RESUMO
To investigate a possible central mechanism of action of Botulinum toxin A (BoNT/A) following injection in the bladder, complementary to the acknowledged peripheral bladder effect, we studied changes in the expression of neuropeptides and receptors involved in lower urinary tract function in the spinal cord (SC) and dorsal root ganglia (DRG) of normal rats following BoNT/A bladder injection. Thirty-six Sprague-Dawley rats, divided into three groups of n = 12, received bladder injections of 2U or 5U OnabotulinumtoxinA (BOTOX®), or saline. Six animals from each group were sacrificed on days 7 and 14. Expression of Tachykinin 1 (Tac1), capsaicin receptor (TRPV1), neuropeptide Y (NPY), proenkephalin (PENK) and muscarinic receptors M1, M2, M3, was evaluated in the bladder, L6-S1 DRG, and SC segments using real-time PCR and Western blotting. Real-time PCR revealed increased expression of NPY in all tissues except for SC, and increased TRPV1 and PENK expression in DRG and SC, whereas expression of Tac1, M1 and M2 was decreased. Less significant changes were noted in protein levels. These findings suggest that bladder injections of OnabotulinumtoxinA may be followed by changes in the expression of sensory, sympathetic and cholinergic bladder function regulators at the DRG/SC level.
Assuntos
Toxinas Botulínicas Tipo A , Animais , Ratos , Toxinas Botulínicas Tipo A/farmacologia , Bexiga Urinária/metabolismo , Ratos Sprague-Dawley , Gânglios Espinais/metabolismo , Medula Espinal/metabolismoRESUMO
PURPOSE: Several studies have shown frequent changes in DNA methylation in bladder cancer (BCa), which vary among different geographical areas. The aim of this study is to examine the diagnostic accuracy of a panel of DNA methylation biomarkers in a Greek clinical setting contributing to the development of a universal panel of urine biomarkers. MATERIALS AND METHODS: Individuals with primary BCa and control individuals matching the gender, age and smoking status of the cancer patients were recruited. DNA methylation was assessed for the gene promoters of RASSF1, RARB, DAPK, TERT and APC in urine samples collected by spontaneous urination using quantitative Methylation Specific PCR (qMSP). All genes had been previously separately associated with BCa. RESULTS: Fifty patients and 35 healthy controls were recruited, with average age of 70.26 years and average smoking status of 44.78 pack-years. In the BCa group, DNA methylation was detected in 27 (61.4%) samples. RASSF1 was methylated in 52.2% of samples. Only 3 (13.6%) samples from the control group were methylated, all in the RASSF1 gene promoter. The specificity and sensitivity of this panel of genes to diagnose BCa was 86% and 61% respectively. The RASSF1 gene could diagnose BCa with specificity 86.4% and sensitivity 52.3%. CONCLUSION: Promoter DNA methylation of this panel of five genes could be further investigated as urine biomarker for the diagnosis of BCa. The RASSF1 could be a single candidate biomarker for predicting BCa patients versus controls. Studies are required in order to develop a geographically adjusted diagnostic biomarker for BCa.
Assuntos
Metilação de DNA , Neoplasias da Bexiga Urinária , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/urina , Adutos de DNA , Humanos , Bexiga Urinária , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/urinaRESUMO
Noroviruses (NoVs) are important human pathogens associated with acute viral gastroenteritis worldwide, displaying significant genetic heterogeneity. Genotype GII.4 is responsible for the majority of outbreaks reported to date. A total of 460 faecal samples from sporadic gastroenteritis cases were screened for the presence of NoV RNA. Four additional human samples collected during a waterborne NoV gastroenteritis outbreak observed in 2005 in northern Greece, were also included in the study. All PCR-positive samples were tested further using a multiplex RT-PCR, which targets the viral capsid VP1 region D. PCR products from all outbreak samples and from 20 randomly selected samples were sequenced. Phylogenetic analysis revealed that GII.4 genotype predominated (70%), while genotypes GII.2 (10%), GII.7 (15%), and GI.1 (5%) were also detected. All the outbreak NoV strains belonged to the GI.1 genotype. The present study provides a first insight into the epidemiology and genetic diversity of NoVs in Greece and shows that various strains are circulating in the country and cause sporadic cases or outbreaks.
Assuntos
Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Gastroenterite/epidemiologia , Gastroenterite/virologia , Norovirus/classificação , Norovirus/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Análise por Conglomerados , Fezes/virologia , Feminino , Genótipo , Grécia/epidemiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Multiplex , Norovirus/isolamento & purificação , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência , Adulto JovemRESUMO
PURPOSE: To study the effect of acute exposure of rabbit eyes to artificial sunlight in vivo, on the integrity of corneal and conjunctival tissue as well as on the gene expression of the receptor for platelet activating factor (PAFR). METHODS: New Zealand albino rabbits were immobilized opposite a 300 W Osram Ultra-Vitalux® light bulb with an emission radiation spectrum similar to that of normal sunlight at noon, and exposed to ultraviolet B radiation in the range of the reported threshold for corneal damage. Corneal and third eyelid tissue samples were removed from exposed eyes at 2, 6 and 24 h following the end of the exposure to the bulb light and were subsequently processed for histochemical staining and RNA extraction. The gene expression of PAFR was detected with real time reverse transcription polymerase chain reaction. RESULTS: Some epithelial shedding was detected in the corneal tissue as a result of acute exposure to artificial sunlight. In the eyelid conjunctiva, a marked accumulation of eosinophils was noticed, as early as 2 h post-exposure, apparently directed toward the upper part of the epithelial layer. This effect appears to subside by hour 24. No statistically significant changes in gene expression were detected in the corneal tissue, whereas in the third eyelid, PAFR gene expression was significantly induced, most prominently at t = 2 and 6 h post-exposure. CONCLUSION: Acute exposure of rabbit eyes to artificial sunlight induced a marked infiltration of eosinophils into the epithelial layer of the conjunctiva but no gross alterations in the cornea or the third eyelid. The gene expression of PAFR was upregulated, as an effect of light exposure, in the third eyelid but not in the cornea.
Assuntos
Túnica Conjuntiva/efeitos da radiação , Córnea/efeitos da radiação , Iluminação/efeitos adversos , Membrana Nictitante/efeitos da radiação , Glicoproteínas da Membrana de Plaquetas/genética , Receptores Acoplados a Proteínas G/genética , Animais , Túnica Conjuntiva/patologia , Túnica Conjuntiva/fisiologia , Córnea/patologia , Córnea/fisiologia , Eosinófilos/patologia , Eosinófilos/efeitos da radiação , Expressão Gênica/efeitos da radiação , Iluminação/métodos , Membrana Nictitante/patologia , Membrana Nictitante/fisiologia , Estimulação Luminosa/métodos , Coelhos , Luz SolarRESUMO
Cancers of the respiratory tract (lung and head and neck) share common aetiologies, risk factors and molecular characteristics. Epigenetic reprogramming is one of the hallmarks of cancer and DNA methylation is currently the best-studied form. There are a number of characteristics of DNA methylation, which seem advantageous in biomarker development. Early detection is still an unmet clinical care need, which guarantees to significantly reduce the mortality of patients with respiratory cancers. The application of such biomarkers in biological fluids being sampled in everyday clinical practice is a long-term demand. In this review we summarise the current literature on DNA methylation detection in bronchial washings, sputum, saliva, plasma and serum and discuss the potential of their clinical implementation. We also discuss important aspects of biomarker development and validation pointing to the appropriate route for a biomarker to reach clinical standards.
Assuntos
Biomarcadores Tumorais/metabolismo , Líquidos Corporais/metabolismo , Metilação de DNA , Detecção Precoce de Câncer/métodos , Neoplasias do Sistema Respiratório/diagnóstico , Neoplasias do Sistema Respiratório/metabolismo , Animais , Sequência de Bases , Humanos , Neoplasias do Sistema Respiratório/genéticaRESUMO
The exceptional high mortality of lung cancer can be instigated to a high degree by late diagnosis. Despite the plethora of studies on potential molecular biomarkers for lung cancer diagnosis, very few have reached clinical implementation. In this study, we developed a panel of DNA methylation biomarkers and validated their diagnostic efficiency in bronchial washings from a large retrospective cohort. Candidate targets from previous high-throughput approaches were examined by pyrosequencing in an independent set of 48 lung tumor/normal paired. Ten promoters were selected and quantitative methylation-specific PCR (qMSP) assays were developed and used to screen 655 bronchial washings from the Liverpool Lung Project (LLP) subjects divided into training (194 cases and 214 controls) and validation (139 cases and 109 controls) sets. Three statistical models were used to select the optimal panel of markers and to evaluate the performance of the discriminatory algorithms. The final logit regression model incorporated hypermethylation at p16, TERT, WT1, and RASSF1. The performance of this 4-gene methylation signature in the validation set showed 82% sensitivity and 91% specificity. In comparison, cytology alone in this set provided 43% sensitivity at 100% specificity. The diagnostic efficiency of the panel did not show any biases with age, gender, smoking, and the presence of a nonlung neoplasm. However, sensitivity was predictably higher in central (squamous and small cell) than peripheral (adenocarcinomas) tumors, as well as in stage 2 or greater tumors. These findings clearly show the impact of DNA methylation-based assays in the diagnosis of cytologically occult lung neoplasms. A prospective trial is currently imminent in the LLP study to provide data on the enhancement of diagnostic accuracy in a clinical setting, including by additional markers.
Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Neoplasias Pulmonares/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Casos e Controles , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
p73 possesses an extrinsic P1 promoter and an intrinsic P2 promoter controlling the expression of the pro-apoptotic TAp73 isoforms and the anti-apoptotic ΔΝp73 isoforms respectively. In this study, we investigated the DNA methylation status of both promoters as a means of epigenetic transcriptional control of their corresponding isoforms in 102 primary non-small cell lung carcinomas (NSCLCs). We demonstrated that while P1 hypermethylation-associated reduction of TAp73 mRNA levels is relatively infrequent, the P2 hypomethylation-associated over-expression of ΔΝp73 mRNA is a frequent event, particularly among squamous cell carcinomas. P2 hypomethylation strongly correlated with LINE-1 element hypomethylation, indicating that ΔΝp73 over-expression may be a passive consequence of global DNA hypomethylation.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Ativação Transcricional , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Proteína Tumoral p73RESUMO
Vanadium exerts a variety of biological effects, including antiproliferative responses through activation of the respective signaling pathways and the generation of reactive oxygen species. As epidermal cells are exposed to environmental insults, human keratinocytes (HaCaT) were used to investigate the mechanism of the antiproliferative effects of vanadyl(IV) sulfate (VOSO(4)). Treatment of HaCaT cells with VOSO(4) inhibited proliferation and induced apoptosis in a dose-dependent manner. Inhibition of proliferation was associated with downregulation of cyclins D1 and E, E2F1, and the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1). Induction of apoptosis correlated with upregulation of the c-fos oncoprotein, changes in the expression of clusterin (CLU), an altered ratio of antiapoptotic to proapoptotic Bcl-2 protein family members, and poly(ADP-ribose) polymerase-1 cleavage. Forced overexpression of c-fos induced apoptosis in HaCaT cells that correlated with secretory CLU downregulation and upregulation of nuclear CLU (nCLU), a pro-death protein. Overexpression of Bcl-2 protected HaCaT cells from vanadium-induced apoptosis, whereas secretory CLU overexpression offered no cytoprotection. In contrast, nCLU sensitized HaCaT cells to apoptosis. Our data suggest that vanadium-mediated apoptosis was promoted by c-fos, leading to alterations in CLU isoform processing and induction of the pro-death nCLU protein.
Assuntos
Apoptose/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Clusterina/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Vanádio/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Clusterina/genética , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Regulação para Cima/efeitos dos fármacosRESUMO
2-Methoxyestradiol (2-ME2) induces leukemia cells to undergo apoptosis in association with Bcl-2 inactivation but the mechanisms whereby Bcl-2 contributes to protection against programmed cell death in this context remain unclear. Here we showed that 2-ME2 inhibited the proliferation of Jurkat leukemia cells by markedly suppressing the levels of cyclins D3 and E, E2F1 and p21(Cip1/Waf1) and up-regulating p16(INK4A). Further, 2-ME2 induced apoptosis of Jurkat cells in association with down-regulation and phosphorylation of Bcl-2 (as mediated by JNK), up-regulation of Bak, activation of caspases-9 and -3 and PARP-1 cleavage. To determine the importance and mechanistic role of Bcl-2 in this process, we enforced its expression in Jurkat cells by retroviral transduction. Enforcing Bcl-2 expression in Jurkat cells abolished 2-ME2-induced apoptosis and instead produced a G1/S phase cell cycle arrest in association with markedly increased levels of p27(Kip1). Bcl-2 and p27(Kip1) were localized mainly in the nucleus in these apoptotic resistant cells. Interestingly, NF-kappaB activity and p50 levels were increased by 2-ME2 and suppression of NF-kappaB signaling reduced p27(Kip1) expression and sensitized cells to 2-ME2-induced apoptosis. Importantly, knocking-down p27(Kip1) in Jurkat Bcl-2 cells sensitized them to spontaneous and 2-ME2-induced apoptosis. Thus, Bcl-2 prevented the 2-ME2-induced apoptotic response by orchestrating a p27(Kip1)-dependent G1/S phase arrest in conjunction with activating NF-kappaB. Thus, we achieved a much better understanding of the penetrance and mechanistic complexity of Bcl-2 dependent anti-apoptotic pathways in cancer cells and why Bcl-2 inactivation is so critical for the efficacy of apoptosis and anti-proliferative inducing drugs like 2-ME2.
Assuntos
Apoptose/efeitos dos fármacos , Estradiol/análogos & derivados , Fase G1/fisiologia , Células Jurkat/efeitos dos fármacos , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Fase S/fisiologia , 2-Metoxiestradiol , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Quinases Ciclina-Dependentes/antagonistas & inibidores , Estradiol/farmacologia , Fase G1/efeitos dos fármacos , Humanos , Células Jurkat/citologia , Fase S/efeitos dos fármacosRESUMO
Normal cells divide for a limited number of generations, after which they enter a state of irreversible growth arrest termed replicative senescence. While replicative senescence is due to telomere erosion, normal human fibroblasts can undergo stress-induced senescence in response to oncogene activation, termed oncogene-induced senescence (OIS). Both, replicative and OIS, initiate a DNA damage checkpoint response (DDR) resulting in the activation of the p53-p21(Cip1/Waf1) pathway. However, while the nuclear factor-kappaB (NF-kappaB) signaling pathway has been implicated in DDR, its role in OIS has not been investigated. Here, we show that oncogenic Ha-RasV12 promoted premature senescence of IMR-90 normal human diploid fibroblasts by activating DDR, hence verifying the classical model of OIS. However, enforced expression of a constitutively active IKKbeta T-loop mutant protein (IKKbetaca), significantly delayed OIS of IMR-90 cells by suppressing Ha-RasV12 instigated DDR. Thus, our experiments have uncovered an important selective advantage in chronically activating canonical NF-kappaB signaling to overcome the anti-proliferative OIS response of normal primary human fibroblasts.
Assuntos
Senescência Celular , Dano ao DNA , Fibroblastos/metabolismo , NF-kappa B/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Transdução de Sinais , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fibroblastos/citologia , Humanos , NF-kappa B/genética , Proteína Oncogênica p21(ras)/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The role of clusterin/apolipoprotein J (Clu/ApoJ) and Bcl-2 on C(2)-ceramide-induced apoptosis of embryonic human diploid fibroblasts, MRC-5 and immortalized adult skin keratinocytes, HaCaT was investigated. C(2)-ceramide-induced apoptosis of HaCaT in a time- and dose-dependent manner, while in MRC-5 only at higher concentrations. There was a dose-dependent accumulation of Clu/ApoJ and downregulation of Bcl-2 which correlated with C(2)-ceramide-induced apoptosis of MRC-5. While overexpression of Bcl-2 suppressed C(2)-ceramide-mediated apoptosis in both cell types, Clu/ApoJ failed to do so, accessed by morphological changes, DNA fragmentation and PARP cleavage. There was no change in the expression of endogenous p53 or p21(Waf1/Cip1) upon C(2)-ceramide treatment of MRC-5. However, mutant p53(143ala) increased the sensitivity of MRC-5 to C(2)-ceramide-induced apoptosis by markedly downregulating Bcl-2, pointing to a role for p53. These results suggested that whereas downregulation of Bcl-2 may be a crucial factor involved in C(2)-ceramide-induced apoptosis, accumulation of Clu/ApoJ may be a signal of stress response. Moreover, the ceramide-activated apoptotic pathway may be regulated by p53.