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Background The process of technology validation and transfer of new molecular diagnostic tests towards the clinic faces challenges and needs to be improved. There is no empirical measure of the chronology and pace of technology transfer of molecular genetic discoveries. Methods We studied these for 29 molecular genetic test discoveries in order to (1) provide estimates of the timeframe between discovery of a clinical application and complete clinical implementation, and (2) compare the trajectories between different new tests to identify common patterns. We identified 11 publicly available "timestamps" for the technology transfer process ranging from discovery of the marker to use in a clinical setting. For each test selected, we searched public databases to identify available timestamps and dates. We plotted and compared trajectories of individual tests, including chronology. Results We show that there is much variability in the chronology of transfer between biomarkers. The median time between discovery of the marker and availability of the clinical test was 9.5 years (minimum 1). There was a median time of 18 years between test discovery and FDA approval (minimum 7 years), and it took a median of 17 years between discovery and the availability of a certified reference material for the 10 assays that have one (minimum 9 years). Conclusions We conclude that new molecular genetic tests take significant time between discovery and clinical implementation, and that further work is needed to pinpoint key factors, including policy and organization factors, that may allow for improving and streamlining this process.
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Biomarcadores de Tumor/genética , Técnicas de Diagnóstico Molecular , Bases de Datos Genéticas , HumanosRESUMEN
Approximately 75 % of extraskeletal myxoid chondrosarcoma tumors (EMC) harbor a t(9;22) chromosome translocation generating an EWS/NR4A3 fusion protein that is thought to be instrumental in the tumoral process. Current evidence suggests that one function of the fusion protein is to overexpress target genes. We have generated an in vitro human cellular model in which the fusion protein is expressed in mesenchymal bone marrow stem cells. We have performed microarray analyses of these cells and identified several genes overexpressed in the presence of EWS/NR4A3 which are also overexpressed in EMC tumors. These genes and their products represent potential therapeutic targets for EMC tumors.
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Condrosarcoma/genética , Proteínas de Unión al ADN/genética , Proteínas de Fusión Oncogénica/genética , Proteína EWS de Unión a ARN/genética , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Transactivadores/genética , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , HumanosRESUMEN
BACKGROUND: The completion of the Human Genome Project has increased the pace of discovery of genetic markers for disease. Despite tremendous efforts in fundamental research, clinical applications still lag behind expectations, partly due to the lack of effective tools to systematically search for and summarize published data relative to the clinical assessment of new diagnostic molecular tests. METHODS: Through a collaborative process using published tools and an expert panel, we developed a detailed checklist of the evidence that needs to be collected or produced to evaluate the potential usefulness of a new molecular diagnostic test. This tool is called GETT, for Genetic testing Evidence Tracking Tool. RESULTS: GETT allows 1) researchers to summarize the current evidence and to identify knowledge gaps for further research and; 2) stakeholders to collect data related to a given molecular test and improve their decision-making process. GETT comprises 72 clearly defined items/questions, grouped into 10 categories and 26 sub-themes, including an overview of disease epidemiology and genetics, the available diagnostic tools, and their analytical and clinical performances, availability of quality control programs, laboratory and clinical best practice guidelines, clinical utility, and impact on health care and psycho-social, ethical and legal implications. It also includes a summary of the evidence available and attempts to prioritise knowledge gaps related to the testing. We also compare GETT to other existing frameworks. CONCLUSIONS: This systematic evidence-based tracking tool, which is more detailed than existing frameworks and provides clear definition for each item, will help streamline collection of the available evidence to appraise the potential for clinical application of new molecular diagnostic tests and prioritize research to produce the evidence-base relative to the clinical implementation of molecular diagnostic tests.
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Pruebas Genéticas/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Laboratorio Clínico , Técnicas de Apoyo para la Decisión , HumanosRESUMEN
We compared clinical validity of two non-invasive prenatal screening (NIPS) methods for fetal trisomies 13, 18, 21, and monosomy X. We recruited prospectively 2203 women at high risk of fetal aneuploidy and 1807 at baseline risk. Three-hundred and twenty-nine euploid samples were randomly removed. The remaining 1933 high risk and 1660 baseline-risk plasma aliquots were assigned randomly between four laboratories and tested with two index NIPS tests, blind to maternal variables and pregnancy outcomes. The two index tests used massively parallel shotgun sequencing (semiconductor-based and optical-based). The reference standard for all fetuses was invasive cytogenetic analysis or clinical examination at birth and postnatal follow-up. For each chromosome of interest, chromosomal ratios were calculated (number of reads for chromosome/total number of reads). Euploid samples' mean chromosomal ratio coefficients of variation were 0.48 (T21), 0.34 (T18), and 0.31 (T13). According to the reference standard, there were 155 cases of T21, 49 T18, 8 T13 and 22 45,X. Using a fetal fraction ≥4% to call results and a chromosomal ratio z-score of ≥3 to report a positive result, detection rates (DR), and false positive rates (FPR) were not statistically different between platforms: mean DR 99% (T21), 100%(T18, T13); 79%(45,X); FPR < 0.3% for T21, T18, T13, and <0.6% for 45,X. Both methods' negative predictive values in high-risk pregnancies were >99.8%, except for 45,X(>99.6%). Threshold analysis in high-risk pregnancies with different fetal fractions and z-score cut-offs suggested that a z-score cutoff to 3.5 for positive results improved test accuracy. Both sequencing platforms showed equivalent and excellent clinical validity.
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Aneuploidia , Ácidos Nucleicos Libres de Células , Feto , Ensayos Analíticos de Alto Rendimiento/métodos , Factor de Transcripción Ikaros/genética , Adolescente , Adulto , Síndrome de Down , Femenino , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular , Embarazo , Síndrome de la Trisomía 13 , Síndrome de la Trisomía 18 , Síndrome de Turner , Adulto JovenRESUMEN
Fragile X-related 1 protein (FXR1P) is a member of a small family of RNA-binding proteins that includes the Fragile X mental retardation 1 protein (FMR1P) and the Fragile X-related 2 protein (FXR2P). These proteins are thought to transport mRNA and to control their translation. While FMR1P is highly expressed in neurons, substantial levels of FXR1P are found in striated muscles and heart, which are devoid of FMRP and FXR2P. However, little is known about the functions of FXR1P. We have isolated cDNAs for Xenopus Fxr1 and found that two specific splice variants are conserved in evolution. Knockdown of xFxr1p in Xenopus had highly muscle-specific effects, normal MyoD expression being disrupted, somitic myotomal cell rotation and segmentation being inhibited, and dermatome formation being abnormal. Consistent with the absence of the long muscle-specific xFxr1p isoform during early somite formation, these effects could be rescued by both the long and short mRNA variants. Microarray analyses showed that xFxr1p depletion affected the expression of 129 known genes of which 50% were implicated in muscle and nervous system formation. These studies shed significant new light on Fxr1p function(s).
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/fisiología , Proteínas de Unión al ARN/fisiología , Somitos/metabolismo , Proteínas de Xenopus/fisiología , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Blastómeros/metabolismo , Fase de Segmentación del Huevo/metabolismo , Embrión no Mamífero/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Variación Genética , Humanos , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/embriología , Proteína MioD/biosíntesis , Proteína MioD/genética , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Somitos/fisiología , Proteínas de Xenopus/metabolismoRESUMEN
Nuclear receptors represent a large family of transcription factors involved in development, differentiation, homeostasis, and cancer. In recent years, a growing number of cofactors has been discovered that participate in the regulation of the transcriptional activity of these proteins. We present in this study the identification of a cofactor, the homeotic protein Six3, which differentially regulates the transcriptional activity of the orphan nuclear receptor NOR-1 (NR4A3). NOR-1 is normally involved in the balance between cell proliferation and cell death, and is implicated in oncogenesis as part of the EWS/NOR-1 fusion protein found in human extraskeletal myxoid chondrosarcoma (EMC) tumors. Reverse transcription-PCR analyses indicate that EMC tumors expressing the EWS/NOR-1 mRNA also express mRNAs encoding NOR-1 and Six3. Glutathione S-transferase fusion protein assays show that Six3 binds in vitro the DNA-binding domain of NOR-1 and the EWS domain of EWS/NOR-1 and that the homeodomain of Six3 is required for these interactions. Mammalian two-hybrid experiments, using immortalized human chondrocytes as a model, indicate that Six3 also interacts with NOR-1 and EWS/NOR-1 in vivo. Cotransfection experiments show that Six3 stimulates the transcriptional activity of NOR-1, whereas it represses that of EWS/NOR-1. Considering the highly specific expression pattern of Six3, our finding that it is expressed in EMC suggests that it plays a pivotal role in the development of these tumors. We propose that Six3 maintains a transcriptional balance between the activities of NOR-1 and EWS/NOR-1, the net effect being to deregulate the expression of specific target genes and push the equilibrium toward uncontrolled cell proliferation.
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Condrosarcoma/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Neoplasias de los Tejidos Blandos/metabolismo , Animales , Condrosarcoma/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas del Ojo , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Desnudos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteína EWS de Unión a ARN/biosíntesis , Proteína EWS de Unión a ARN/genética , Receptores de Esteroides , Receptores de Hormona Tiroidea , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de los Tejidos Blandos/genética , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , Proteína Homeobox SIX3RESUMEN
In approximately 70% of human extraskeletal myxoid chondrosarcoma (EMC) tumors, a t(9;22) chromosome translocation gives rise to a fusion protein, named EWS/NOR1, containing the amino-terminal domain of EWS fused to the complete amino acid sequence of the nuclear receptor NOR1. Several observations suggest that one role of EWS/NOR1 in EMC may be to deregulate the expression of specific genes involved in the tumoral process. In order to identify these genes, we have used a CFK2 chondrogenic cell line over-expressing EWS/NOR1. A differential display analysis has identified the PLAGL1 gene as being down-regulated in the CFK2(EWS/NOR1) cell line compared to native CFK2 cells. RT-PCR analyses show that whereas the PLAGL1 mRNAs encoding the two isoforms of the protein are highly expressed in four human chondrocyte immortalized cell lines and two human chondrocyte primary cultures, they are strongly down-regulated in six EMC tumors. We conclude that down-regulation of PLAGL1 may be a significant contributing factor in the development of EMC tumors.
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Proteínas de Ciclo Celular/metabolismo , Condrosarcoma/metabolismo , Genes Supresores de Tumor/fisiología , Proteína EWS de Unión a ARN/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Ciclo Celular/genética , Condrosarcoma/genética , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Proteínas de Transporte de Membrana , Neoplasias Nasofaríngeas , Isoformas de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína EWS de Unión a ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
Holoprosencephaly (HPE) is a relatively common brain malformation resulting in an incomplete separation of the two cerebral hemispheres. A number of mutations in different genes have been linked to this malformation, including three missense mutations in the homeodomain of the transcription factor SIX3. In this study, we investigated the functional consequences of these SIX3 mutations with respect to the ability of the protein to interact with and stimulate the transcriptional activity of the nuclear receptor NOR1 (NR4A3). Using glutathione S-transferase fusion protein pull-down assays and transient cotransfections of Neuro-2a cells with expression and reporter vectors, we found that one mutation, c.676C>G (p.L226V), does not alter the properties of SIX3 toward NOR1. Another mutation, c.749T>C (p.V250A), results in the production of a highly unstable protein in Neuro-2a cells. The third mutation, c.770G>C (p.R257P), results in a mutant SIX3 protein that no longer interacts with NOR1 in vivo. These observations suggest that different SIX3 mutations in HPE2 may affect different signaling pathways, and that one of these pathways may involve the nuclear receptor NOR1.
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Proteínas de Unión al ADN/metabolismo , Proteínas del Ojo/genética , Holoprosencefalia/genética , Proteínas de Homeodominio/genética , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Línea Celular , Proteínas del Ojo/metabolismo , Holoprosencefalia/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Activación Transcripcional , Proteína Homeobox SIX3RESUMEN
The EWS/TEC fusion protein encoded by the t(9:22) chromosomal translocation in human extraskeletal myxoid chondrosarcoma tumors is thought to participate in the tumoral process at least in part by deregulating the expression of specific target genes involved in the control of cell proliferation. In this work we show that the activation function-2 (AF2) domain of TEC is essential for the transcriptional activity of the EWS/TEC fusion protein. Significantly, deleting only the last 15 amino acids of the fusion protein, which contains 949 amino acids in its full form, results in a loss of over 70% of its transcriptional activity in transfected human chondrocyte cell lines. Point mutation analyses indicate that within the AF2 domain, amino acid residues I939, D940 and F943 all play a crucial role in the activity of EWS/TEC. Comparable results were obtained with the native TEC receptor. These results suggest that EWS/TEC interacts at least in part with the same transcriptional coactivators as the native TEC receptor, and that these coactivators may be involved in the tumoral process leading to human chondrosarcoma tumors.
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Proteínas de Unión al ADN/genética , Proteínas del Tejido Nervioso , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/genética , Transcripción Genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Neoplasias Óseas/genética , Condrosarcoma/genética , Cromosomas Humanos Par 22 , Cromosomas Humanos Par 9 , Clonación Molecular , Proteínas de Unión al ADN/química , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteína EWS de Unión a ARN , Proteínas de Unión al ARN/química , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides , Receptores de Hormona Tiroidea , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Translocación GenéticaRESUMEN
The fragile X mental retardation (FXMR) syndrome is one of the most frequent causes of mental retardation. Affected individuals display a wide range of additional characteristic features including behavioural and physical phenotypes, and the extent to which individuals are affected is highly variable. For these reasons, elucidation of the pathophysiology of this disease has been an important challenge to the scientific community. 1991 marks the year of the discovery of both the FMR1 gene mutations involved in this disease, and of their dynamic nature. Although a mouse model for the disease has been available for 16 years and extensive research has been performed on the FMR1 protein (FMRP), we still understand little about how the disease develops, and no treatment has yet been shown to be effective. In this review, we summarise current knowledge on FXMR with an emphasis on the technical challenges of molecular diagnostics, on its prevalence and dynamics among populations, and on the potential of screening for FMR1 mutations.
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BACKGROUND: p21(WAF1/CIP1) is a well known cyclin-dependent kinase inhibitor induced by various stress stimuli. Depending on the stress applied, p21 upregulation can either promote apoptosis or prevent against apoptotic injury. The stress-mediated induction of p21 involves not only its transcriptional activation but also its posttranscriptional regulation, mainly through stabilization of p21 mRNA levels. We have previously reported that the proteasome inhibitor MG132 induces the stabilization of p21 mRNA, which correlates with the formation of cytoplasmic RNA stress granules. The mechanism underlying p21 mRNA stabilization, however, remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We identified the stress granules component CUGBP1 as a factor required for p21 mRNA stabilization following treatment with bortezomib (â=â PS-341/Velcade). This peptide boronate inhibitor of the 26S proteasome is very efficient for the treatment of myelomas and other hematological tumors. However, solid tumors are sometimes refractory to bortezomib treatment. We found that depleting CUGBP1 in cancer cells prevents bortezomib-mediated p21 upregulation. FISH experiments combined to mRNA stability assays show that this effect is largely due to a mistargeting of p21 mRNA in stress granules leading to its degradation. Altering the expression of p21 itself, either by depleting CUGBP1 or p21, promotes bortezomib-mediated apoptosis. CONCLUSIONS/SIGNIFICANCE: We propose that one key mechanism by which apoptosis is inhibited upon treatment with chemotherapeutic drugs might involve upregulation of the p21 protein through CUGBP1.
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Apoptosis/efectos de los fármacos , Ácidos Borónicos/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Gránulos Citoplasmáticos/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Pirazinas/farmacología , Proteínas de Unión al ARN/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Bortezomib , Proteínas CELF1 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Gránulos Citoplasmáticos/efectos de los fármacos , Células HeLa , Humanos , Unión Proteica/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Fisiológico/efectos de los fármacosAsunto(s)
Cuerpo Carotídeo/crecimiento & desarrollo , Cuerpo Carotídeo/metabolismo , Hipoxia/genética , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/genética , Animales , Secuencia de Bases , Cartilla de ADN/genética , Regulación del Desarrollo de la Expresión Génica , Hipoxia/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Factores de TiempoRESUMEN
OBJECTIVE: To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. DESIGN: Prospective study. SETTING: Human reproduction research laboratory. PATIENT(S): Nine women with endometriotic lesions. INTERVENTION(S): Endometriotic lesions were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. RESULT(S): This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. CONCLUSION(S): This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.
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Endometriosis/genética , Interleucina-1beta/farmacología , Factores Inhibidores de la Migración de Macrófagos/genética , FN-kappa B/fisiología , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Técnicas de Cultivo de Célula , Endometriosis/patología , Femenino , Humanos , Embarazo , Embarazo Ectópico/genética , ARN Mensajero/genéticaRESUMEN
Phosphorylation is a very common post-translational modification event known to modulate a wide range of biological responses. Beyond the regulation of protein activity, the interrelation of phosphorylation with other post-translational mechanisms is responsible for the control of diverse signaling pathways. Several observations suggest that phosphorylation of poly(ADP-ribose) polymerase-1 (PARP-1) regulates its activity. There is also accumulating evidence to suggest the establishment of phosphorylation-dependent assembly of PARP-1-associated multiprotein complexes. Although it is relatively straightforward to demonstrate phosphorylation of a defined target, identification of the actual amino acids involved still represents a technical challenge for many laboratories. With the use of a combination of bioinformatics-based predictions tools for generic and kinase-specific phosphorylation sites, in vitro phosphorylation assays and mass spectrometry analysis, we investigated the phosphorylation profile of PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), two major enzymes responsible for poly(ADP-ribose) turnover. Mass spectrometry analysis revealed the phosphorylation of several serine/threonine residues within important regulatory domains and motifs of both enzymes. With the use of in vivo microirradiation-induced DNA damage, we show that altered phosphorylation at specific sites can modify the dynamics of assembly and disassembly of PARP-1 at sites of DNA damage. By documenting and annotating a collection of known and newly identified phosphorylation sites, this targeted proteomics study significantly advances our understanding of the roles of phosphorylation in the regulation of PARP-1 and PARG.
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Glicósido Hidrolasas/química , Poli(ADP-Ribosa) Polimerasas/química , Secuencia de Aminoácidos , Animales , Daño del ADN , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Humanos , Espectrometría de Masas , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Datos de Secuencia Molecular , Fosfopéptidos/química , Fosfopéptidos/genética , Fosfopéptidos/metabolismo , Fosforilación , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Transducción de SeñalRESUMEN
Poly(ADP-ribose) polymerase-1 (PARP-1) hyper-activation promotes cell death but the signaling events downstream of PARP-1 activation are not fully identified. To gain further information on the implication of PARP-1 activation and PAR synthesis on signaling pathways influencing cell death, we exposed HeLa cells to the DNA alkylating agent N-methyl-N'-methyl-nitro-N-nitrosoguanidine (MNNG). We found that massive PAR synthesis leads to down-regulation of ERK1/2 phosphorylation, Bax translocation to the mitochondria, release of cytochrome c and AIF and subsequently cell death. Inhibition of massive PAR synthesis following MNNG exposure with the PARP inhibitor PJ34 prevented those events leading to cell survival, whereas inhibition of ERK1/2 phosphorylation by inhibiting MEK counteracted the cytoprotective effect of PJ34. Together, our results provide evidence that PARP-1-induced cell death by MNNG exposure in HeLa cells is mediated in part through inhibition of the MEK/ERK signaling pathway and that inhibition of massive PAR synthesis by PJ34, which promotes sustained activation of ERK1/2, leads to cytoprotection.
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Apoptosis/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metilnitronitrosoguanidina/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasas/fisiología , Neoplasias del Cuello Uterino/enzimología , Neoplasias del Cuello Uterino/patología , Apoptosis/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Femenino , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Poli(ADP-Ribosa) Polimerasa-1RESUMEN
The NR4A3 nuclear receptor (also known as NOR1) is involved in tumorigenesis by the t(9;22) chromosome translocation encoding the EWS/NOR1 fusion protein found in approximately 75% of all cases of extraskeletal myxoid chondrosarcomas (EMC). Several observations suggest that one role of EWS/NOR1 in tumorigenesis may be to deregulate the expression of specific target genes. We have shown previously that constitutive expression of EWS/NOR1 in CFK2 fetal rat chondrogenic cells induces their transformation as measured by growth beyond confluency and growth in soft agar. To identify genes regulated by the fusion protein in this model, we have generated a CFK2 cell line in which the expression of EWS/NOR1 is controlled by tetracycline. Using the differential display technique, we have identified the serum- and glucocorticoid-regulated kinase 1 (SGK1) mRNA as being up-regulated in the presence of EWS/NOR1. Co-immunocytochemistry confirmed over-expression of the SGK1 protein in cells expressing EWS/NOR1. Significantly, immunohistochemistry of 10 EMC tumors positive for EWS/NOR1 showed that all of them over-express the SGK1 protein in contrast to non-neoplastic cells in the same biopsies and various other sarcoma types. These results strongly suggest that SGK1 may be a genuine in vivo target of EWS/NOR1 in EMC.
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Proteínas de Unión al ADN/fisiología , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas del Tejido Nervioso/fisiología , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteína EWS de Unión a ARN/fisiología , Animales , Neoplasias Óseas/fisiopatología , Línea Celular , Condrosarcoma/fisiopatología , Inducción Enzimática , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Proteínas Recombinantes de Fusión/farmacología , Regulación hacia ArribaRESUMEN
The EWS/NOR-1 fusion protein is encoded by the t(9;22) chromosomal translocation found in approximately 75% of extraskeletal myxoid chondrosarcoma (EMC) tumors. The lack of cellular models in which the oncogenic properties of this fusion protein are expressed has seriously hampered the study of its role in the development of EMC. To generate such a cellular model, we have used the chondrogenic cell line CFK2. We show in this study that although stable expression of EWS/NOR-1 does not alter the population doubling time and the cell cycle distribution of CFK2 cells in subconfluent cultures, it induces their transformation as measured by growth beyond confluency and anchorage-independent growth in soft agarose medium. Glycosaminoglycan accumulation in CFK2(EWS/NOR-1) cell lines indicate that the fusion protein does not appear to interfere with the ability of CFK2 cells to differentiate into chondrocyte-like cells in vitro. These results support the hypothesis that the role of EWS/NOR-1 in EMC may be to disrupt the proliferation properties of cells involved in chondrogenesis.
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Neoplasias Óseas/metabolismo , Condrocitos/citología , Proteínas de Unión al ADN/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Sarcoma de Ewing/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Neoplasias Óseas/genética , Diferenciación Celular , Línea Celular , Transformación Celular Neoplásica , Cromosomas Humanos Par 22 , Cromosomas Humanos Par 9 , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Glicosaminoglicanos/biosíntesis , Humanos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína , Ratas , Receptores de Esteroides , Receptores de Hormona Tiroidea , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma de Ewing/química , Sarcoma de Ewing/genética , Translocación GenéticaRESUMEN
PROBLEM: Tumor necrosis factor-alpha (TNF-alpha) is secreted mainly during the menstrual phase and has been suggested to play a role in induction of apoptosis in endometrial cells and menstrual shedding. TNF-alpha receptor type 2 (TNF-RII) is believed to play a central role in TNFalpha-mediated cytotoxic, mitogenic, anti-proliferative and apoptotic effects. The aim of this study was to assess whether TNF-RII maybe expressed differentially in the endometrium of women with different degrees of endometriosis. METHOD OF STUDY: TNF-RII expression in the endometrial tissue of women with and without endometriosis was investigated by immunohistochemical techniques and in situ hybridization. RESULTS: In histological sections, we observed TNF-RII mRNA and the corresponding protein localized mainly in endometrial glandular cells, with only very faint immunostaining in the surrounding stromal cells. Statistical analysis of our data showed a significant decrease in protein and mRNA expression of TNF-RII in endometrial glandular cells of patients with stages I and II endometriosis compared to normal subjects. TNF-RII expression was also found to decrease significantly in the secretory phase of the menstrual cycle in women with early endometriosis stages (I and II). CONCLUSIONS: In view of the relevant role of TNF-RII in the modulation of the inflammatory and the proapoptotic effects of TNFalpha, deficient expression of TNF-RII mRNA in the endometrium of women at the earliest stages of endometriosis may play a significant role in the pathophysiology of this disease.
Asunto(s)
Antígenos CD/metabolismo , Endometriosis/metabolismo , Endometrio/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Adulto , Antígenos CD/genética , Endometriosis/patología , Endometrio/química , Endometrio/patología , Femenino , Expresión Génica , Humanos , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral , Células U937RESUMEN
This study determined: (1) whether dopamine (DA) D2-receptor (R) and D1-R mRNA levels in the carotid body (CB), petrosal ganglion (PG) and superior cervical ganglion (SCG) are modulated by hypoxia; (2) the role of hypoxia intensity and exposure duration, and (3) whether the pattern of modulation differs between newborn (1-day-old) and adult rabbits. Rabbits were exposed to five FiO2 conditions: 21% (control); 15%/6 h; 15%/24 h; 8%/6 h and 8%/24 h. D2- and D1-R mRNA expression levels were calculated for each hypoxia condition relative to control using RT-PCR analysis. In the CB of 1-day-old rabbits, D2- and D1-R transcript levels increased and decreased after exposure to 15 and 8% O2, respectively. In the adult CB, D2- and D1-R transcript levels decreased independently of hypoxia intensities. Only changes in D1-R mRNA levels were dependent on exposure time. In the PG, both hypoxia intensities decreased the D2-R transcript levels in 1-day-old and adult rabbits; but for the D1-R mRNA levels, hypoxia decreased its level in 1-day-old rabbits and increased it in adults regardless of exposure duration. In the SCG, hypoxia had no determinant effect on the D2-R mRNA levels either in newborn or adult rabbits while it induced a decrease of D1-R transcript levels in 1-day-old rabbits and an increase in adult rabbits. It is concluded that hypoxia affects DA D2- or D1-R mRNA levels in the chemoreflex pathway in an age-dependent and site-specific manner.
Asunto(s)
Envejecimiento , Arterias/fisiopatología , Células Quimiorreceptoras/fisiopatología , Hipoxia/fisiopatología , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Reflejo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Cuerpo Carotídeo/metabolismo , Cuerpo Estriado/metabolismo , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Ganglios Sensoriales/metabolismo , Hipoxia/metabolismo , Oxígeno/administración & dosificación , ARN Mensajero/metabolismo , Conejos , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/genética , Ganglio Cervical Superior/metabolismoRESUMEN
Fragile X Mental Retardation protein (FMRP) is an RNA-binding protein that contains multiple domains with apparently differential affinity to mRNA and to the ribonucleotide homopolymer poly(G). Attempts have been made to map the RNA-binding sites along the protein sequence with a view to determining which of the KH1, KH2 and RGG domains are required to recognize and bind to RNA. While these studies have greatly contributed to the delineation of domains that bind homopolymers or mRNA in vitro, little is known concerning their implications in FMRP function(s) in vivo. To address this question, we have prepared a series of FMRP versions, in which each known in vitro functional domain has been individually deleted, leaving the rest of the protein intact. Constructs with deletions in the protein-protein interaction and RNA-binding as well as in the phosphorylation domains were expressed in STEK-KO cells lacking FMRP and their recruitment into polyribosomal mRNPs and their intra-cellular localization were determined. Our results indicate that the KH RNA-binding domains and the Protein-Protein Interacting domain are essential for FMRP to associate with polyribosomal mRNPs, while the RGG box and the phosphorylated domains are dispensable.