RESUMO
MicroRNAs (miRNAs) in urine are examined as potential biomarkers. We examined the urine samples from 70 individuals (45 males, 25 females, mean age 65 years, range 20-84 years). Of the urine donors, 15 were healthy volunteers, 5 were patients with non-cancer diseases, 50 were patients with different stages of bladder cancer. To examine the spectrum of miRNAs in the cell-free fraction of urine, TaqMan Human miRNA Array Card A v.2.1 was used. A set of 30 miRNAs were found that are constantly present in urine supernatants independently of sex, age and health status of the subjects. We compared this set with miRNAs found in plasma, expressed in kidney and genito-urinary tract. Our results indicate that some miRNA could be transferred from the circulation into urine.
Assuntos
Biomarcadores Tumorais/genética , Rim/metabolismo , MicroRNAs/genética , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/urina , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/sangue , MicroRNAs/urina , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Bexiga Urinária/diagnóstico , Adulto JovemRESUMO
INTRODUCTION: Concentration of urinary cell-free DNA (ucfDNA) belongs to potential bladder cancer markers, but the reported results are inconsistent due to the use of various non-standardised methodologies. The aim of the study was to standardise the methodology for ucfDNA quantification as a potential non-invasive tumour biomarker. MATERIAL AND METHODS: In total, 66 patients and 34 controls were enrolled into the study. Volumes of each urine portion (V) were recorded and ucfDNA concentrations (c) were measured using real-time PCR. Total amounts (TA) of ucfDNA were calculated and compared between patients and controls. Diagnostic accuracy of the TA of ucfDNA was determined. RESULTS: The calculation of TA of ucfDNA in the second urine portion was the most appropriate approach to ucfDNA quantification, as there was logarithmic dependence between the volume and the concentration of a urine portion (p = 0.0001). Using this methodology, we were able to discriminate between bladder cancer patients and subjects without bladder tumours (p = 0.0002) with area under the ROC curve of 0.725. Positive and negative predictive value of the test was 90 and 45%, respectively. CONCLUSION: Quantification of ucf DNA according to our modified method could provide a potential non-invasive biomarker for diagnosis of patients with bladder cancer.
Assuntos
Biomarcadores Tumorais/urina , DNA/urina , Urinálise/normas , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/urina , Idoso , Estudos de Casos e Controles , Sistema Livre de Células , DNA/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Therefore, the present study has been designed to evaluate the cytochrome P450 (CYP)-mediated oxidative detoxification and reductive activation of AAI in a rat model. METHODS: DNA adduct formation was investigated by the nuclease P1 version of the 32P-postlabeling method. The CYP-mediated formation of a detoxication metabolite of AAI, 8-hydroxyaristolochic acid I (AAIa), in vitro in rat hepatic microsomes was determined by HPLC. RESULTS: Rat hepatic CYPs both detoxicate AAI by its oxidation to AAIa and reductively activate this carcinogen to a cyclic N-acylnitrenium ion forming AAI-DNA adducts in vitro. To define the role of hepatic CYPs in AAI demethylation and activation, the modulation of AAIa and AAI-DNA adduct formation by CYP inducers and selective CYP inhibitors was investigated. Based on these studies, we attribute the major role of CYP1A1 and 1A2 in AAI detoxication by its demethylation to AAIa, and, under hypoxic conditions also to AAI activation to species forming DNA adducts. Using microsomes of Baculovirus transfected insect cells (Supersomes™) containing recombinantly expressed rat CYPs, NADPH:CYP reductase and/or cytochrome b5, a major role of CYP1A1 and 1A2 in both reactions in vitro was confirmed. CONCLUSION: Based on the results found in this and former studies we propose that AAI activation and detoxication in rats are dictated mainly by AAI binding affinity to CYP1A1/2 or NADPH(P)H:quinone oxidoreductase, by their turnover and by the balance between oxidation and reduction of AAI by CYP1A.
Assuntos
Ácidos Aristolóquicos/farmacocinética , Sistema Enzimático do Citocromo P-450/fisiologia , Fígado/efeitos dos fármacos , Animais , Biotransformação , Carcinógenos/farmacocinética , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2 , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos/genética , Citocromos/metabolismo , Adutos de DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inativação Metabólica , Fígado/enzimologia , Fígado/metabolismo , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Ratos , Ratos WistarRESUMO
Clonality was tested in 86 tumours from 25 patients with recurrent and multifocal superficial bladder transitional cell carcinomas (TCCs) using the analysis of TP53 mutations and of LOH in the 17p13 and 9p21 regions. Tumours from the majority of individuals showed either absence or presence of the same TP53 mutation and/or an identical LOH pattern, with the same allele lost in all tumours. Only two pairs of tumours from two patients had discordant findings, which were incompatible with monoclonality. Therefore, our results rather support the monoclonal model of development of highly recurrent superficial bladder TCCs.
Assuntos
Carcinoma de Células de Transição/genética , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 9 , Perda de Heterozigosidade , Mutação , Proteína Supressora de Tumor p53/biossíntese , Neoplasias da Bexiga Urinária/genética , Idoso , Anticorpos Monoclonais/química , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Proteína Supressora de Tumor p53/genéticaRESUMO
Ingestion of aristolochic acid (AA) is associated with development of urothelial tumors linked with AA nephropathy and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. We investigated the efficiency of human NAD(P)H:quinone oxidoreductase (NQO1) to activate aristolochic acid I (AAI) and used in silico docking, using soft-soft (flexible) docking procedure, to study the interactions of AAI with the active site of human NQO1. AAI binds to the active site of NQO1 indicating that the binding orientation allows for direct hydride transfer (i.e., two electron reductions) to the nitro group of AAI. NQO1 activated AAI, generating DNA adduct patterns reproducing those found in urothelial tissues from humans exposed to AA. Because reduced aromatic nitro-compounds are often further activated by sulfotransferases (SULTs) or N,O-acetlytransferases (NATs), their roles in AAI activation were investigated. Our results indicate that phase II reactions do not play a major role in AAI bioactivation; neither native enzymes present in human hepatic or renal cytosols nor human SULT1A1, -1A2, -1A3, -1E, or -2A nor NAT1 or NAT2 further enhanced DNA adduct formation by AAI. Instead under the in vitro conditions used, DNA adducts arise by enzymatic reduction of AAI through the formation of a cyclic hydroxamic acid (N-hydroxyaristolactam I) favored by the carboxy group in peri position to the nitro group without additional conjugation. These results emphasize the major importance of NQO1 in the metabolic activation of AAI and provide the first evidence that initial nitroreduction is the rate limiting step in AAI activation.
Assuntos
Acetiltransferases/metabolismo , Ácidos Aristolóquicos/metabolismo , Adutos de DNA/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Sulfotransferases/metabolismo , Ácidos Aristolóquicos/química , Humanos , Estrutura MolecularRESUMO
Aristolochic acid (AA) causes aristolochic acid nephropathy, Balkan endemic nephropathy, and their urothelial malignancies. To identify enzymes involved in the metabolism of aristolochic acid I (AAI), the major toxic component of AA we used HRN (hepatic cytochrome P450 [Cyp] reductase null) mice, in which NADPH:Cyp oxidoreductase (Por) is deleted in hepatocytes. AAI was demethylated by hepatic Cyps in vitro to 8-hydroxy-aristolochic acid I (AAIa), indicating that less AAI is distributed to extrahepatic organs in wild-type (WT) mice. Indeed, AAI-DNA-adduct levels were significantly higher in organs of HRN mice, having low hepatic AAI demethylation capacity, than in WT mice. Absence of AAI demethylation in HRN mouse liver was confirmed in vitro; hepatic microsomes from WT, but not from HRN mice, oxidized AAI to AAIa. To define the role of hepatic Cyps in AAI demethylation, modulation of AAIa formation by CYP inducers was investigated. We conclude that AAI demethylation is attributable mainly to Cyp1a1/2. The higher AAI-DNA adduct levels in HRN than WT mice were the result of the lack of hepatic AAI demethylation concomitant with a higher activity of cytosolic NAD(P)H:quinone oxidoreductase (Nqo1), which activates AAI. Mouse hepatic Cyp1a1/2 also activated AAI to DNA adducts under hypoxic conditions in vitro, but in renal microsomes, Por and Cyp3a are more important than Cyp1a for AAI-DNA adduct formation. We propose that AAI activation and detoxication in mice are dictated mainly by AAI binding affinity to Cyp1a1/2 or Nqo1, by their turnover, and by the balance between oxidation and reduction of AAI by Cyp1a.
Assuntos
Ácidos Aristolóquicos/farmacocinética , Carcinógenos/farmacocinética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Fígado/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão , Adutos de DNA , Humanos , Fígado/enzimologia , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH-Ferri-Hemoproteína Redutase/genética , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Shb (Src homology 2 protein B) is an adapter protein downstream of the vascular endothelial growth factor receptor receptor-2 (VEGFR-2). Previous experiments have suggested a role for Shb in endothelial cell function. Recently, the Shb gene was inactivated and Shb null mice were obtained on a mixed genetic background, but not on C57Bl6 mice. The present study was performed to address endothelial function in the Shb knockout mouse and its relevance for tumor angiogenesis. Tumor growth was retarded in Shb mutant mice, and this correlated with decreased angiogenesis both in tumors and in Matrigel plugs. Shb null mice display an abnormal endothelial ultrastructure in liver sinusoids and heart capillaries with cytoplasmic extensions projecting toward the lumen. Shb null heart VE-cadherin staining was less distinct than that of control heart, exhibiting in the former case a wavy and punctuate pattern. Experiments on isolated endothelial cells suggest that these changes could partly reflect cytoskeletal abnormalities. Vascular permeability was increased in Shb null mice in heart, kidney, and skin, whereas VEGF-stimulated vascular permeability was reduced in Shb null mice. It is concluded that Shb plays an important role in maintaining a functional vasculature in adult mice, and that interference with Shb signaling may provide novel means to regulate tumor angiogenesis.
Assuntos
Neoplasias Experimentais/irrigação sanguínea , Proteínas Proto-Oncogênicas/fisiologia , Animais , Antígenos CD/análise , Caderinas/análise , Permeabilidade Capilar , Células Endoteliais/fisiologia , Células Endoteliais/ultraestrutura , Fígado/irrigação sanguínea , Fígado/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/ultraestrutura , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Proteínas Proto-Oncogênicas/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
SHB is an Src homology 2 domain-containing adapter protein that has been found to be involved in numerous cellular responses. We have generated an Shb knockout mouse. No Shb-/- pups or embryos were obtained on the C57Bl6 background, indicating an early defect as a consequence of Shb- gene inactivation on this genetic background. Breeding heterozygotes for Shb gene inactivation (Shb+/-) on a mixed genetic background (FVB/C57Bl6/129Sv) reveals a distorted transmission ratio of the null allele with reduced numbers of Shb+/+ and Shb-/- animals, but increased number of Shb+/- animals. The Shb- allele is associated with various forms of malformations, explaining the relative reduction in the number of Shb-/- offspring. Shb-/- animals that were born were viable, fertile, and showed no obvious defects. However, Shb+/- female mice ovulated preferentially Shb- oocytes explaining the reduced frequency of Shb+/+ mice. Our study suggests a role of SHB during reproduction and development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Anormalidades Múltiplas/genética , Alelos , Animais , Blastocisto/metabolismo , Feminino , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , OvulaçãoRESUMO
OBJECTIVES: The individual recurrence-free period after primary surgery of patients with Ta urothelial cell carcinoma (UCC) cannot be predicted accurately. This study aims at discriminating between patients with primary Ta UCC and long or short recurrence-free periods. METHODS: We investigated mRNA expression of 23 genes in 44 primary Ta tumours (23 and 21 tumours were from patients with long [>or=4 yr] or short [Assuntos
Carcinoma de Células de Transição/genética
, Carcinoma de Células de Transição/cirurgia
, Perfilação da Expressão Gênica
, Recidiva Local de Neoplasia/genética
, Neoplasias da Bexiga Urinária/genética
, Neoplasias da Bexiga Urinária/cirurgia
, Adulto
, Idoso
, Idoso de 80 Anos ou mais
, Feminino
, Humanos
, Masculino
, Pessoa de Meia-Idade
, Valor Preditivo dos Testes
, RNA Neoplásico/biossíntese
RESUMO
Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context.
Assuntos
Diferenciação Celular , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/citologia , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Ensaio de Unidades Formadoras de Colônias , Técnicas de Cultura Embrionária , Embrião de Mamíferos/metabolismo , Proteínas Fetais/metabolismo , Marcação de Genes , Hematopoese , Técnicas Imunoenzimáticas , Integrases , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Accurate prediction of tumor recurrence in patients with superficial urothelial cell carcinoma (UCC) might result in a significant reduction of invasive follow-up cystoscopies. A recent study identified a panel of 26 genes from a large cDNA microarray analysis of bladder tumors that discriminated between early- and late-recurring patients with superficial Ta tumors (Dyrskjøt et al., Nat Genet 2003;33:90-6). We aimed to validate this panel of genes in 44 primary Ta UCCs (23 and 21 tumors from patients with short or prolonged recurrence-free periods, respectively), by real-time quantitative PCR. Statistical analysis showed marginal significant different mRNA expression levels between the 2 patient groups. To evaluate a supplementary effect of genes for the identification of patients with short or prolonged recurrence-free intervals, forward logistic regression analysis was applied. This revealed that a combination of the expression profiles of the genes HNRPK, LTB4DH and ANP32B resulted in the best performance, although the combination only marginally increased the predictive value of HNRPK alone. Comparing the receiver-operating-characteristic curves for HNRPK expression among patients with short or prolonged recurrence-free periods, revealed an area under the curve of 0.696 (95% CI, 0.537-0.855). Using the median HNRPK expression level as cut-off, a sensitivity of 69.6% and a specificity of 71.4% were obtained for the identification of patients with short or prolonged recurrence-free periods, respectively. In conclusion, we were not able to confirm the microarray gene expression pattern of the 26 genes shown by Dyrskjøt et al. The discovery of accurate recurrence predictive markers, therefore, remains a challenge.
Assuntos
Perfilação da Expressão Gênica/métodos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/genética , Humanos , Recidiva Local de Neoplasia/classificação , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Neoplasias da Bexiga Urinária/classificação , Urotélio/metabolismo , Urotélio/patologiaRESUMO
The SH2 domain-containing adapter protein SHB transmits signals from receptor tyrosine kinases regulating diverse processes such as apoptosis and differentiation. To elucidate a role for SHB in cell differentiation, wild-type and R522K (inactive SH2 domain-mutant) SHB were transfected and expressed in mouse embryonic stem (ES) cells. Microarray analysis using Affymetrix U74A chips on undifferentiated ES cells and expression of selected differentiation markers after generation of embryoid bodies were subsequently assessed. Wild-type SHB altered the expression of 16 genes in undifferentiated ES cells, many of which have been found to relate to neural cell function. R522K-SHB altered the expression of 128 genes in undifferentiated ES cells, the majority of which were decreased, including several transcription factors related to development. When grown as embryoid bodies, after 4 days R522K-SHB ES cells were already found to display a different morphological appearance, with an impaired cavity formation that occurred in the absence of altered OCT4 expression. This impairment was reversed by exogenous addition of Matrigel. In addition, R522K-SHB embryoid bodies displayed reduced mRNA contents of the liver protein albumin, the pancreatic proteins amylase, glucagon and insulin after 20 days of differentiation. Matrigel did not restore the impaired expression of albumin in the R522K-SHB cells. Expression of the mesodermal marker cardiac actin and the neural marker neurofilament heavy chain alpha was not affected by wild-type or R522K-SHB overexpression. It is concluded that SHB is required for efficient differentiation of ES cells into embryoid bodies with normal cavities and cells belonging to endodermal lineages.