RESUMEN
BACKGROUND & AIMS: Transforming growth factor beta (TGF-ß) suppresses early stages of tumorigenesis, but also contributes to migration and metastasis of cancer cells. A large number of human tumors contain mutations that inactivate its receptors, or downstream proteins such as Smad transcription factors, indicating that the TGF-ß signaling pathway prevents tumor growth. We investigated the effects of TGF-ß inhibition on liver tumorigenesis in mice. METHODS: C57BL/6 mice received hydrodynamic tail-vein injections of transposons encoding HRASG12V and a short hairpin RNA (shRNA) to down-regulate p53, or those encoding HRASG12V and MYC, or those encoding HRASG12V and TAZS89A, to induce liver tumor formation; mice were also given injections of transposons encoding SMAD7 or shRNA against SMAD2, SMAD3, SMAD4, or SNAI1 (Snail), with or without ectopic expression of Snail. Survival times were compared, and livers were weighted and examined for tumors. Liver tumor tissues were analyzed by quantitative reverse-transcription PCR, RNA sequencing, immunoblots, and immunohistochemistry. We analyzed gene expression levels in human hepatocellular carcinoma samples deposited in The Cancer Genome Atlas. A cell proliferation assay was performed using human liver cancer cell lines (HepG2 and Huh7) stably expressing Snail or shRNA against Snail. RESULTS: TGF-ß inhibition via overexpression of SMAD7 (or knockdown of SMAD2, SMAD3, or SMAD4) consistently reduced formation and growth of liver tumors in mice that expressed activated RAS plus shRNA against p53, or in mice that expressed activated RAS and TAZ. TGF-ß signaling activated transcription of the Snail gene in liver tumors induced by HRASG12V and shRNA against p53, and by activated RAS and TAZ. Knockdown of Snail reduced liver tumor formation in both tumor models. Ectopic expression of Snail restored liver tumorigenesis suppressed by disruption of TGF-ß signaling. In human hepatocellular carcinoma, Snail expression correlated with TGF-ß activation. Ectopic expression of Snail increased cellular proliferation, whereas Snail knockdown led to reduced proliferation in human hepatocellular carcinoma cells. CONCLUSIONS: In analyses of transgenic mice, we found TGF-ß signaling to be required for formation of liver tumors upon expression of activated RAS and shRNA down-regulating p53, and upon expression of activated RAS and TAZ. Snail is the TGF-ß target that is required for hepatic tumorigenesis in these models.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Transformación Celular Neoplásica/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Hígado/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Regulación Neoplásica de la Expresión Génica , Genes myc , Genes ras , Predisposición Genética a la Enfermedad , Células Hep G2 , Humanos , Hígado/patología , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factores de Transcripción de la Familia Snail/genética , Factores de Tiempo , Transfección , Factor de Crecimiento Transformador beta/genética , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Regulación hacia ArribaRESUMEN
Mutation in one of three RAS genes (i.e., HRAS, KRAS, and NRAS) leading to constitutive activation of RAS signaling pathways is considered a key oncogenic event in human carcinogenesis. Whether activated RAS isoforms possess different oncogenic potentials remains an unresolved question. Here, we compared oncogenic properties among RAS isoforms using liver-specific transgenesis in mice. Hydrodynamic transfection was performed using transposons expressing short hairpin RNA downregulating p53 and an activated RAS isoform, and livers were harvested at 23 days after gene delivery. No differences were found in the hepatocarcinogenic potential among RAS isoforms, as determined by both gross examination of livers and liver weight per body weight ratio (LW/BW) of mice expressing HRASQ61L, KRAS4BG12V and NRASQ61K. However, the tumorigenic potential differed significantly between KRAS splicing variants. The LW/BW ratio in KRAS4AG12V mice was significantly lower than in KRAS4BG12V mice (p < 0.001), and KRAS4AG12V mice lived significantly longer than KRRAS4BG12V mice (p < 0.0001). Notably, tumors from KRAS4AG12V mice displayed higher expression of the p16INK4A tumor suppressor when compared with KRAS4BG12V tumors. Forced overexpression of p16INK4A significantly reduced tumor growth in KRAS4BG12V mice, suggesting that upregulation of p16INK4A by KRAS4AG12V presumably delays tumor development driven by the latter oncogene.
Asunto(s)
Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica/patología , GTP Fosfohidrolasas/genética , Neoplasias Hepáticas/patología , Proteínas de la Membrana/genética , Mutación/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Apoptosis , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , GTP Fosfohidrolasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Liver fibrosis and its end-stage disease, cirrhosis, are major risk factors for hepatocellular carcinoma (HCC) and present in 80 to 90 % of patients with HCC. Current genetically engineered mouse models for HCC, however, generally do not feature liver fibrosis, which is a critical discrepancy between human HCC and murine models thereof. In this study, we developed a simple transgenic mouse model of HCC within the context of a fibrotic liver. METHODS: Employing hydrodynamic transfection (HT), coupled with the Sleeping Beauty (SB) transposon system, liver was stably transfected with transposons expressing cMyc and a short hairpin RNA down-regulating p53 (shp53). A chronic liver injury model, induced by hepatotoxic carbon tetrachloride (CCl4), was applied to the transgenic mice, allowing cells expressing cMyc plus shp53 to become malignant in the background of liver fibrosis. RESULTS: Livers harvested about 3 months after HT had excessive collagen deposition and activated hepatic stellate cells surrounding the tumors. Hepatocarcinogenesis was significantly accelerated in the fibrotic livers compared to those of the control, significantly decreasing the life span of the mice. The tumor incidence and average number of tumors per mouse were significantly higher in the group treated with CCl4 compared to the vehicle-treated control mice, following HT (p < 0.01). CONCLUSIONS: Considering the simplicity and efficiency in generating HCC for fibrotic livers, the transgenic HCC model has the potential to be effectively used in preclinical testing of HCC anticancer therapy and in studies of hepatocarcinogenesis in fibrotic livers.
Asunto(s)
Carcinoma Hepatocelular/etiología , Cirrosis Hepática Experimental/complicaciones , Neoplasias Hepáticas Experimentales/etiología , Neoplasias Hepáticas/etiología , Animales , Tetracloruro de Carbono , Carcinogénesis/metabolismo , Carcinoma Hepatocelular/metabolismo , Modelos Animales de Enfermedad , Genes myc/genética , Genes p53 , Células Estrelladas Hepáticas/metabolismo , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Ratones , Ratones Transgénicos , ARN Interferente Pequeño , Transposasas/metabolismoRESUMEN
Calorie restriction or a low-carbohydrate diet (LCD) can increase life span in normal cells while inhibiting carcinogenesis. Various phytochemicals also have calorie restriction-mimetic anticancer properties. We investigated whether an isocaloric carbohydrate-restriction diet and AMP-activated protein kinase (AMPK)-activating phytochemicals induce synergic tumor suppression. We used a mixture of AMPK-activating phytochemical extracts including curcumin, quercetin, catechins, and resveratrol. Survival analysis was carried out in a B16F10 melanoma model fed a control diet (62.14% kcal carbohydrate, 24.65% kcal protein and 13.2% kcal fat), a control diet with multiple phytochemicals (MP), LCD (16.5, 55.2, and 28.3% kcal, respectively), LCD with multiple phytochemicals (LCDmp), a moderate-carbohydrate diet (MCD, 31.9, 62.4, and 5.7% kcal, respectively), or MCD with phytochemicals (MCDmp). Compared with the control group, MP, LCD, or MCD intervention did not produce survival benefit, but LCDmp (22.80±1.58 vs. 28.00±1.64 days, P=0.040) and MCDmp (23.80±1.08 vs. 30.13±2.29 days, P=0.008) increased the median survival time significantly. Suppression of the IGF-1R/PI3K/Akt/mTOR signaling, activation of the AMPK/SIRT1/LKB1pathway, and NF-κB suppression were the critical tumor-suppression mechanisms. In addition, SIRT1 suppressed proliferation of the B16F10 and A375SM cells under a low-glucose condition. Alterations in histone methylation within Pten and FoxO3a were observed after the MCDmp intervention. In the transgenic liver cancer model developed by hydrodynamic transfection of the HrasG12V and shp53, MCDmp and LCDmp interventions induced significant cancer-prevention effects. Microarray analysis showed that PPARα increased with decreased IL-6 and NF-κB within the hepatocytes after an MCDmp intervention. In conclusion, an isocaloric carbohydrate-restriction diet and natural AMPK-activating agents induce synergistic anticancer effects. SIRT1 acts as a tumor suppressor under a low-glucose condition.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Carbohidratos de la Dieta/administración & dosificación , Suplementos Dietéticos , Neoplasias Hepáticas Experimentales/prevención & control , Melanoma Experimental/prevención & control , Fitoquímicos/administración & dosificación , Sirtuina 1/metabolismo , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Carbohidratos de la Dieta/farmacología , Sinergismo Farmacológico , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/mortalidad , Neoplasias Hepáticas Experimentales/patología , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/mortalidad , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Fitoquímicos/farmacocinética , Transducción de Señal , Sirtuina 1/genética , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide; however, the genetic mechanisms underlying its pathogenesis are incompletely understood. Genetically engineered mouse (GEM) models of HCC have been developed to elucidate the role of individual cancer-related genes in hepatocarcinogenesis. However, the expensive and time-consuming processes related to generating a GEM model discourage the development of diverse genotype models. Recently, a simple and inexpensive liver-specific transgenic approach was developed, in which a hydrodynamics-based transfection (HT) method was coupled with the Sleeping Beauty transposase system. Various HT models in which different oncogenic pathways are activated and/or tumor-suppressing pathways inactivated have been developed in recent years. The applicability of HT models in liver cancer research is expected to broaden and ultimately elucidate the cooperation between oncogenic signaling pathways and aid in designing molecular therapy to target altered pathways.
Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas Experimentales/genética , Transfección/métodos , Animales , Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Hidrodinámica , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Ratones , Ratones Transgénicos , Terapia Molecular DirigidaRESUMEN
BACKGROUND & AIMS: Liver fibrosis is an increasing health concern worldwide and a major risk factor for hepatocellular carcinoma (HCC). Although the involvement of Hedgehog signaling in hepatic fibrosis has been known for some time, the causative role of activated Hedgehog signaling in liver fibrosis has not been verified in vivo. METHODS: Using hydrodynamics-based transfection, a transgenic mouse model has been developed that expresses Sonic Hedgehog (SHH), a ligand for Hedgehog signaling, in the liver. Levels of hepatic fibrosis and fibrosis-related gene expression were assessed in the model. Hepatic expression of SHH was induced in a murine model for hepatocellular adenoma (HCA) and tumor development was subsequently investigated. RESULTS: The transgenic mice revealed SHH expression in 2-5% of hepatocytes. Secreted SHH activated Hedgehog signaling in numerous cells of various types in the tissues. Hepatic expression of SHH led to fibrosis, activation of hepatic stellate cells, and an upregulation of various fibrogenic genes. Liver injury and hepatocyte apoptosis were observed in SHH mice. Persistent expression of SHH for up to 13months failed to induce tumors in the liver; however, it promoted liver tumor development induced by other oncogenes. By employing a HCA model induced by P53(R172H) and KRAS(G12D), we found that the SHH expression promoted the transition from HCA to HCC. CONCLUSIONS: SHH expression in the liver induces liver fibrosis with concurrent activation of hepatic stellate cells and fibrogenic genes. It can also enhance hepatocarcinogenesis induced by other oncogenes.
Asunto(s)
Proteínas Hedgehog/fisiología , Cirrosis Hepática Experimental/etiología , Neoplasias Hepáticas Experimentales/etiología , Animales , Apoptosis , Transición Epitelial-Mesenquimal , Proteínas Hedgehog/análisis , Cirrosis Hepática Experimental/patología , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal , Factor de Crecimiento Transformador beta1/fisiologíaRESUMEN
BACKGROUND: Plasmodium vivax apical membrane antigen-1 (PvAMA-1) is a leading candidate antigen for blood stage malaria vaccine. However, antigenic variation is a major obstacle in the development of an effective vaccine based on this antigen. In this study, the genetic structure and the effect of natural selection of PvAMA-1 among Korean P. vivax isolates were analysed. METHODS: Blood samples were collected from 66 Korean patients with vivax malaria. The entire PvAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvAMA-1 sequence of each isolate was sequenced and the polymorphic characteristics and effect of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs. RESULTS: Thirty haplotypes of PvAMA-1, which were further classified into seven different clusters, were identified in the 66 Korean P. vivax isolates. Domain II was highly conserved among the sequences, but substantial nucleotide diversity was observed in domains I and III. The difference between the rates of non-synonymous and synonymous mutations suggested that the gene has evolved under natural selection. No strong evidence indicating balancing or positive selection on PvAMA-1 was identified. Recombination may also play a role in the resulting genetic diversity of PvAMA-1. CONCLUSIONS: This study is the first comprehensive analysis of nucleotide diversity across the entire PvAMA-1 gene using a single population sample from Korea. Korean PvAMA-1 had limited genetic diversity compared to PvAMA-1 in global isolates. The overall pattern of genetic polymorphism of Korean PvAMA-1 differed from other global isolates and novel amino acid changes were also identified in Korean PvAMA-1. Evidences for natural selection and recombination event were observed, which is likely to play an important role in generating genetic diversity across the PvAMA-1. These results provide useful information for the understanding the population structure of P. vivax circulating in Korea and have important implications for the design of a vaccine incorporating PvAMA-1.
Asunto(s)
Antígenos de Protozoos/genética , Variación Genética , Genotipo , Proteínas de la Membrana/genética , Plasmodium vivax/clasificación , Plasmodium vivax/genética , Proteínas Protozoarias/genética , Selección Genética , ADN Protozoario/química , ADN Protozoario/genética , Vectores Genéticos , Genética de Población , Humanos , Corea (Geográfico) , Malaria Vivax/parasitología , Datos de Secuencia Molecular , Plasmodium vivax/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Recombinación Genética , Análisis de Secuencia de ADNRESUMEN
Paramyosin of Clonorchis sinensis (CsPmy) is a myofibrillar protein localized in subtegumental muscle, tegument, and the muscle layer surrounding the intestine of the parasite. Previously, we have identified that CsPmy reacted with sera of human clonorchiasis and this protein had a potential as a candidate antigen for serodiagnosis of clonorchiasis. However, we also found that CsPmy is able to bind to human immunoglobulin G (IgG) in non-specific manners, which can affect the diagnostic value of the protein. Here, we mapped CsPmy-specific IgG binding site on CsPmy to analyze the putative epitopes recognized by CsPmy-specific IgG in sera of human clonorchiasis. The fragmental expression of CsPmy followed by immunoblot analyses with sera from patients with clonorchiasis and non-specific human IgG revealed that the middle portion of CsPmy (CsPmyC: 301-600 amino acid residues) had epitopes responsible for CsPmy-specific IgG recognition. The precise CsPmy-specific IgG binding site was further narrowed down to a fragment (CsPmyC-2), which harbors 151 amino acid residues (375-525) of CsPmy. Specific antibodies for CsPmyC-2 were produced in rats after two-weeks of post-experimental infection. The CsPmyC-2 showed low levels of cross reactivity against the sera from patients with other helminth parasites. Our results suggested that CsPmyC-2 has real epitopes recognized by CsPmy-specific IgG in sera of human clonorchiasis and the fragment can be useful as a reliable serodiagnostic antigen to develop a serodiagnostic method for clonorchiasis.
Asunto(s)
Antígenos Helmínticos/inmunología , Clonorquiasis/inmunología , Clonorchis sinensis/inmunología , Mapeo Epitopo , Epítopos/inmunología , Inmunoglobulina G/inmunología , Tropomiosina/inmunología , Animales , Clonorquiasis/diagnóstico , Humanos , Immunoblotting , Ratas , Pruebas Serológicas/métodosRESUMEN
Asparaginyl endopeptidases (AEP: EC 3.4.22.34) are a family of cysteine proteases classified into the MEROPS clan CD, family C13. In this study, we characterized the biochemical and antigenic properties of an AEP of Clonorchis sinensis (CsAEP). The recombinant CsAEP showed hydrolytic activity at pH values ranging from acidic to neutral with optimum activity at pH 6.0. While the recombinant CsAEP was stable at neutral pHs, it was unstable at acidic pHs and resulted in loss of enzymatic activity. The recombinant enzyme was effectively inhibited by iodoacetic acid and N-ethylmaleimide, but not by E-64. The partially purified native CsAEP showed biochemical properties similar to the recombinant enzyme. Native CsAEP is likely to be cleaved into an N-terminal mature enzyme and a C-terminal fragment via autocatalytic activation at acidic pHs. Polyclonal antibody raised against the recombinant CsAEP recognized three forms of CsAEP, proenzyme, the N-terminal mature enzyme and the C-terminal fragment, in the worm extract (WE) of C. sinensis. However, only the C-terminal fragment was mainly found in the excretory and secretory (ES) products of the parasite. Strong CsAEP activity was found in the WE, but only a trace level of CsAEP activity was detected in the ES products of the parasite. CsAEP was expressed in various developmental stages of C. sinensis, from metacercariae to adults, and was found to be localized in the intestine of the parasite as well as in intestinal contents. Sera from rats experimentally infected with C. sinensis reacted with CsAEP beginning 4 weeks after infection. These results suggest that CsAEP is a gut-associated enzyme synthesized in the intestine of C. sinensis and subsequently secreted into the intestinal lumen of the parasite.
Asunto(s)
Clonorquiasis/parasitología , Clonorchis sinensis/enzimología , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Proteínas del Helminto/química , Secuencia de Aminoácidos , Animales , Clonación Molecular , Clonorchis sinensis/química , Clonorchis sinensis/genética , Clonorchis sinensis/crecimiento & desarrollo , Cisteína Endopeptidasas/genética , Femenino , Tracto Gastrointestinal/enzimología , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Alineación de SecuenciaRESUMEN
AIM: Hepatocellular carcinoma (HCC), one of the most common malignancies in adults displays aberrant miRNA expression during its pathogenesis. We assessed expression of miRNA in surgically resected human HCC of an early stage and murine HCC with a high malignancy in order to find miRNA overexpressed in HCC regardless of tumor stage and underlying etiology. Further, the role of the deregulated miRNA in HCC pathogenesis was investigated. METHODS: miRNA were isolated from HCC tissues and surrounding non-tumorous tissues from HCC patients and a murine transgenic model of HCC. A quantitative reverse transcription polymerase chain reaction was performed to determine expression levels of miRNA. Human HCC cell lines stably expressing individual miRNA were generated to investigate the biological function of overexpressed miRNA. RESULTS: We found that levels of miR-221, -181b-1, -155-5p, -25 and -17-5p were significantly upregulated in both human and murine HCC regardless of tumor stage, underlying etiology or the presence of fibrosis. Using HCC cell lines stably expressing respective miRNA, we found that miR-221 increased the proliferation of hepatoma cells, while miR-17-5p induced cell migration. CONCLUSION: We identified miRNA that are consistently upregulated in HCC. The overexpressed miRNA could potentially be used as a bona fide biomarker for HCC.
RESUMEN
Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53(R172H) and KRAS(G12D), are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53(R172H) and KRAS(G12D) in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53(R172H) and KRAS(G12D).
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Neoplasias Hepáticas/patología , Luciferasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Luciferasas/genética , Mediciones Luminiscentes , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Sistemas de Lectura Abierta/genética , Imagen Óptica , Proteínas Proto-Oncogénicas p21(ras)/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Piel/metabolismo , Piel/patología , Tasa de Supervivencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Methionine aminopeptidases (MetAPs), ubiquitous enzymes that play an important role in nascent protein maturation, have been recognized as attractive targets for the development of drugs against pathogenic protozoa including Plasmodium spp. Here, we characterized partial biochemical properties of a type I MetAP of Plasmodium vivax (PvMetAP1). PvMetAP1 had the typical amino acid residues essential for metal binding and substrate binding sites, which are well conserved in the type I MetAP family enzymes. Recombinant PvMetAP1 showed activity in a broad range of neutral pHs, with optimum activity at pH 7.5. PvMetAP1 was stable under neutral and alkaline pHs, but was relatively unstable under acidic conditions. PvMetAP1 activity was highly increased in the presence of Mn(2+), and was effectively inhibited by a metal chelator, EDTA. Fumagillin and aminopeptidase inhibitors, amastatin and bestatin, also showed an inhibitory effect on PvMetAP1. The enzyme had a highly specific hydrolytic activity for N-terminal methionine. These results collectively suggest that PvMetAP1 belongs to the family of type I MetAPs and may play a pivotal role for the maintenance of P. vivax physiology by mediating protein maturation and processing of the parasite.
Asunto(s)
Expresión Génica , Metionil Aminopeptidasas/biosíntesis , Metionil Aminopeptidasas/química , Plasmodium vivax/enzimología , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Leucina/análogos & derivados , Leucina/química , Manganeso/química , Metionil Aminopeptidasas/genética , Péptidos/química , Plasmodium vivax/genética , Proteínas Protozoarias/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Otubains are a recently discovered family of cysteine proteases that participate in the ubiquitin pathway. Here, we partially characterized the biochemical properties of a cysteine protease of Cryptosporidium parvum, which is closely related to otubains. The gene encoding otubain-like cysteine protease of C. parvum (CpOTU) contained the aspartate, cysteine and histidine residues that form the catalytic triad of otubains. The modified ubiquitin-associated domain and LxxL motif were identified in CpOTU. The recombinant CpOTU showed the isopeptidase activity at neutral pH values and its activity was effectively inhibited by ubiquitin aldehyde, N-ethylmaleimide and iodoacetic acid. Interestingly, CpOTU had an unusual C-terminal extension of 217 amino acids compared to mammalian otubains, and the C-terminal extension is essential for the activity of the enzyme. Expression of CpOTU peaked in the oocyst stage of the parasite, which suggested its potential physiological role for the oocyst stage.
Asunto(s)
Cryptosporidium parvum/enzimología , Cryptosporidium parvum/genética , Proteasas de Cisteína/genética , Proteasas de Cisteína/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Cryptosporidium parvum/metabolismo , Proteasas de Cisteína/química , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Protozoarias/químicaRESUMEN
BACKGROUND: The merozoite surface protein-3ß of Plasmodium vivax (PvMSP-3ß) is one of the candidate antigens for blood stage malaria vaccine development. The polymorphisms in PvMSP-3ß have been reported in certain P. vivax isolates. However, the diversity of PvMSP-3ß throughout its global distribution has not been well understood. In this study, the genetic diversity and the effects of natural selection in PvMSP-3ß among P. vivax Korean isolates were analysed. METHODS: Blood samples were collected from 95 patients with vivax malaria in Korea. The region flanking full-length PvMSP-3ß was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvMSP-3ß sequence of each isolate was determined and the polymorphic characteristics and effects of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs. RESULTS: Five different subtypes of PvMSP-3ß were identified based on single nucleotide polymorphisms (SNPs), insertions, and deletions. Although a high level of sequence diversity was observed in the PvMSP-3ß gene, the coiled-coil tertiary structure of the PvMSP-3ß protein was well conserved in all of the sequences. The PvMSP-3ß of Korean isolates is under natural selection. DNA polymerase slippage and intragenic recombination likely contributed to PvMSP-3ß diversity in Korean P. vivax isolates. CONCLUSIONS: The PvMSP-3ß of Korean P. vivax isolates displayed polymorphisms, with SNPs, insertions and deletions scattered throughout of the gene. These results of parasite heterogeneity are relevant to the development of a PvMSP-3ß based vaccine against P. vivax and the implementation of malaria control programmes in Korea.
Asunto(s)
Antígenos de Protozoos/genética , Malaria Vivax/parasitología , Plasmodium vivax/clasificación , Plasmodium vivax/genética , Polimorfismo Genético , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Antígenos de Protozoos/química , Clonación Molecular , Análisis por Conglomerados , ADN Protozoario/química , ADN Protozoario/genética , ADN Protozoario/aislamiento & purificación , Evolución Molecular , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Filogenia , Plasmodium vivax/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Protozoarias/química , República de Corea , Selección Genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
CsStefin-2, the second cysteine protease inhibitor of Clonorchis sinensis, was identified and characterized. CsStefin-2 is a cysteine protease inhibitor that belongs to family 1 stefins based on its phylogenetic and structural properties. However, CsStefin-2 had a QIVSG cystatin motif distinct from the common QVVAG cystatin motif that is well conserved in family 1 stefins. Mutagenesis analysis revealed that the two amino acid substitutions in the QIVSG cystatin motif of CsStefin-2 did not affect its inhibitory activity. Molecular modeling also indicated that no critical change was induced in the interaction between CsStefin-2 and its target enzyme. CsStefin-2 showed broad inhibitory activities against several cysteine proteases, including human cathepsins B and L, papain, and cathepsin Fs of C. sinensis (CsCFs), and effectively inhibited the autocatalytic maturation of CsCF-6. Native CsStefin-2 was assembled into a homo-tetramer, in which intermolecular disulfide bonds are not involved in the assembly of the tetramer. CsStefin-2 was expressed throughout the various developmental stages of the parasite and was localized in the intestinal epithelium, where CsCFs are actively synthesized. These results suggest that CsStefin-2 is the second active cysteine protease inhibitor of C. sinensis that shares functional redundancy with CsStefin-1 to modulate the activity and processing of CsCFs.
Asunto(s)
Clonorchis sinensis/genética , Cistatinas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Proteínas del Helminto/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Catepsina B/antagonistas & inhibidores , Catepsina L/antagonistas & inhibidores , Clonación Molecular , Clonorchis sinensis/metabolismo , Cistatinas/genética , Inhibidores de Cisteína Proteinasa/genética , Proteínas del Helminto/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Papaína/antagonistas & inhibidores , Filogenia , Estructura Terciaria de ProteínaRESUMEN
CsCF-6 is a member of the multigene family of cathepsin F cysteine proteases of Clonorchis sinensis. Similar to other papain family proteases, CsCF-6 is synthesized as a proenzyme and is converted to the mature form by autocatalytic removal of the prodomain. Here, we analyzed the regulatory and inhibitory elements within the CsCF-6 prodomain to understand the regulatory mechanism of CsCF-6 by its prodomain. The CsCF-6 prodomain played an essential role in the folding of CsCF-6. Particularly, the ERFNAQ motif within the prodomain was essential, and the minimum segment required for this event was the C-terminal part of the prodomain, including Asn(58) and downstream residues. The CsCF-6 prodomain effectively inhibited CsCF-6, in which the ERFNAQ motif played a critical role in the inhibition, but the GTFD motif was also required for complete inhibition of CsCF-6. The CsCF-6 prodomain showed broad inhibitory activity against several cysteine proteases. These results suggest that the CsCF-6 prodomain plays bi-functional roles in correct folding and inhibition of its cognate enzyme.
Asunto(s)
Catepsinas/metabolismo , Clonorchis sinensis/enzimología , Precursores Enzimáticos/metabolismo , Proteínas del Helminto/metabolismo , Procesamiento Proteico-Postraduccional , Secuencias de Aminoácidos , Animales , Catepsinas/antagonistas & inhibidores , Análisis Mutacional de ADN , Inhibidores Enzimáticos/metabolismo , Proteínas del Helminto/antagonistas & inhibidores , Pliegue de Proteína , Eliminación de SecuenciaRESUMEN
BACKGROUND: Transmission-blocking vaccines (TBVs), which target the sexual stages of malaria parasites to interfere with and/or inhibit the parasite's development within mosquitoes, have been regarded as promising targets for disrupting the malaria transmission cycle. In this study, genetic diversity of four TBV candidate antigens, Pvs25, Pvs28, Pvs48/45, and PvWARP, among Plasmodium vivax Korean isolates was analysed. METHODS: A total of 86 P. vivax-infected blood samples collected from patients in Korea were used for analyses. Each of the full-length genes encoding four TBV candidate antigens, Pvs25, Pvs28, Pvs48/45, and PvWARP, were amplified by PCR, cloned into T&A vector, and then sequenced. Polymorphic characteristics of the genes were analysed using the DNASTAR, MEGA4, and DnaSP programs. RESULTS: Polymorphism analyses of the 86 Korean P. vivax isolates revealed two distinct haplotypes in Pvs25 and Pvs48/45, and three different haplotypes in PvWARP. In contrast, Pvs28 showed only a single haplotype. Most of the nucleotide substitutions and amino acid changes identified in all four TBV candidate antigens were commonly found in P. vivax isolates from other geographic areas. The overall nucleotide diversities of the TBV candidates were much lower than those of blood stage antigens. CONCLUSIONS: Limited sequence polymorphisms of TBV candidate antigens were identified in the Korean P. vivax population. These results provide baseline information for developing an effective TBV based on these antigens, and offer great promise for applications of a TBV against P. vivax infection in regions where the parasite is most prevalent.
Asunto(s)
Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Vacunas contra la Malaria/genética , Vacunas contra la Malaria/inmunología , Plasmodium vivax/genética , Plasmodium vivax/inmunología , Polimorfismo Genético , Sangre/parasitología , ADN Protozoario/química , ADN Protozoario/genética , Humanos , Malaria Vivax/parasitología , Datos de Secuencia Molecular , Plasmodium vivax/aislamiento & purificación , Reacción en Cadena de la Polimerasa , República de Corea , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Liver cancer is a complex multistep process requiring genetic alterations in multiple proto-oncogenes and tumor suppressor genes. Although hundreds of genes are known to play roles in hepatocarcinogenesis, oncogenic collaboration among these genes is still largely unknown. Here, we report a simple methodology by which oncogenic cooperation between cancer-related genes can be efficiently investigated in the liver. We developed various non-germline transgenic mouse models using hydrodynamics-based transfection which express HrasG12V, SmoM2, and a short-hairpin RNA down-regulating p53 (shp53) individually or in combination in the liver. In this transgenic system, firefly luciferase was co-expressed with the oncogenes as a reporter, allowing tumor growth in the liver to be monitored over time without an invasive procedure. Very strong bioluminescence imaging (BLI) signals were observed at 4 weeks post-hydrodynamic injection (PHI) in mice co-expressing HrasG12V and shp53, while only background signals were detected in other double or single transgenic groups until 30 weeks PHI. Consistent with the BLI data, tumors were observed in the HrasG12V plus shp53 group at 4 weeks PHI, while other transgenic groups failed to exhibit a hyperplastic nodule at 30 weeks PHI. In the HrasG12V plus shp53 transgenic group, BLI signals were well-correlated with actual tumor growth in the liver, confirming the versatility of BLI-based monitoring of tumor growth in this organ. The methodology described here is expected to accelerate and facilitate in vivo studies of the hepatocarcinogenic potential of cancer-related genes by means of oncogenic cooperation.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Imagen de Cuerpo Entero , Animales , Hidrodinámica , Neoplasias Hepáticas/metabolismo , Luciferasas , Luminiscencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Células 3T3 NIH , Oncogenes , Plásmidos , Factores de Tiempo , TransfecciónRESUMEN
The Sleeping Beauty transposon system is used as a tool for insertional mutagenesis and oncogenesis. However, little is known about the exact histological phenotype of the tumors induced. Thus, we used immunohistochemical markers to enable histological identification of the type of tumor induced by subcutaneous injection of the HRAS, c-Myc and shp53 oncogenes in female C57BL/6 mice. The tumor was removed when it reached 100 mm3 in volume. Subsequently, we used 13 immunohistochemical markers to histologically identify the tumor type. The results suggested that the morphology of the tumor was similar to that of sarcomatoid carcinoma.