoRESUMEN
Field trial studies have been performed with captan and fenitrothion on cauliflower to propose maximum residue limits and to study the dissipation of the pesticides. Residue levels have been determined at different times following good laboratory practice using gas chromatography with mass spectrometric detection. The behaviour of residue levels of these compounds after household processing has been analysed using gas chromatography with electron capture detection. Seven days after treatment, residue levels of captan could be detected, but not of fenitrothion. The half-lives of dissipation for captan and fenitrothion were calculated as 0.9 and 1.8 days respectively. Washing did not significantly affect the residual amounts of captan and fenitrothion observed in raw vegetables; however, after cooking, captan had degraded completely, whereas residue levels of fenitrothion were not modified significantly.
Asunto(s)
Brassica , Captano/análisis , Fenitrotión/análisis , Contaminación de Alimentos , Fungicidas Industriales/análisis , Insecticidas/análisis , Residuos de Plaguicidas/análisis , Captano/química , Cromatografía de Gases/métodos , Exposición a Riesgos Ambientales , Fenitrotión/química , Manipulación de Alimentos , Fungicidas Industriales/química , Semivida , Calor , Concentración de Iones de Hidrógeno , Insecticidas/química , Espectrometría de Masas/métodos , Residuos de Plaguicidas/química , Medición de RiesgoRESUMEN
Senescent cells and cancer cells recruit immunosuppressive myeloid cells. In this issue of Cancer Cell, Eggert et al. report that senescent cells recruit immature myeloid cells (iMCs) through the secretion of the CCL2 cytokine and that these iMCs have pro- or anti-tumorigenic activities, depending on the cellular context.
Asunto(s)
Tolerancia Inmunológica , Neoplasias , Senescencia Celular , Citocinas , Humanos , Células MieloidesRESUMEN
We have identified a novel mechanism of regulation of the protein CDKN1A (also known as p21) by the serine/threonine kinase complex mammalian target of rapamycin complex 1 (mTORC1). Our results demonstrate that the mTORC1 substrate EIF4E-binding protein 1 (4E-BP1) in its non-phosphorylated state interacts with p21 and promotes p21 degradation. In addition, we demonstrate the prevalence of this mechanism in head and neck squamous cell carcinomas and show that it strongly and significantly associates with improved disease-specific survival, providing evidence for its clinical relevance.
RESUMEN
The levels, regulation and prognostic value of p21 in head and neck squamous cell carcinomas (HNSCC) has been puzzling for years. Here, we report a new mechanism of regulation of p21 by the mTORC1/4E-BP1 pathway. We find that non-phosphorylated 4E-BP1 interacts with p21 and induces its degradation. Accordingly, hyper-activation of mTORC1 results in phosphorylation of 4E-BP1 and stabilization of p21. In HNSCC, p21 levels strongly correlate with mTORC1 activity but not with p53 status. Finally, clinical data indicate that HNSCC patients with p21 and phospho-S6-double-positive tumours present a better disease-specific survival. We conclude that over-activation of the mTORC1/4E-BP1/p21 pathway is a frequent and clinically relevant alteration in HNSCC.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma de Células Escamosas/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Complejos Multiproteicos/metabolismo , Fosfoproteínas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Neoplasias de Cabeza y Cuello/genética , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Persona de Mediana Edad , Complejos Multiproteicos/genética , Fosfoproteínas/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
The small basic protein p14ARF, encoded by one of the alternative transcripts from the human INK4A/ARF locus, interferes with MDM2-mediated ubiquitination of the p53 tumour suppressor protein. The resultant stabilization of p53 leads to increased expression of p53-regulated genes, such as MDM2 itself and the cyclin-dependent kinase inhibitor p21(CIP1). Here we relate physical interactions between p14ARF and MDM2, as determined using synthetic peptides and systematic deletions of p14ARF, with consequential effects on p53 stabilization and transcriptional activity. The data imply that the amino terminal half of p14ARF, encoded by the alternative first exon (exon 1beta) contacts MDM2 through multiple domains that can independently impede MDM2-mediated degradation of p53, provided that they are localized in the cell nucleus. As well as identifying previously unrecognized functional domains, our findings offer an explanation for the relative paucity of missense mutations in exon 1beta in human tumours.
Asunto(s)
Proteínas Nucleares , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteína p14ARF Supresora de Tumor/química , Proteína p14ARF Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/metabolismo , Exones/genética , Humanos , Datos de Secuencia Molecular , Mapeo Peptídico , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Eliminación de Secuencia , Transcripción Genética , Transfección , Células Tumorales Cultivadas , Proteína p14ARF Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Fusion proteins containing the amino-terminal domain of human p14(ARF) linked to green fluorescent protein are able to bind MDM2 and stabilize p53 without localization in the nucleolus. However, these fusion proteins are inherently unstable, with half-lives considerably shorter than either authentic ARF or chimaeras containing the entire coding domain, both of which are predominantly nucleolar. We present evidence that the unstable fusion proteins are significantly stabilized if redirected to the nucleolus by addition of a basic motif based on the nuclear localization signal of SV40 T-antigen. Moreover, the stability of these proteins can be enhanced by modulating the functions of MDM2 and p53. These data are consistent with a model in which ARF interacts with MDM2 in the nucleoplasm but is consequently subject to proteasomal degradation. Nucleolar localization may serve to store or stabilize ARF.
Asunto(s)
Nucléolo Celular/metabolismo , Proteína p14ARF Supresora de Tumor/metabolismo , Secuencia de Bases , Línea Celular , Proteínas Fluorescentes Verdes , Semivida , Humanos , Proteínas Luminiscentes/metabolismo , Señales de Localización Nuclear , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , ARN Interferente PequeñoRESUMEN
ASPP1 and ASPP2 are both proteins that interact with p53 and enhance its ability to induce apoptosis by selectively elevating the expression of proapoptotic p53-responsive genes. iASPP(RAI) is a third member of the family that is the most conserved inhibitor of p53-mediated apoptosis. Here, we have described iASPP, a longer form of iASPP(RAI), which at 828 amino acids is more than twice the size of iASPP(RAI). Using two antibodies that recognize both iASPP and iASPP(RAI), we report that this longer form of iASPP is the predominant form of the molecule expressed in cells. Like iASPP(RAI), iASPP also binds to p53 and inhibits apoptosis induced by p53 overexpression. However, whereas iASPP(RAI) is predominantly nuclear, the N-terminus of iASPP is entirely cytoplasmic, and the longer iASPP is located in both the cytoplasm and the nucleus. The effect upon subcellular localization of the longer N-terminus of iASPP means that this new, longer form of the molecule may be subject to greater regulation and provides another layer in the control of p53-induced apoptosis.
Asunto(s)
Proteínas Portadoras/metabolismo , Citoplasma/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Proteínas Portadoras/química , Línea Celular Tumoral , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Homología de Secuencia de Aminoácido , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Diamond-Blackfan anemia (DBA) is characterized by anemia and cancer susceptibility and is caused by mutations in ribosomal genes, including RPL11. Here, we report that Rpl11-heterozygous mouse embryos are not viable and that Rpl11 homozygous deletion in adult mice results in death within a few weeks, accompanied by bone marrow aplasia and intestinal atrophy. Importantly, Rpl11 heterozygous deletion in adult mice results in anemia associated with decreased erythroid progenitors and defective erythroid maturation. These defects are also present in mice transplanted with inducible heterozygous Rpl11 bone marrow and, therefore, are intrinsic to the hematopoietic system. Additionally, heterozygous Rpl11 mice present increased susceptibility to radiation-induced lymphomagenesis. In this regard, total or partial deletion of Rpl11 compromises p53 activation upon ribosomal stress or DNA damage in fibroblasts. Moreover, fibroblasts and hematopoietic tissues from heterozygous Rpl11 mice present higher basal cMYC levels. We conclude that Rpl11-deficient mice recapitulate DBA disorder, including cancer predisposition.
Asunto(s)
Anemia de Diamond-Blackfan/metabolismo , Linfoma/metabolismo , Proteínas Ribosómicas/metabolismo , Anemia de Diamond-Blackfan/genética , Animales , Proliferación Celular , Células Cultivadas , Células Eritroides/metabolismo , Linfoma/genética , Ratones , Ratones Noqueados , Proteínas Ribosómicas/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Field trials have been carried out to determine the variability of residue levels of fenitrothion and its main metabolites fenitrothion-oxon and 3-methyl-4-nitrophenol in individual kaki fruits versus composite samples, in peel versus flesh, and in whole uncooked versus whole cooked fruits. Residue levels have been determined by gas chromatography with thermionic specific detection after extraction with ethyl acetate and without further cleanup. At harvest, residue levels of fenitrothion were below maximum residue levels (MRLs) and the two metabolites 3-methyl-4-nitrophenol and fenitrothion-oxon could be quantified with average amounts of 0.080 and 0.012 mg/kg, respectively. Levels of fenitrothion decreased 88% after peeling, whereas temperature did not result in a high variation. The ratios of the highest residue level in the individual fruits to the corresponding mean of residue levels in the composite samples for fenitrothion were <3. This value is lower than that recommended by the World Health Organization as default value for consumer risk assessment.
Asunto(s)
Diospyros/química , Fenitrotión/análisis , Manipulación de Alimentos/métodos , Frutas/química , Insecticidas/análisis , Residuos de Plaguicidas/análisis , Cromatografía de Gases/métodosRESUMEN
Ribosome biogenesis is the most demanding energetic process in proliferating cells and it is emerging as a critical sensor of cellular homeostasis. Upon disturbance of ribosome biogenesis, specific free ribosomal proteins, most notably L11, bind and inhibit Mdm2, resulting in activation of the tumor suppressor p53. This pathway has been characterized in somatic and cancer cells, but its function in embryonic pluripotent cells has remained unexplored. Here, we show that treatment with low doses of Actinomycin D or depletion of ribosomal protein L37, two well-established inducers of ribosomal stress, activate p53 in an L11-dependent manner in mouse embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). Activation of p53 results in transcriptional induction of p53 targets, including p21, Mdm2, Pidd, Puma, Noxa and Bax. Finally, ribosomal stress elicits L11- and p53-dependent apoptosis in ESCs/iPSCs. These results extend to pluripotent cells the functionality of the ribosomal stress pathway and we speculate that this could be a relevant cellular checkpoint during early embryogenesis.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Proteínas Ribosómicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Dactinomicina/farmacología , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Células Madre Pluripotentes/efectos de los fármacos , Unión Proteica , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Ribosomas/patología , Transcripción Genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
CREBZF is a member of the mammalian ATF/CREB family of transcription factors. Here, we describe a novel functional interaction between CREBZF and the tumor suppressor p53. CREBZF was identified in a yeast two-hybrid screen using HEY1, recently characterized as an indirect p53 activator, as bait. CREBZF interacts in vitro with both HEY1 and p53, and CREBZF expression stabilizes and activates p53. Moreover, CREBZF cooperates synergistically with HEY1 to enhance p53 transcriptional activity. On the other hand, partial depletion of endogenous CREBZF diminishes p53 protein levels and inhibits HEY1-mediated activation of p53. CREBZF-positive effects on p53 signaling may reflect, at least in part, an observed induction of posttranslational modifications in p53 known to prevent its degradation. CREBZF expression protects HCT116 cells from UV radiation-induced cell death. In addition, CREBZF expression confers sensitivity to 5-fluorouracil, a p53-activating chemotherapeutic drug. Our study suggests that CREBZF may participate in the modulation of p53 tumor suppressor function.