RESUMEN

In recent years platinum (Pt) drugs have been found to be especially efficient to treat patients with cancers that lack a proper DNA damage response, e.g. due to dysfunctional BRCA1. Despite this knowledge, we are still missing helpful markers to predict Pt response in the clinic. We have previously shown that volume-regulated anion channels, containing the subunits LRRC8A and LRRC8D, promote the uptake of cisplatin and carboplatin in BRCA1-proficient cell lines. Here, we show that the loss of LRRC8A or LRRC8D significantly reduces the uptake of cis- and carboplatin in BRCA1;p53-deficient mouse mammary tumor cells. This results in reduced DNA damage and in vivo drug resistance. In contrast to Lrrc8a, the deletion of the Lrrc8d gene does not affect the viability and fertility of mice. Interestingly, Lrrc8d-/- mice tolerate a two-fold cisplatin maximum-tolerable dose. This allowed us to establish a mouse model for intensified Pt-based chemotherapy, and we found that an increased cisplatin dose eradicates BRCA1;p53-deficient tumors, whereas eradication is not possible in WT mice. Moreover, we show that decreased expression of LRRC8A/D in head and neck squamous cell carcinoma patients, who are treated with a Pt-based chemoradiotherapy, leads to decreased overall survival of the patients. In particular, high cumulative cisplatin dose treatments lost their efficacy in patients with a low LRRC8A/D expression in their cancers. Our data therefore suggest that LRRC8A and LRRC8D should be included in a prospective trial to predict the success of intensified cis- or car-boplatin-based chemotherapy.

Asunto(s)

Cisplatino , Platino (Metal) , Ratones , Animales , Cisplatino/farmacología , Carboplatino/farmacología , Platino (Metal)/metabolismo , Proteína p53 Supresora de Tumor/genética , Estudios Prospectivos , Proteínas de la Membrana/genética , Aniones/metabolismo

RESUMEN

BACKGROUND: The prognosis of patients with HPV-negative advanced stage head and neck squamous cell carcinoma (HNSCC) remains poor. No prognostic markers other than TNM staging are routinely used in clinic. Epithelial-to-mesenchymal transition (EMT) has been shown to be a strong prognostic factor in other cancer types. The purpose of this study was to determine the role of EMT in HPV-negative HNSCC outcomes. METHODS: Pretreatment tumor material from patients of two cohorts, totalling 174 cisplatin-based chemoradiotherapy treated HPV-negative HNSCC patients, was RNA-sequenced. Seven different EMT gene expression signatures were used for EMT status classification and generation of HNSCC-specific EMT models using Random Forest machine learning. RESULTS: Mesenchymal classification by all EMT signatures consistently enriched for poor prognosis patients in both cohorts of 98 and 76 patients. Uni- and multivariate analyses show important HR of 1.6-5.8, thereby revealing EMT's role in HNSCC outcome. Discordant classification by these signatures prompted the generation of an HNSCC-specific EMT profile based on the concordantly classified samples in the first cohort (cross-validation AUC > 0.98). The independent validation cohort confirmed the association of mesenchymal classification by the HNSCC-EMT model with poor overall survival (HR = 3.39, p < 0.005) and progression free survival (HR = 3.01, p < 0.005) in multivariate analysis with TNM. Analysis of an additional HNSCC cohort from PET-positive patients with metastatic disease prior to treatment further supports this relationship and reveals a strong link of EMT to the propensity to metastasize. CONCLUSIONS: EMT in HPV-negative HNSCC co-defines patient outcome after chemoradiotherapy. The generated HNSCC-EMT prediction models can function as strong prognostic biomarkers.

Asunto(s)

Neoplasias de Cabeza y Cuello , Biomarcadores de Tumor , Quimioradioterapia , Transición Epitelial-Mesenquimal , Neoplasias de Cabeza y Cuello/terapia , Humanos , Pronóstico , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia

RESUMEN

DNA copy number alterations (CNAs) are frequent in cancer, and recently developed CNA signatures revealed their value in molecular tumor stratification for patient prognosis and platinum resistance prediction in ovarian cancer. Head and neck squamous cell carcinoma (HNSCC) is also characterized by high CNAs. In this study, we determined CNA in 173 human papilloma virus-negative HNSCC from a Dutch multicenter cohort by low-coverage whole genome sequencing and tested the prognostic value of seven cancer-derived CNA signatures for these cisplatin- and radiotherapy-treated patients. We find that a high CNA signature 1 (s1) score is associated with low values for all other signatures and better patient outcomes in the Dutch cohorts and The Cancer Genome Atlas HNSCC data set. High s5 and s7 scores are associated with increased distant metastasis rates and high s6 scores with poor overall survival. High cumulative cisplatin doses result in improved outcomes in chemoradiotherapy-treated HNSCC patients. Here we find that tumors high in s1 or low in s6 are most responsive to a change in cisplatin dose. High s5 values, however, significantly increase the risk for metastasis in patients with low cumulative cisplatin doses. Together this suggests that the processes causing these CNA signatures affect cisplatin response in HNSCC. In conclusion, CNA signatures derived from a different cancer type were prognostic and associated with cisplatin response in HNSCC, suggesting they represent underlying molecular processes that define patient outcome.

Asunto(s)

Biomarcadores de Tumor/genética , Quimioradioterapia , Neoplasias de Cabeza y Cuello/terapia , Neoplasias Ováricas/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Cisplatino/farmacología , Cisplatino/uso terapéutico , Variaciones en el Número de Copia de ADN , Conjuntos de Datos como Asunto , Resistencia a Antineoplásicos/genética , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/patología , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Pronóstico , Supervivencia sin Progresión , RNA-Seq , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/mortalidad , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Transcriptoma/genética , Secuenciación Completa del Genoma

RESUMEN

Head and neck squamous cell carcinoma (HNSCC) is characterized by the frequent manifestation of DNA crosslink repair defects. We established novel expression-based DNA repair defect markers to determine the clinical impact of such repair defects. Using hypersensitivity to the DNA crosslinking agents, mitomycin C and olaparib, as proxies for functional DNA repair defects in a panel of 25 HNSCC cell lines, we applied machine learning to define gene expression models that predict repair defects. The expression profiles established predicted hypersensitivity to DNA-damaging agents and were associated with mutations in crosslink repair genes, as well as downregulation of DNA damage response and repair genes, in two independent datasets. The prognostic value of the repair defect prediction profiles was assessed in two retrospective cohorts with a total of 180 patients with advanced HPV-negative HNSCC, who were treated with cisplatin-based chemoradiotherapy. DNA repair defects, as predicted by the profiles, were associated with poor outcome in both patient cohorts. The poor prognosis association was particularly strong in normoxic tumor samples and was linked to an increased risk of distant metastasis. In vitro, only crosslink repair-defective HNSCC cell lines are highly migratory and invasive. This phenotype could also be induced in cells by inhibiting rad51 in repair competent and reduced by DNA-PK inhibition. In conclusion, DNA crosslink repair prediction expression profiles reveal a poor prognosis association in HNSCC. SIGNIFICANCE: This study uses innovative machine learning-based approaches to derive models that predict the effect of DNA repair defects on treatment outcome in HNSCC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5597/F1.large.jpg.

Asunto(s)

Antineoplásicos/farmacología , Reparación del ADN/efectos de los fármacos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Quimioradioterapia/métodos , Cisplatino/farmacología , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Reparación del ADN/genética , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Neoplasias de Cabeza y Cuello/genética , Humanos , Mutación/efectos de los fármacos , Mutación/genética , Fenotipo , Pronóstico , Proteína Recombinante y Reparadora de ADN Rad52/genética , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética

RESUMEN

About half of advanced stage head and neck squamous cell carcinoma (HNSCC) patients can be cured by chemoradiotherapy. Patient outcome may be partially determined by the genetic alterations in HNSCC, rendering these alterations promising candidate prognostic factors and/or therapeutic targets. However, their relevance in patient outcome prognosis remains to be assessed in patients that receive standard-of-care chemoradiotherapy. We therefore tested whether frequent genetic alterations were associated with progression free survival (PFS) in advanced stage HNSCC patients who were uniformly treated with definitive platinum-based chemoradiotherapy. To this end, we performed targeted DNA sequencing on frozen pre-treatment tumor biopsy material from 77 patients with advanced stage oro- and hypopharyngeal carcinoma. This provided somatic point mutation and copy number aberration data of 556 genes. The most frequently mutated genes, TP53 (62%), CCND1 (51%), CDKN2A (30%) and PIK3CA (21%), were not associated with PFS. However, co-occurring CCND1 and CDKN2A mutations were associated with short PFS (HR 2.24, p = 0.028) in HPV-negative tumors. Furthermore, tumor mutational burden (sum of somatic point mutations) showed a trend towards decreased PFS (HR 1.9, p = 0.089), and chromosomal instability (CIN) was associated with shorter PFS (HR 2.3, p = 0.023), independent of HPV status. Our results show that tumor mutational burden, CIN markers, and co-occurring CCND1 and CDKN2A mutations are associated with chemoradiotherapy outcomes in advanced stage oro- and hypopharyngeal HNSCC patients, thereby highlighting their prognostic potential. Given their poor prognosis association and link to biological targets, they may also identify patients for novel targeted therapies and immunotherapies.

RESUMEN

Purpose: Tumor markers that are related to hypoxia, proliferation, DNA damage repair and stem cell-ness, have a prognostic value in advanced stage HNSCC patients when assessed individually. Here we aimed to evaluate and validate this in a multifactorial context and assess interrelation and the combined role of these biological factors in determining chemo-radiotherapy response in HPV-negative advanced HNSCC. Methods: RNA sequencing data of pre-treatment biopsy material from 197 HPV-negative advanced stage HNSCC patients treated with definitive chemoradiotherapy was analyzed. Biological parameter scores were assigned to patient samples using previously generated and described gene expression signatures. Locoregional control rates were used to assess the role of these biological parameters in radiation response and compared to distant metastasis data. Biological factors were ranked according to their clinical impact using bootstrapping methods and multivariate Cox regression analyses that included clinical variables. Multivariate Cox regression analyses comprising all biological variables were used to define their relative role among all factors when combined. Results: Only few biomarker scores correlate with each other, underscoring their independence. The different biological factors do not correlate or cluster, except for the two stem cell markers CD44 and SLC3A2 (r = 0.4, p < 0.001) and acute hypoxia prediction scores which correlated with T-cell infiltration score, CD8+ T cell abundance and proliferation scores (r = 0.52, 0.56, and 0.6, respectively with p < 0.001). Locoregional control association analyses revealed that chronic (Hazard Ratio (HR) = 3.9) and acute hypoxia (HR = 1.9), followed by stem cell-ness (CD44/SLC3A2; HR = 2.2/2.3), were the strongest and most robust determinants of radiation response. Furthermore, multivariable analysis, considering other biological and clinical factors, reveal a significant role for EGFR expression (HR = 2.9, p < 0.05) and T-cell infiltration (CD8+T-cells: HR = 2.2, p < 0.05; CD8+T-cells/Treg: HR = 2.6, p < 0.01) signatures in locoregional control of chemoradiotherapy-treated HNSCC. Conclusion: Tumor acute and chronic hypoxia, stem cell-ness, and CD8+ T-cell parameters are relevant and largely independent biological factors that together contribute to locoregional control. The combined analyses illustrate the additive value of multifactorial analyses and support a role for EGFR expression analysis and immune cell markers in addition to previously validated biomarkers. This external validation underscores the relevance of biological factors in determining chemoradiotherapy outcome in HNSCC.

RESUMEN

Lowered activity of the insulin/IGF signalling (IIS) network can ameliorate the effects of ageing in laboratory animals and, possibly, humans. Although transcriptome remodelling in long-lived IIS mutants has been extensively documented, the causal mechanisms contributing to extended lifespan, particularly in specific tissues, remain unclear. We have characterized the proteomes of four key insulin-sensitive tissues in a long-lived Drosophila IIS mutant and control, and detected 44% of the predicted proteome (6,085 proteins). Expression of ribosome-associated proteins in the fat body was reduced in the mutant, with a corresponding, tissue-specific reduction in translation. Expression of mitochondrial electron transport chain proteins in fat body was increased, leading to increased respiration, which was necessary for IIS-mediated lifespan extension, and alone sufficient to mediate it. Proteasomal subunits showed altered expression in IIS mutant gut, and gut-specific over-expression of the RPN6 proteasomal subunit, was sufficient to increase proteasomal activity and extend lifespan, whilst inhibition of proteasome activity abolished IIS-mediated longevity. Our study thus uncovered strikingly tissue-specific responses of cellular processes to lowered IIS acting in concert to ameliorate ageing.

Asunto(s)

Envejecimiento/metabolismo , Drosophila/metabolismo , Redes Reguladoras de Genes , Proteómica/métodos , Animales , Proteínas de Drosophila , Cuerpo Adiposo/metabolismo , Insulina/metabolismo , Mucosa Intestinal/metabolismo , Modelos Animales , Mutación , Especificidad de Órganos , Proteínas Ribosómicas/metabolismo

RESUMEN

Down-regulation of insulin/insulin-like growth factor signaling (IIS) can increase lifespan in C. elegans, Drosophila and mice. In C. elegans, reduced IIS results in down-regulation of translation, which itself can extend lifespan. However, the effect of reduced IIS on translation has yet to be determined in other multicellular organisms. Using two long-lived IIS models, namely Drosophila lacking three insulin-like peptides (dilp2-3,5(-/-)) and mice lacking insulin receptor substrate 1 (Irs1(-/-)), and two independent translation assays, polysome profiling and radiolabeled amino acid incorporation, we show that reduced IIS lowers translation in these organisms. In Drosophila, reduced IIS decreased polysome levels in fat body and gut, but reduced the rate of protein synthesis only in the fat body. Reduced IIS in mice decreased protein synthesis rate only in skeletal muscle, without reducing polysomes in any tissue. This lowered translation in muscle was independent of Irs1 loss in the muscle itself, but a secondary effect of Irs1 loss in the liver. In conclusion, down-regulation of translation is an evolutionarily conserved response to reduced IIS, but the tissues in which it occurs can vary between organisms. Furthermore, the mechanisms underlying lowered translation may differ in mice, possibly associated with the complexity of the regulatory processes.

Asunto(s)

Proteínas de Drosophila/genética , Proteínas Sustrato del Receptor de Insulina/genética , Insulinas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Longevidad/genética , Biosíntesis de Proteínas , Animales , Drosophila/genética , Insulina/genética , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Estrés Oxidativo/genética , Transducción de Señal/genética

RESUMEN

The biogenesis of ribosomes and their coordination of protein translation consume an enormous amount of cellular energy. As such, it has been established that the inhibition of either process can extend eukaryotic lifespan. Here, we used next-generation sequencing to compare ribosome-associated RNAs from normal strains of Caenorhabditis elegans to those carrying the life-extending daf-2 mutation. We found a long noncoding RNA (lncRNA), transcribed telomeric sequence 1 (tts-1), on ribosomes of the daf-2 mutant. Depleting tts-1 in daf-2 mutants increases ribosome levels and significantly shortens their extended lifespan. We find tts-1 is also required for the longer lifespan of the mitochondrial clk-1 mutants but not the feeding-defective eat-2 mutants. In line with this, the clk-1 mutants express more tts-1 and fewer ribosomes than the eat-2 mutants. Our results suggest that the expression of tts-1 functions in different longevity pathways to reduce ribosome levels in a way that promotes life extension.

RESUMEN

BACKGROUND: Long noncoding RNAs (lncRNAs) form an abundant class of transcripts, but the function of the majority of them remains elusive. While it has been shown that some lncRNAs are bound by ribosomes, it has also been convincingly demonstrated that these transcripts do not code for proteins. To obtain a comprehensive understanding of the extent to which lncRNAs bind ribosomes, we performed systematic RNA sequencing on ribosome-associated RNA pools obtained through ribosomal fractionation and compared the RNA content with nuclear and (non-ribosome bound) cytosolic RNA pools. RESULTS: The RNA composition of the subcellular fractions differs significantly from each other, but lncRNAs are found in all locations. A subset of specific lncRNAs is enriched in the nucleus but surprisingly the majority is enriched in the cytosol and in ribosomal fractions. The ribosomal enriched lncRNAs include H19 and TUG1. CONCLUSIONS: Most studies on lncRNAs have focused on the regulatory function of these transcripts in the nucleus. We demonstrate that only a minority of all lncRNAs are nuclear enriched. Our findings suggest that many lncRNAs may have a function in cytoplasmic processes, and in particular in ribosome complexes.

Asunto(s)

Citosol/química , ARN Largo no Codificante/genética , Ribosomas/genética , Línea Celular Tumoral , Núcleo Celular/química , Núcleo Celular/genética , Biblioteca de Genes , Humanos , ARN Largo no Codificante/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ribosomas/metabolismo , Análisis de Secuencia de ARN

RESUMEN

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered in RPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests that RP mutations affect the synthesis of specific proteins involved in regulating amino acid levels that are important for maintaining the normal proliferative capacity of hematopoietic cells.

Asunto(s)

Anemia de Diamond-Blackfan/metabolismo , Haploinsuficiencia , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo , Transaminasas/biosíntesis , Transcripción Genética , Regiones no Traducidas 5'/genética , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patología , Humanos , Polirribosomas/genética , Polirribosomas/metabolismo , Proteínas Ribosómicas/genética , Transaminasas/genética

RESUMEN

Nucleostemin (NS) is an essential protein for the growth and viability of developmental stem cells. Its functions are multi-faceted, including important roles in ribosome biogenesis and in the p53-induced apoptosis pathway. While NS has been well studied, the functions of its family members GNL2 and GNL3-like (GNL3L) remain relatively obscure despite a high degree of sequence and domain homology. Here, we use zebrafish lines carrying mutations in the ns family to compare and contrast their functions in vertebrates. We find the loss of zebrafish ns or gnl2 has a major impact on 60S large ribosomal subunit formation and/or function due to cleavage impairments at distinct sites of pre-rRNA transcript. In both cases this leads to a reduction of total protein synthesis. In contrast, gnl3l loss shows relatively minor rRNA processing delays that ultimately have no appreciable effects on ribosome biogenesis or protein synthesis. However, the loss of gnl3l still results in p53 stabilization, apoptosis, and lethality similarly to ns and gnl2 loss. The depletion of p53 in all three of the mutants led to partial rescues of the morphological phenotypes and surprisingly, a rescue of the 60S subunit collapse in the ns mutants. We show that this rescue is due to an unexpected effect of p53 loss that even in wild type embryos results in an increase of 60S subunits. Our study presents an in-depth description of the mechanisms through which ns and gnl2 function in vertebrate ribosome biogenesis and shows that despite the high degree of sequence and domain homology, gnl3l has critical functions in development that are unrelated to the ribosome.

Asunto(s)

Proteínas Nucleares/fisiología , Ribosomas/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Genes Letales , Genes p53 , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Pez Cebra/embriología

RESUMEN

Mutations in the daf-2 gene of the conserved Insulin/Insulin-like Growth Factor (IGF-1) pathway double the lifespan of the nematode Caenorhabditis elegans. This phenotype is completely suppressed by deletion of Forkhead transcription factor daf-16. To uncover regulatory mechanisms coordinating this extension of life, we employed a quantitative proteomics strategy with daf-2 mutants in comparison with N2 and daf-16; daf-2 double mutants. This revealed a remarkable longevity-specific decrease in proteins involved in mRNA processing and transport, the translational machinery, and protein metabolism. Correspondingly, the daf-2 mutants display lower amounts of mRNA and 20S proteasome activity, despite maintaining total protein levels equal to that observed in wild types. Polyribosome profiling in the daf-2 and daf-16;daf-2 double mutants confirmed a daf-16-dependent reduction in overall translation, a phenotype reminiscent of Dietary Restriction-mediated longevity, which was independent of germline activity. RNA interference (RNAi)-mediated knockdown of proteins identified by our approach resulted in modified C. elegans lifespan confirming the importance of these processes in Insulin/IGF-1-mediated longevity. Together, the results demonstrate a role for the metabolism of proteins in the Insulin/IGF-1-mediated extension of life.

Asunto(s)

Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Factor I del Crecimiento Similar a la Insulina/genética , Insulina/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Receptor de Insulina/genética , Factores de Transcripción/genética , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/metabolismo , Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Genotipo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Longevidad/genética , Mutación , Fenotipo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Receptor de Insulina/antagonistas & inhibidores , Receptor de Insulina/metabolismo , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo

RESUMEN

Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells such as stem and cancer cells and is involved in the control of cell cycle progression. Both depletion and overexpression of NS result in stabilization of the tumor suppressor p53 protein in vitro. Although it has been previously suggested that NS has p53-independent functions, these to date remain unknown. Here, we report two zebrafish mutants recovered from forward and reverse genetic screens that carry loss of function mutations in two members of this nucleolar protein family, Guanine nucleotide binding-protein-like 2 (Gnl2) and Gnl3/NS. We demonstrate that these proteins are required for correct timing of cell cycle exit and subsequent neural differentiation in the brain and retina. Concomitantly, we observe aberrant expression of the cell cycle regulators cyclinD1 and p57kip2. Our models demonstrate that the loss of Gnl2 or NS induces p53 stabilization and p53-mediated apoptosis. However, the retinal differentiation defects are independent of p53 activation. Furthermore, this work demonstrates that Gnl2 and NS have both non-cell autonomously and cell-autonomous function in correct timing of cell cycle exit and neural differentiation. Finally, the data suggest that Gnl2 and NS affect cell cycle exit of neural progenitors by regulating the expression of cell cycle regulators independently of p53.

Asunto(s)

Ciclo Celular/fisiología , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica/fisiología , Neurogénesis/fisiología , Proteínas Nucleares/metabolismo , Retina/embriología , Pez Cebra/embriología , Animales , Western Blotting , Bromodesoxiuridina , Ciclina D1/metabolismo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al GTP/genética , Inmunohistoquímica , Hibridación in Situ , Análisis por Micromatrices , Microscopía Fluorescente , Mutación/genética , Proteínas Nucleares/genética , Oligonucleótidos/genética , Plásmidos/genética

RESUMEN

Nitric oxide (NO) is known to depress ribosome biogenesis in vitro. In this study we analyzed the influence of exogenous NO on ribosome biogenesis in vivo using a proven antihypertensive model of perinatal NO administration in genetically hypertensive rats. Fawn-hooded hypertensive rat (FHH) dams were supplied with the NO-donor molsidomine in drinking water from 2 weeks before to 4 weeks after birth, and the kidneys were subsequently collected from 2 day, 2 week, and 9 to 10-month-old adult offspring. Although the NO-donor increased maternal NO metabolite excretion, the NO status of juvenile renal (and liver) tissue was unchanged as assayed by EPR spectroscopy of NO trapped with iron-dithiocarbamate complexes. Nevertheless, microarray analysis revealed marked differential up-regulation of renal ribosomal protein genes at 2 days and down-regulation at 2 weeks and in adult males. Such differential regulation of renal ribosomal protein genes was not observed in females. These changes were confirmed in males at 2 weeks by expression analysis of renal ribosomal protein L36a and by polysome profiling, which also revealed a down-regulation of ribosomes in females at that age. However, renal polysome profiles returned to normal in adults after early exposure to molsidomine. No direct effects of molsidomine were observed on cellular proliferation in kidneys at any age, and the changes induced by molsidomine in renal polysome profiles at 2 weeks were absent in the livers of the same rats. Our results suggest that the previously found prolonged antihypertensive effects of perinatal NO administration may be due to epigenetically programmed alterations in renal ribosome biogenesis during a critical fetal period of renal development, and provide a salient example of a drug-induced reduction of ribosome biogenesis that is accompanied by a beneficial long-term health effect in both males and females.

RESUMEN

PURPOSE: Urothelium has 2 main functions. It is a barrier to urine and has a sensory role. In response to stretch urothelium releases various substances that modulate afferent nerve activity. Recent data on the localization of cyclooxygenase type 1, the enzyme responsible for prostaglandin production, suggests that prostaglandin may have complex local action. MATERIALS AND METHODS: The bladders of 7 guinea pigs were stained for prostaglandin receptors type 1 and 2, and costained for vimentin and cyclooxygenase I. RESULTS: Prostaglandin receptor type 1 staining was seen in urothelial cells and in the suburothelium. Urothelial staining, which was often punctuate and weak, was detected in all urothelial cell layers, including suburothelial cells. In contrast, strong prostaglandin receptor type 2 staining was seen in the urothelium and in suburothelial cells. Cyclooxygenase I was absent in interstitial cells and umbrella cells with the highest concentration in the basal cell layer. CONCLUSIONS: Interstitial cells express prostaglandin receptor types 1 and 2, indicating that they can respond to prostaglandin. Umbrella cells do not express cyclooxygenase I. Cyclooxygenase I was present in basal urothelial cells, making them a possible site of prostaglandin synthesis. Thus, prostaglandin produced by urothelium may target prostaglandin receptor types 1 and 2 in the urothelium and suburothelium. Therefore prostaglandin is hypothesized to have a role in signal regulation in the bladder wall.

Asunto(s)

Receptores de Prostaglandina E/análisis , Vejiga Urinaria/química , Animales , Cobayas , Masculino , Membrana Mucosa/química , Subtipo EP1 de Receptores de Prostaglandina E , Subtipo EP2 de Receptores de Prostaglandina E , Urotelio/química

RESUMEN

OBJECTIVE: To examine the expression of ubiquitin hydrolase (UH), an enzyme which is part of the ubiquitin-proteasome system involved in the regulation of cell growth and differentiation, to gain an insight into the cell types and processes underlying the tissue remodelling that occur after bladder neck damage. MATERIALS AND METHODS: Three groups of male guinea pigs were used, comprising controls (not operated, four), sham (five) and obstructed (six). The bladder outlet was obstructed by implanting a silver ring around the urethra, which was left in situ for 2-4 weeks. Sham-operated guinea pigs had the same operative procedure but no ring was implanted. The bladders were removed and incubated in Krebs' solution at 36 degrees C, gassed with 95% O2 and 5% CO2, Tissues were then fixed in 4% depolymerized paraformaldehyde and processed for immunohistochemistry. We used antibodies raised against UH, cyclooxygenase type I and vimentin. Specific antibody binding was visualized using the appropriate secondary antibodies. RESULTS: Staining with an antibody to UH showed the presence of both sensory and motor nerves in control, sham and obstructed bladders. In the control bladders this was the predominant staining pattern. In the sham and obstructed bladders UH staining revealed additional positive cell types; cells associated with the outermost layers of the urothelium (the umbrella cells), in the lamina propria (the lamina propria interstitial cells (lp-ICs), on the surface of the muscle bundles (surface muscle, sm-ICs) and on the serosal surface (muscle coat, mc-ICs). All ICs stained with vimentin. The ICs within the muscle bundles (intramuscular, im-ICs) did not stain with UH. The number and density of the UH-positive cells was greater in the obstructed than in the sham bladders, suggesting a change in relation to the severity of damage to the bladder neck. CONCLUSION: The expression of UH implies the re-targeting of proteins marked for degradation in the proteasome. Increased expression of UH in the lp-ICs and sm-ICs shows that these cells are active in the early and late stages of the tissue remodelling resulting from obstruction. These results show a further subset of ICs that might be involved in the increased deposition of extracellular material and tissue remodelling.

Asunto(s)

Ubiquitina Tiolesterasa/metabolismo , Obstrucción del Cuello de la Vejiga Urinaria/enzimología , Vejiga Urinaria/enzimología , Animales , Diferenciación Celular , Ciclooxigenasa 1/metabolismo , Cobayas , Inmunohistoquímica , Masculino , Regulación hacia Arriba , Obstrucción del Cuello de la Vejiga Urinaria/patología , Urotelio/enzimología

RESUMEN

OBJECTIVE: To identify the cells expressing the M(3) muscarinic receptor subtype in the lamina propria of the bladder. MATERIALS AND METHODS: The bladders from five normal guinea pigs were isolated and fixed in 4% paraformaldehyde. Tissues sections (10 microm) were then cut and stained with antibodies to the type 3 muscarinic receptor (M(3)), the interstitial cell marker vimentin and the nonspecific nerve marker PGP 9.5. The specificity of the antibody to the M(3) receptor was established using the complementary blocking peptide and Western blot analysis of human embryonic kidney (HEK) cells transfected to express the M(3) receptor protein. RESULTS The M(3) antibody pre-incubated with its blocking peptide showed no immunohistochemical staining. Investigating this antibody using HEK cells transfected to express the M(3) receptor protein and control HEK cells showed a single band in the transfected cells and no band in the control cells. M(3) receptor immunoreactivity (M(3)-IR) was detected primarily on a dense network of vimentin-positive (vim(+)) cells lying immediately below the urothelium, i.e. the suburothelial interstitial cells (Su-ICs). The M(3)-IR was punctate and appeared to be located on the cell surface. The diffuse network of cells in the remaining regions of the lamina propria showed no M(3)-IR. Few nerve fibres were associated with the M(3)-IR Su-ICs. The M(3)-IR Su-ICs were most numerous and prominent in the lateral wall. The number of M(3)-IR/vim(+) cells diminished towards the bladder base and were absent in the bladder urethral junction. In the base and urethral junction there were vim(+) cells that were not M(3)-IR. A population of umbrella cells in the lateral wall also showed weak punctate M(3)-IR. CONCLUSIONS Using a well-characterized M(3) antibody, these results show for the first time that the M(3) muscarinic receptor in the lamina propria is located specifically on the Su-ICs. The physiological role of these cells is unknown and consequently the significance of what appears to be a cholinergic signalling system is unclear. Previously published data showed that these cells respond to nitric oxide and atrial natriuretic peptide with an increase in cGMP and possibly prostaglandin. All of these observations, taken together, suggest that the Su-ICs receive multiple inputs and that they must be part of a complex signalling system in this region of the bladder wall.

Asunto(s)

Receptor Muscarínico M3/metabolismo , Vejiga Urinaria/metabolismo , Animales , Biomarcadores/metabolismo , Western Blotting , Comunicación Celular/fisiología , Cobayas , Inmunohistoquímica , Masculino , Transducción de Señal/fisiología , Ubiquitina Tiolesterasa/metabolismo , Obstrucción del Cuello de la Vejiga Urinaria/diagnóstico , Urotelio/metabolismo , Vimentina/metabolismo