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Deubiquitinases (DUBs) are specialized proteases that remove ubiquitin from substrates or cleave within ubiquitin chains to regulate ubiquitylation and therefore play important roles in eukaryotic biology. Dysregulation of DUBs is implicated in several human diseases, highlighting the importance of DUB function. In addition, many pathogenic bacteria and viruses encode and deploy DUBs to manipulate host immune responses and establish infectious diseases in humans and animals. Hence, therapeutic targeting of DUBs is an increasingly explored area that requires an in-depth mechanistic understanding of human and pathogenic DUBs. In this review, we summarize the multiple layers of regulation that control autoinhibition, activation, and substrate specificity of DUBs. We discuss different strategies to inhibit DUBs and the progress in developing selective small-molecule DUB inhibitors. Finally, we propose a classification system of DUB inhibitors based on their mode of action.
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Tratamiento Farmacológico de COVID-19 , COVID-19 , Enzimas Desubicuitinizantes , Inhibidores Enzimáticos/uso terapéutico , SARS-CoV-2 , Ubiquitinación/efectos de los fármacos , COVID-19/enzimología , Enzimas Desubicuitinizantes/antagonistas & inhibidores , Enzimas Desubicuitinizantes/metabolismo , HumanosRESUMEN
Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2.
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Enzimas Desubicuitinizantes/metabolismo , Poliubiquitina/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía , Enzimas Desubicuitinizantes/genética , Activación Enzimática , Humanos , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Dispersión del Ángulo Pequeño , Relación Estructura-Actividad , Ubiquitina Tiolesterasa/genética , UbiquitinaciónRESUMEN
Aberrant expression of miR-145-5p has been observed in prostate cancer where is has been suggested to play a tumor suppressor role. In other cancers, miR-145-5p acts as an inhibitor of epithelial-to-mesenchymal transition (EMT), a key molecular process for tumor progression. However, the interaction between miR-145-5p and EMT remains to be elucidated in prostate cancer. In this paper the link between miR-145-5p and EMT in prostate cancer was investigated using a combination of in silico and in vitro analyses. miR-145-5p expression was significantly lower in prostate cancer cell lines compared to normal prostate cells. Bioinformatic analysis of The Cancer Genome Atlas prostate adenocarcinoma (TCGA PRAD) data showed significant downregulation of miR-145-5p in prostate cancer, correlating with disease progression. Functional enrichment analysis significantly associated miR-145-5p and its target genes with EMT. MYO6, an EMT-associated gene, was identified and validated as a novel target of miR-145-5p in prostate cancer cells. In vitro manipulation of miR-145-5p levels significantly altered cell proliferation, clonogenicity, migration and expression of EMT-associated markers. Additional TCGA PRAD analysis suggested miR-145-5p tumor expression may be useful predictor of disease recurrence. In summary, this is the first study to report that miR-145-5p may inhibit EMT by targeting MYO6 in prostate cancer cells. The findings suggest miR-145-5p could be a useful diagnostic and prognostic biomarker for prostate cancer.
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Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , MicroARNs , Cadenas Pesadas de Miosina , Neoplasias de la Próstata , Humanos , Masculino , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , MicroARNs/genética , MicroARNs/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismoRESUMEN
The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.
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Antivirales/química , Antivirales/farmacología , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/farmacología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Compuestos de Anilina/farmacología , Animales , Benzamidas/farmacología , Chlorocebus aethiops , Proteasas Similares a la Papaína de Coronavirus/genética , Proteasas Similares a la Papaína de Coronavirus/aislamiento & purificación , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Sinergismo Farmacológico , Pruebas de Enzimas , Flavinas/farmacología , Transferencia Resonante de Energía de Fluorescencia , Furanos/farmacología , Ensayos Analíticos de Alto Rendimiento , Concentración 50 Inhibidora , Naftalenos/farmacología , Fenantrenos/farmacología , Quinonas/farmacología , Reproducibilidad de los Resultados , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/crecimiento & desarrollo , Bibliotecas de Moléculas Pequeñas/química , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
With a plethora of molecularly targeted agents under investigation in cancer, a clear need exists to understand which pathways can be targeted simultaneously with multiple agents to elicit a maximal killing effect on the tumour. Combination therapy provides the most promise in difficult to treat cancers such as pancreatic. Ref-1 is a multifunctional protein with a role in redox signalling that activates transcription factors such as NF-κB, AP-1, HIF-1α and STAT3. Formerly, we have demonstrated that dual targeting of Ref-1 (redox factor-1) and STAT3 is synergistic and decreases cell viability in pancreatic cancer cells. Data presented here extensively expands upon this work and provides further insights into the relationship of STAT3 and Ref-1 in multiple cancer types. Using targeted small molecule inhibitors, Ref-1 redox signalling was blocked along with STAT3 activation, and tumour growth evaluated in the presence and absence of the relevant tumour microenvironment. Our study utilized qPCR, cytotoxicity and in vivo analysis of tumour and cancer-associated fibroblasts (CAF) response to determine the synergy of Ref-1 and STAT3 inhibitors. Overall, pancreatic tumours grown in the presence of CAFs were sensitized to the combination of STAT3 and Ref-1 inhibition in vivo. In vitro bladder and pancreatic cancer demonstrated the most synergistic responses. By disabling both of these important pathways, this combination therapy has the capacity to hinder crosstalk between the tumour and its microenvironment, leading to improved tumour response.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Factor de Transcripción STAT3/metabolismo , Animales , Benzofuranos/farmacología , Western Blotting , Línea Celular Tumoral , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Células HCT116 , Humanos , Inmunohistoquímica , Ratones , Naftoquinonas/farmacología , Nitrilos , Neoplasias Pancreáticas/genética , Pirazoles/farmacología , Pirimidinas , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/genética , Microambiente Tumoral/efectos de los fármacosRESUMEN
Primary open-angle glaucoma (POAG) is a progressive optic neuropathy with a complex, multifactorial aetiology. Raised intraocular pressure (IOP) is the most important clinically modifiable risk factor for POAG. All current pharmacological agents target aqueous humour dynamics to lower IOP. Newer therapeutic agents are required as some patients with POAG show a limited therapeutic response or develop ocular and systemic side effects to topical medication. Elevated IOP in POAG results from cellular and molecular changes in the trabecular meshwork driven by increased levels of transforming growth factor ß (TGFß) in the anterior segment of the eye. Understanding how TGFß affects both the structural and functional changes in the outflow pathway and IOP is required to develop new glaucoma therapies that target the molecular pathology in the trabecular meshwork. In this study, we evaluated the effects of TGF-ß1 and -ß2 treatment on miRNA expression in cultured human primary trabecular meshwork cells. Our findings are presented in terms of specific miRNAs (miRNA-centric), but given miRNAs work in networks to control cellular pathways and processes, a pathway-centric view of miRNA action is also reported. Evaluating TGFß-responsive miRNA expression in trabecular meshwork cells will further our understanding of the important pathways and changes involved in the pathogenesis of glaucoma and could lead to the development of miRNAs as new therapeutic modalities in glaucoma.
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MicroARNs , Malla Trabecular , Malla Trabecular/metabolismo , Malla Trabecular/efectos de los fármacos , Malla Trabecular/patología , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Glaucoma de Ángulo Abierto/genética , Glaucoma de Ángulo Abierto/metabolismo , Glaucoma de Ángulo Abierto/patología , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Presión Intraocular/efectos de los fármacosRESUMEN
Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding of branched Ub function in cell signaling has been stunted by the absence of accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins and devise approaches to probe K48-K63-branched Ub function. We establish a method to monitor cleavage of linkages within complex Ub chains and unveil ATXN3 and MINDY as debranching enzymes. We engineer a K48-K63 branch-specific nanobody and reveal the molecular basis of its specificity in crystal structures of nanobody-branched Ub chain complexes. Using this nanobody, we detect increased K48-K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition and after DNA damage. Together with our discovery that multiple VCP/p97-associated proteins bind to or debranch K48-K63-linked Ub, these results suggest a function for K48-K63-branched chains in VCP/p97-related processes.
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[This corrects the article DOI: 10.1371/journal.pone.0253364.].
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An altered expression of miR-143-3p has been previously reported in prostate cancer where it is purported to play a tumor suppressor role. Evidence from other cancers suggests miR-143-3p acts as an inhibitor of epithelial-to-mesenchymal transition (EMT), a key biological process required for metastasis. However, in prostate cancer the interaction between miR-143-3p and EMT-associated mechanisms remains unclear. Therefore, this paper investigated the link between miR-143-3p and EMT in prostate cancer using in vitro and in silico analyses. PCR detected that miR-143-3p expression was significantly decreased in prostate cancer cell lines compared to normal prostate cells. Bioinformatic analysis of The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA PRAD) data showed a significant downregulation of miR-143-3p in prostate cancer, correlating with pathological markers of advanced disease. Functional enrichment analysis confirmed the significant association of miR-143-3p and its target genes with EMT. The EMT-linked gene AKT1 was subsequently shown to be a novel target of miR-143-3p in prostate cancer cells. The in vitro manipulation of miR-143-3p levels significantly altered the cell proliferation, clonogenicity, migration and expression of EMT-associated markers. Further TCGA PRAD analysis suggested miR-143-3p tumor expression may be a useful predictor of disease recurrence. In summary, this is the first study to report that miR-143-3p overexpression in prostate cancer may inhibit EMT by targeting AKT1. The findings suggest miR-143-3p could be a useful diagnostic and prognostic biomarker for prostate cancer.
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Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a poor response to current treatment regimens. The multifunctional DNA repair-redox signaling protein Ref-1 has a redox signaling function that activates several transcriptional factors (TFs) including NF-κB (RelA), STAT3, AP-1. These have been implicated in signaling in PDAC and associated with cancer progression and therapy resistance. Numerous studies have shown a role for RelA in PDAC inflammatory responses and therapy resistance, little is known as to how these inflammatory responses are modulated through Ref-1 redox signaling pathways during pancreatic pathogenesis. RelA and STAT3 are two major targets of Ref-1 and are important in PDAC pathogenesis. To decipher the mechanistic role of RelA in response to Ref-1 inhibition, we used PDAC cells (KC3590) from a genetically engineered Kras G12D-driven mouse model that also is functionally deficient for RelA (Parent/Vector) or KC3590 cells with fully functional RelA added back (clone 13; C13). We demonstrated that RelA deficient cells are more resistant to Ref-1 redox inhibitors APX3330, APX2009, and APX2014, and their sensitivity is restored in the RelA proficient cells. Knockdown of STAT3 did not change cellular sensitivity to Ref-1 redox inhibitors in either cell type. Gene expression analysis demonstrated that Ref-1 inhibitors significantly decreased IL-8, FOSB, and c-Jun when functional RelA is present. We also demonstrated that PRDX1, a known Ref-1 redox modulator, contributes to Ref-1 inhibitor cellular response. Knockdown of PRDX1 when functional RelA is present resulted in dramatically increased PDAC killing in response to Ref-1 inhibitors. The enhanced cell killing was not due to increased intracellular ROS production. Although Ref-1 inhibition decreased the NADP/NADPH ratio in the cells, the addition of PRDX1 knockdown did not further this redox imbalance. This data suggests that the mechanism of cell killing following Ref-1 inhibition is at least partially mediated through RelA and not STAT3. Further imbalancing of the redox signaling through disruption of the PRDX1-Ref-1 interaction may have therapeutic implications. Our data further support a pivotal role of RelA in mediating Ref-1 redox signaling in PDAC cells with the Kras G12D genotype and provide novel therapeutic strategies to combat PDAC drug resistance.
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Apurinic/apyrimidinic (AP) endonuclease-reduction/oxidation factor 1 (APE1/Ref-1, also called APE1) is a multifunctional enzyme with crucial roles in DNA repair and reduction/oxidation (redox) signaling. APE1 was originally described as an endonuclease in the Base Excision Repair (BER) pathway. Further study revealed it to be a redox signaling hub regulating critical transcription factors (TFs). Although a significant amount of focus has been on the role of APE1 in cancer, recent findings support APE1 as a target in other indications, including ocular diseases [diabetic retinopathy (DR), diabetic macular edema (DME), and age-related macular degeneration (AMD)], inflammatory bowel disease (IBD) and others, where APE1 regulation of crucial TFs impacts important pathways in these diseases. The central responsibilities of APE1 in DNA repair and redox signaling make it an attractive therapeutic target for cancer and other diseases.
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Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Descubrimiento de Drogas , Humanos , Terapia Molecular Dirigida/métodos , Oxidación-Reducción/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.
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Antivirales/farmacología , Inhibidores de Proteasas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Anticuerpos de Cadena Única/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Células A549 , Complejo Antígeno-Anticuerpo , Humanos , Concentración 50 Inhibidora , ARN Polimerasa Dependiente del ARN/inmunología , ARN Polimerasa Dependiente del ARN/metabolismo , Anticuerpos de Cadena Única/inmunología , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismoRESUMEN
BACKGROUND: Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia. METHODS: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1's role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo. RESULTS: Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. CONCLUSION: Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Neoplasias Pancreáticas/genética , Animales , Humanos , Ratones , Neoplasias Pancreáticas/patología , TransfecciónRESUMEN
Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA 3'-end processing enzyme that repairs topoisomerase 1B-induced DNA damage. We use a new tool combining site-specific DNA-protein cross-linking with mass spectrometry to identify Tdp1 interactions with DNA. A conserved phenylalanine (F259) of Tdp1, required for efficient DNA processing in biochemical assays, cross-links to defined positions in DNA substrates. Crystal structures of Tdp1-DNA complexes capture the DNA repair machinery after 3'-end cleavage; these reveal how Tdp1 coordinates the 3'-phosphorylated product of nucleosidase activity and accommodates duplex DNA. A hydrophobic wedge splits the DNA ends, directing the scissile strand through a channel towards the active site. The F259 side-chain stacks against the -3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel. Our results explain why Tdp1 cleavage is non-processive and provide a molecular basis for DNA 3'-end processing by Tdp1.
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Daño del ADN , Reparación del ADN , ADN/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , ADN/química , ADN/genética , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Hidrolasas Diéster Fosfóricas/química , Unión Proteica , Dominios ProteicosRESUMEN
A possible mechanism for poor perinatal outcomes in singleton pregnancies conceived following assisted reproductive technologies (ART) and those conceived naturally following a period of infertility (>12 months) is thought to be placental dysfunction. This was investigated by measuring plasma concentrations of biochemical markers: (i) soluble fms-like tyrosine kinase1 (sFlt1); (ii) placental growth factor (PlGF); (iii) leptin; and (iv) plasminogen activator inhibitor 2 (PAI-2), serially at four antenatal time points. Baseline concentrations of each marker after delivery were also measured. The control group was naturally conceived singleton pregnancies with no history of infertility. Non-smoking, age-matched nulliparous women with no significant medical history were recruited to all groups. The ART group had significantly lower mean plasma concentrations of PlGF at all antenatal time points compared to the control group (p < 0.001). The subfertility (SF) group had significantly higher mean serum concentrations of leptin than the other groups at all time points (p < 0.001), even after correction for body mass index. There were no significant differences in sFlt1 and PAI-2 concentrations between the groups. Low plasma PlGF concentrations in the ART group might suggest abnormal placentation and/or abnormal function in ART pregnancies with relevance to pathogenesis of pregnancy complications in these women.
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Infertilidad Femenina/sangre , Leptina/sangre , Placenta/fisiopatología , Inhibidor 2 de Activador Plasminogénico/sangre , Proteínas Gestacionales/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre , Adulto , Biomarcadores/sangre , Femenino , Humanos , Infertilidad Femenina/fisiopatología , Factor de Crecimiento Placentario , Embarazo , Técnicas Reproductivas AsistidasRESUMEN
OBJECTIVES: International cooperative group trials require specific, sensitive biomarker assays that are validated between continents. Neurokinin A (NKA) has been shown to be a powerful independent predictor of a poor prognosis in well-differentiated midgut neuroendocrine tumors. We hypothesized that NKA concentrations of clinical specimens evaluated in NKA assays in the United States and the United Kingdom would be equivalent, even though assay techniques were significantly different. METHODS: Frozen clinical specimen aliquots were shipped from the United States to the United Kingdom (n = 67), and from United Kingdom to the United States (n = 50). In addition, spiked plasma standards and medium-spiked standards were exchanged. Samples from the United States were directly assayed in a radioimmunoassay, whereas the UK specimens were extracted, and the reconstituted specimens assayed in the radioimmunoassay. Neurokinin A values from the 2 studies were analyzed by regression analysis. RESULTS: The NKA values from the US and UK laboratories were essentially identical (United States to United Kingdom, r = 0.88, P < 0.0001; and United Kingdom to United States, r = 0.96, P < 0.0001). CONCLUSIONS: Validation of biomarker assays across continents will ensure that laboratory observations made by researchers are equivalent and that prediction of clinical outcomes based on these assays is also reliable.
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Biomarcadores de Tumor/sangre , Técnicas de Laboratorio Clínico/normas , Tumores Neuroendocrinos/sangre , Neuroquinina A/sangre , Radioinmunoensayo/normas , Calibración , Humanos , Estimación de Kaplan-Meier , Tumores Neuroendocrinos/mortalidad , Variaciones Dependientes del Observador , Valor Predictivo de las Pruebas , Pronóstico , Estándares de Referencia , Análisis de Regresión , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Manejo de Especímenes/normasRESUMEN
Prader-Willi syndrome (PWS) is a contiguous gene syndrome characterized by uncontrollable eating or hyperphagia. Several studies have confirmed that plasma ghrelin levels are markedly elevated in PWS adults and children. The study of anorexigenic hormones is of interest because of their regulation of appetite by negative signals. To study the pattern and response of the anorexigenic hormones such as cholecystokinin (CCK) and peptide YY (PYY) to a meal in PWS, we measured the plasma CCK, PYY, ghrelin and serum insulin levels in PWS patients (n=4) and in controls (n=4) hourly for a day, and analyzed hormone levels and hormonal responses to meals. Repeated measures of ANOVA of hormone levels demonstrated that only insulin levels decreased (p=0.013) and CCK (p=0.005) and ghrelin (p=0.0007) increased in PWS over 24 hr. However, no significant group x time interactions (ghrelin: p=0.89, CCK: p=0.93, PYY: p=0.68 and insulin: p=0.85) were observed; in addition, there were no differences in an assessment of a three-hour area under the curve after breakfast. These results suggest that the response pattern of hormones to meals in PWS patients parallels that of normal controls. In addition, the decrease of insulin levels over 24 hr, in spite of obesity and elevated ghrelin levels, suggests that the baseline insulin level, not the insulin response to meals, may be abnormal in patients with PWS.