RESUMO
The rapidSTRIPE H1N1 test, based on a nucleic acid lateral-flow assay, has been developed for diagnosis of a swine-origin influenza A (H1N1) virus. This test is simple and cost-effective and allows specific detection of the S-OIV A (H1N1) virus from swab sampling to final detection on a lateral-flow stripe within 2 to 3 h.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/diagnóstico , Influenza Humana/virologia , Técnicas de Diagnóstico Molecular/métodos , Virologia/métodos , Humanos , Técnicas de Diagnóstico Molecular/economia , Sensibilidade e Especificidade , Fatores de Tempo , Virologia/economiaRESUMO
OBJECTIVES: To examine erythrocyte band III transport protein (SLC4A1), erythrocyte oxalate flux, and plasmatic, cellular, and urine oxalate concentrations and blood gas analyses in calcium oxalate monohydrate stone-forming patients (COM) in comparison with normal controls (NC). METHODS: Isolated red cells from 51 NC and 25 COM cases were divided for cellular oxalate measurement and for measurement of transcellular erythrocyte oxalate flux (pH 7.48-8.24). SLC4A1 protein levels were determined by Western blot analyses. Plasmatic and urinary oxalate levels and the venous blood gas analysis were measured simultaneously. RESULTS: SLC4A1 protein levels were significantly higher in COM (8.76 ± 2.12) than in NC (4.17 ± 0.61; P < .02). Cellular oxalate and venous HCO(3)(-) were significantly lower in COM (2.35 ± 0.26 µmol/L) and (24.06 ± 0.24 mmo/l) than in NC (4.03 ± 0.49 µmol/L; P < .05) and (24.93 ± 0.17 mmol/L; P < .01). Urinary oxalate was significantly higher in COM (0.31 ± 0.02 mmol/L) than in NC (0.25 ± 0.01 mmol/L; P < .04). The erythrocyte transmembrane oxalate flux correlated with the pH value and with the urinary oxalate in both groups (r = .25-.55; P = .01). With increased pH values, the oxalate flux showed inverse effects in both groups. CONCLUSIONS: SLC4A1 associated changes of HCO(3)(-) and pH levels influenced the cellular oxalate levels and urinary oxalate clearance. Under normal conditions (pH 7.55) the oxalate efflux in COM was comparable with the acid stimulated oxalate efflux in NC. The addition of HCO(3)(-) compensated the flux of COM stone formers to the levels of normal controls.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/fisiologia , Oxalato de Cálcio , Oxalatos/metabolismo , Cálculos Urinários/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Due to their particular magnetic properties, nanoparticles of metallic iron are promising candidates for magnetic fluid hyperthermia when compared to the commonly used iron oxides. However, the difficulty of handling these structures in ambient conditions without oxidation hinders its practical application. In this work, iron filled carbon nanotubes non-covalently functionalized by human serum albumin are studied as potential agents for hyperthermia. Here the iron is encapsulated inside of the carbon shells and protected from reactions with its environment. Besides protecting the iron and biological environment against each other, the carbon shells can also work as an interface for conjugation with other biological molecules of interest. In order to assess if such structures could induce any toxic effect in human cell cultures, we have probed its biocompatibility on a dosage and time dependent manner by measuring metabolic activity, cell proliferation, cell cycle distribution and apoptosis. Our results have shown that those nanotubes strongly associate with cells within a short incubation period and do not pose any significant toxic effect. The magnetic properties of iron filled carbon nanotubes in biological environment, i.e., associated to cells, have been studied and a possible rotation as a function of the applied magnetic field is discussed. Our initial findings encourage the further study of these structures as potential hyperthermia agents.
Assuntos
Materiais Biocompatíveis , Ferro , Nanotubos de Carbono , Albumina Sérica/administração & dosagem , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Febre/terapia , Humanos , Técnicas In Vitro , Magnetismo , Masculino , Microscopia Eletrônica de TransmissãoRESUMO
PURPOSE: The aim of this study was to evaluate the clinical relevance of the presence of disseminated tumor cells in peripheral blood (so-called circulating tumor cells) for renal cell carcinoma patients. METHODS: Two hundred thirty-three peripheral blood samples from 154 renal cell carcinoma patients were investigated for the presence of disseminated tumor cells by autoMACS technique and immunocytochemical staining of cytokeratin. The frequency of circulating tumor cells was analyzed statistically for correlation with relevant clinical data. RESULTS: Two kinds of tumor cells were detected: those with expression of cytokeratin 8/18 (CK+) and cells without a detectable cytokeratin expression, which we called large blue-stained cells with a tumorlike morphology. After following the CD45 autoMACS depletion protocol, we identified circulating tumor cells in 96 (41%) of 233 peripheral blood samples, which originated from 81 (53%) of 154 renal cell carcinoma patients. A significant correlation between the detection of circulating tumor cells and positive lymph node status (P < 0.001; chi(2) test) and the presence of synchronous metastases at the time of primary tumor resection (P = 0.014; chi(2) test) was found. In a multivariate Cox's regression hazard model, presence of CK+ circulating tumor cells was significantly correlated with poor overall survival for renal cell carcinoma patients (relative risk, 2.3; P = 0.048). CONCLUSIONS: The presence of circulating tumor cells correlated to lymph node status and presence of synchronous metastases in renal cell carcinoma. It is important to evaluate CK+ and blue-stained tumor cells together to determine the role of circulating tumor cells in tumor behavior and disease progression. Detection of CK+ circulating tumor cells in peripheral blood is a significant and independent prognostic factor for renal cell carcinoma.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Renais/sangue , Neoplasias Renais/sangue , Células Neoplásicas Circulantes/patologia , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Queratina-18/metabolismo , Queratina-8/metabolismo , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Metástase Linfática/patologia , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes/metabolismo , PrognósticoRESUMO
Our understanding of metastatic spread is limited and molecular mechanisms causing particular characteristics of metastasis are largely unknown. Herein, transcriptome-wide expression profiles of a unique cohort of 20 laser-resected pulmonary metastases (Mets) of 18 patients with clear-cell renal cell carcinoma (RCC) were analyzed to identify expression patterns associated with two important prognostic factors in RCC: the disease-free interval (DFI) after nephrectomy and the number of Mets per patient. Differentially expressed genes were identified by comparing early (DFI < or = 9 months) and late (DFI > or = 5 years) Mets, and Mets derived from patients with few (< or =8) and multiple (> or =16) Mets. Early and late Mets could be separated by the expression of genes involved in metastasis-associated processes, such as angiogenesis, cell migration and adhesion (e.g., PECAM1, KDR). Samples from patients with multiple Mets showed an elevated expression of genes associated with cell division and cell cycle (e.g., PBK, BIRC5, PTTG1) which indicates that a high number of Mets might result from an increased growth potential. Minimal sets of genes for the prediction of the DFI and the number of Mets per patient were identified. Microarray results were confirmed by quantitative PCR by including nine further pulmonary Mets of RCC. In summary, we showed that subgroups of Mets are distinguishable based on their expression profiles, which reflect the DFI and the number of Mets of a patient. To what extent the identified molecular factors contribute to the development of these characteristics of metastatic spread needs to be analyzed in further studies.
Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Neoplasias Pulmonares/genética , Metástase Neoplásica , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Pulmonares/secundário , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Análise Serial de TecidosRESUMO
OBJECTIVES: To examine the cellular, plasma, and urinary oxalate and erythrocyte oxalate flux in patients with calcium oxalate monohydrate (COM) stone formation vs normal controls. Pathologic oxalate clearance in humans is mostly integrated in calcium oxalate stone formation. An underlying cause of deficient oxalate clearance could be defective transmembrane oxalate transport, which, in many tissues, is regulated by an anion exchanger (SLC26). METHODS: We studied 2 groups: 40 normal controls and 41 patients with COM stone formation. Red blood cells were divided for cellular oxalate measurement and for resuspension in a buffered solution (pH 7.40); 0.1 mmol/L oxalate was added. The supernatant was measured for oxalate immediately and 1 hour after incubation. The plasma and urinary oxalate were analyzed in parallel. RESULTS: The mean cellular oxalate concentrations were significantly greater in the normal controls (5.25 +/- 0.47 micromol/L) than in those with COM stone formation (2.36 +/- 0.28 micromol/L; P < .01). The mean urinary oxalate concentrations were significantly greater in those with COM stone formation (0.31 +/- 0.02 mmol/L) than in the controls (0.24 +/- 0.02 mmol/L; P < .01). The cellular oxalate concentrations correlated significantly with the plasma (r = 0.49-0.63; P < .01) and urinary oxalate (r = -0.29-0.41; P < .03) concentrations in both groups. The plasma oxalate concentrations correlated significantly with the urinary oxalate concentrations (r = -0.30; P < .03) in the controls and with the erythrocyte oxalate flux (r = 0.25; P < .05) in those with COM stone formation. CONCLUSIONS: Our data implicate the presence of a cellular oxalate buffer to stabilize plasma and urinary oxalate concentrations in normal controls.
Assuntos
Oxalato de Cálcio , Oxalatos/metabolismo , Cálculos Urinários/metabolismo , Oxalato de Cálcio/análise , Células/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cálculos Urinários/químicaRESUMO
BACKGROUND: The knockdown of XIAP, BCL2 and BCL-X(L) by siRNAs represents a promising treatment option for bladder cancer (BCa) since the overexpression of antiapoptotic genes is often associated with tumor progression and treatment resistance. MATERIALS AND METHODS: EJ28 BCa cells were transfected with siRNAs--separately and combined--followed by analysis of target expression, viability, clonogenic survival, apoptosis and cell cycle. Furthermore, a possible chemosensitization by siRNA pretreatment was investigated. RESULTS: The siRNA-mediated inhibition of these targets--either separately or combined--reduced the targets' expression, reduced cell growth and sensitized cells to a subsequent chemotherapy. CONCLUSION: Since tumor cells may bypass the inhibition of a single gene by changing their expression profile, e.g. switch from BCL2 to BCL-X(L), the combined knockdown of multiple genes of the same pathway might be more effective in killing cancer cells. The siRNAs used represent appropriate tools for this aim since they reduced their targets' expression significantly and long-lastingly.
Assuntos
Apoptose/genética , Terapia Genética/métodos , RNA Interferente Pequeno/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/terapia , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Humanos , Mitomicina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transfecção , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/biossíntese , Proteína bcl-X/genéticaRESUMO
Genetic aberrations are crucial in renal tumor progression. In this study, we describe loss of heterozygosity (LOH) and DNA-copy number abnormalities in clear cell renal cell carcinoma (cc-RCC) discovered by genome-wide single nucleotide polymorphism (SNP) arrays. Genomic DNA from tumor and normal tissue of 22 human cc-RCCs was analyzed on the Affymetrix GeneChip Human Mapping 10K Array. The array data were validated by quantitative polymerase chain reaction and immunohistochemistry. Reduced DNA copy numbers were detected on chromosomal arm 3p in 91%, on chromosome 9 in 32%, and on chromosomal arm 14q in 36% of the tumors. Gains were detected on chromosomal arm 5q in 45% and on chromosome 7 in 32% of the tumors. Copy number abnormalities were found not only in FHIT and VHL loci, known to be involved in renal carcinogenesis, but also in regions containing putative new tumor suppressor genes or oncogenes. In addition, microdeletions were detected on chromosomes 1 and 6 in genes with unknown impact on renal carcinogenesis. In validation experiments, abnormal protein expression of FOXP1 (on 3p) was found in 90% of tumors (concordance with SNP array data in 85%). As assessed by quantitative polymerase chain reaction, PARK2 and PACRG were down-regulated in 57% and 100%, respectively, and CSF1R was up-regulated in 69% of the cc-RCC cases (concordance with SNP array data in 57%, 33%, and 38%). Genome-wide SNP array analysis not only confirmed previously described large chromosomal aberrations but also detected novel microdeletions in genes potentially involved in tumor genesis of cc-RCC.
Assuntos
Carcinoma de Células Renais/genética , Aberrações Cromossômicas , Mapeamento Cromossômico/métodos , Dosagem de Genes , Neoplasias Renais/genética , Perda de Heterozigosidade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Mapeamento Cromossômico/instrumentação , Cromossomos Humanos , Cromossomos Humanos Par 3 , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Since cancer cells are characterised by multiple genetic alterations the single inhibition of one tumour- associated gene might not be sufficient as a therapeutic strategy. We examined the effects of a combined inhibition of survivin, human telomerase reverse transcriptase (hTERT) and vascular endothelial growth factor (VEGF) with antisense oligodeoxynucleotides (AS-ODNs) and small interfering RNAs (siRNAs) in EJ28 and 5637 bladder cancer (BCa) cells. Following verification of the uptake of intraperitoneally applied fluorescence-labelled AS-ODNs and siRNAs in subcutaneous BCa xenografts, the target-directed constructs were tested as single agents in SCID mice bearing subcutaneous EJ28. Simultaneous inhibition of two of the selected transcripts significantly enhanced cell viability reduction compared to the controls consisting of a target directed construct and an appropriate control construct without any homology to the human genome. The uptake of both antisense inhibitor types in the subcutaneous BCa was achieved even without a carrier. In vivo studies with 9 consecutive intraperitoneal injections with 20 mg/kg AS-ODNs or 4.6 mg/kg siRNAs revealed the biocompatibility of both antisense inhibitor types and showed anti-tumoural activity of the AS-ODNs used.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Telomerase/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Humanos , Proteínas Inibidoras de Apoptose , Camundongos , Camundongos Nus , Camundongos SCID , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , RNA Mensageiro/metabolismo , Survivina , Telomerase/genética , Fatores de Tempo , Transfecção , Transplante Heterólogo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
PURPOSE: To evaluate the pure prognostic impact of the uPA-receptor splice variant uPAR-del4/5 for lymph node-negative breast cancer patients, and to identify differentially expressed genes associated with high or low uPAR-del4/5 mRNA levels. PATIENTS AND METHODS: mRNA transcript levels were measured by real-time PCR in tumor samples from 280 node-negative breast cancer patients who had not received adjuvant systemic therapy. Endpoints were distant metastasis-free survival (DMFS) and overall survival (OS). Gene expression analysis was performed with RNA isolated from breast cancer tissue and breast cancer cell lines using Affymetrix U133a GeneChips. RESULTS: In multivariate analysis, uPAR-del4/5 significantly contributed to the base model of traditional prognostic factors for DMFS (HR = 3.29, P < 0.001) and OS (HR = 2.87, P = 0.002). Using microarrays, seven genes were found to be up-regulated in tumor samples and cancer cell lines with high uPAR-del4/5 mRNA expression. The gene encoding rab31, a member of the Ras oncogene family, was selected for quantitative analysis of mRNA expression in the set of 280 patients. High rab31 values were significantly associated with worse outcome of patients for DMFS (HR = 2.27, P < 0.001) and OS (HR = 2.01, P = 0.008) in multivariate analysis, independent from uPAR-del4/5. The patient subgroup with high uPAR-del4/5 and rab31 levels showed the worst DMFS and OS (P < 0.001, both) compared with tumors with low values of both factors. CONCLUSIONS: Our results suggest that uPAR-del4/5 and rab31 mRNA represent independent prognostic markers in breast cancer and may be components of different, but possibly associated, tumor-relevant signaling pathways.
Assuntos
Neoplasias da Mama/genética , Receptores de Superfície Celular/genética , Proteínas rab de Ligação ao GTP/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Análise Multivariada , Metástase Neoplásica , Prognóstico , RNA Mensageiro/análise , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
The knockdown of genes that are over-expressed in cancer, and function in tumor onset and/or progression, is an attractive tool to impair the growth of tumor cells. Synthetic nucleic acids such as antisense oligodeoxynucleotides (AS-ODNs) or small-interfering RNAs (siRNAs) were applied against different tumor-associated transcripts, including the human telomerase reverse transcriptase (hTERT), to inhibit the proliferation of tumor cells and to sensitize them against chemotherapeutic (CT) agents. The efficacy of nucleic acid-based inhibitors was evaluated in vitro by determining the extent of down-regulation of the respective target mRNA and protein expression as well as by extensively investigating growth properties (e.g., viability, proliferation, apoptosis, and cell-cycle distribution) of the affected tumor cells. Methods for a successful down-regulation of hTERT and for the quantitative determination of resulting effects on cellular growth were described herein.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Biologia Molecular/métodos , Oligonucleotídeos Antissenso/farmacologia , RNA Interferente Pequeno/farmacologia , Telomerase/genética , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/genética , Contagem de Células , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Repressão Enzimática/efeitos dos fármacos , Fluorescência , Humanos , Oligonucleotídeos Antissenso/genética , RNA Interferente Pequeno/genética , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Transfecção , Ensaio Tumoral de Célula-TroncoRESUMO
OBJECTIVES: Abnormal gene activation in human tumours including bladder cancers (bCAs) may cause altered proliferation, maturation, and apoptosis as well as development of resistance to therapeutic interventions. Therefore, silencing of abnormally activated genes appears to be a rational approach for specific target-directed and sensitising therapies. METHODS: Of the available strategies for gene silencing, antisense-based techniques have attracted much attention and are the focus of this review. Putative target genes should be involved in essential tumour-promoting pathways, such as growth signalling, immortalisation, cell cycle regulation, apoptosis, angiogenesis, and development of therapy resistances. This review gives an overview of selected studies performed on bCA-derived cell lines and xenografts reporting down-regulation of potential target genes by antisense-based synthetic nucleic acids such as antisense oligodeoxynucleotides (AS-ODNs) and small interfering RNAs (siRNAs). Effects on proliferation of bCA cells and enhancement of the cytotoxic action of different chemotherapeutics were evaluated. RESULTS: Knock-down of the selected target genes frequently caused an impairment of growth of different bCA cell lines originating from cell cycle arrest or increased apoptosis. In numerous studies, the pretreatment with AS-ODNs or siRNAs provoked strong enhancement of subsequent chemotherapies, emphasising the effectiveness of these inhibition approaches. CONCLUSIONS: The application of antisense-based inhibitors in combination with chemotherapeutics might represent an alternative strategy for the adjuvant treatment of superficial bCA. Nevertheless, translation of this technology to the clinic might be hampered by inestimable off-target effects caused by AS-ODNs and their behaviour after intravesical instillation has to be evaluated in preclinical and clinical trials.
Assuntos
Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Células Cultivadas , HumanosRESUMO
Earlier investigations in our laboratory have demonstrated that UVB irradiation of cultured human keratinocytes induces the conversion of 7-dehydrocholesterol (7-DHC) to hormonally active 1alpha,25-dihydroxyvitamin D3 (calcitriol). In the research presented here, we have investigated the influence of UVB-triggered calcitriol production on gene expression of the vitamin D3 hydroxylating enzymes catabolic 25-hydroxyvitamin-D3-24-hydroxylase (CYP24A1), active vitamin-D3-25-hydroxylase (CYP27A1), and 25-hydroxyvitamin-D3-1alpha-hydroxylase (CYP27B1) using real-time PCR. Our results demonstrate a marked and wavelength-dependent induction of CYP24A1-mRNA in cultured human keratinocytes supplemented with 7-DHC, which parallels the spectral optimum at about 300 nm of calcitriol production as detected by HPLC and radioimmunoassay. Owing to the high sensitivity of real-time PCR, we provide evidence of a wavelength-dependent induction of CYP24A1-mRNA even in 7-DHC-deficient keratinocytes. Interestingly, we have found a strong but transient induction of CYP24A1-mRNA in non-irradiated keratinocytes, followed by accelerated cell proliferation. In contrast, UVB and calcitriol had no effect on gene expression of CYP27A1 and CYP27B1. We conclude from these experiments a constitutive gene expression of the vitamin D3 hydroxylases, whereas the catabolic enzyme CYP24A1 is markedly regulated by UVB, calcitriol, and perhaps cell proliferation. If confirmed at protein level, these findings could have an impact on epidermal vitamin D3 metabolism and its modulation by UVB in health and disease.
Assuntos
Calcitriol/biossíntese , Regulação da Expressão Gênica/efeitos da radiação , Queratinócitos/efeitos da radiação , RNA Mensageiro/análise , Esteroide Hidroxilases/genética , Raios Ultravioleta , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Proliferação de Células , Células Cultivadas , Colestanotriol 26-Mono-Oxigenase/genética , Humanos , Queratinócitos/metabolismo , Vitamina D3 24-HidroxilaseRESUMO
BACKGROUND: This study describes the evaluation of the expression patterns of prostate-related transcripts in 106 matched prostate tissues from prostatectomies as predictors for prostate cancer (PCa). METHODS: Quantitative PCR (QPCR) assays with site-specific hybridization probes were established for four housekeeping genes (GAPDH, HPRT, PBGD, TBP) and nine prostate-related genes (AibZIP, D-GPCR, EZH2, PCA3, PDEF, prostein, PSA, PSCA, TRPM8). RESULTS: The relative mRNA expression levels of AibZIP, D-GPCR, EZH2, PCA3, PDEF, PSA, TRPM8 (all P < 0.001) and prostein (P = 0.019) normalized to the TBP reference gene were significantly higher in malignant compared to non-malignant prostate tissues. Employing receiver-operating characteristic (ROC) analyses, PCA3 was the best single tumor marker with the highest area-under-the-curve (AUC = 0.85). A multivariate logit model for the predictability of the tumor was developed, which employed the relative expression levels of EZH2, PCA3, prostein, and TRPM8 and yielded an AUC of 0.90. CONCLUSIONS: The transcript marker PCA3 is a powerful predictor of primary PCa but the inclusion of EZH2, prostein, and TRPM8 adds even more to the diagnostic power. The finding of a significantly higher mRNA expression of three different genes (prostein, PSA, TRPM8) in organ-confined tumors compared to non-organ-confined tumors as well as the multi-marker PCa prediction model developed in the retrospective model system on prostatectomies could be of clinical importance for diagnostic purposes, and should be verified in diagnostic biopsies.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Testes Genéticos/métodos , Neoplasias da Próstata/genética , Idoso , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Biópsia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Análise Multivariada , Complexo Repressor Polycomb 2 , Valor Preditivo dos Testes , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Canais de Cátion TRPM/genética , Fatores de Transcrição/genética , Falha de TratamentoRESUMO
Human C13orf19 was previously identified to be downregulated in prostate cancer (PCa) but its function is unknown to date. In the present study, C13orf19 mRNA expression was inhibited by siRNA transfection. Furthermore, a possible regulation by androgens and the previously postulated interaction with p38 MAP kinase (p38MAPK) was investigated. The siRNA-mediated downregulation of the C13orf19 mRNA expression in the prostate cell lines PC-3 and BPH-1 was examined by quantitative PCR. Cellular viability, apoptosis, cell cycle distribution and clonogenic survival were investigated. In addition, the effects of C13orf19 downregulation in combination with chemotherapy on overall cell survival were studied. The inhibition of C13orf19 mRNA expression to 12% (after 12 h) and 55% (after 96 h) in PC-3 cells attested to a strong and persistent molecular effect provoked by the siRNA-D5 construct. However, no obvious effects on doubling time and cellular morphology were observed. Cell cycle distribution, clonogenic survival, apoptosis and cell viability showed no alterations, even after combining siRNA transfection with chemotherapy. Therefore, it can be concluded that the reduced expression of C13orf19 in PCa is not involved in the malignant transformation of the cells. A possible androgen dependence of C13orf19 mRNA expression was investigated by treating LNCaP cells with the androgen R1881 and in combination with the antiandrogen, bicalutamide. C13orf19 is expressed independently of the androgen. To analyze the putative interaction between C13orf19 and p38MAPK, PC-3 and BPH-1 cells were treated with the p38MAPK inhibitor, SB203580, and C13orf19 mRNA expression was examined. Additionally, the expression and phosphorylation status of p38MAPK after the inhibition of C13orf19 was investigated by Western blotting. No interaction between C13orf19 and p38MAPK was identified. Therefore, the gene should forthwith be named C13orf19 or Fam48A and not P38IP.
Assuntos
Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Apoptose/genética , Ciclo Celular/genética , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Humanos , Imidazóis/farmacologia , Masculino , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Piridinas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The human telomerase reverse transcriptase (hTERT) contributes to the immortal phenotype of the majority of cancers. Targeting hTERT by transfection with antisense oligonucleotides (AS-ODNs) induced immediate growth inhibition in human bladder cancer (BCa) cells. The molecular basis of the antiproliferative capacity of hTERT AS-ODNs was investigated by oligonucleotide microarray analyses and was compared to effects caused by siRNA-mediated knock-down of hTERT in EJ28 BCa cells. Two different AS-ODNs -- both down-regulated the expression of hTERT -- changed the expression of different genes mainly involved in stress response (including EGR1, ATF3 and GDF15), but without an association to telomerase function. This indicates that the immediate growth inhibition was caused, at least in part, by off-target effects. In comparison to that the blockade of the expression of hTERT using 2 different siRNAs was accompanied by the down-regulation of the oncogenes FOS-like antigen 1 (FOSL1) and epidermal growth factor receptor (EGFR), known to be overexpressed in BCa. We show here for the first time that repression of the hTERT transcript number decreased the expression of EGFR both at the mRNA and protein levels, suggesting a potential new function of hTERT in the regulation of EGFR-stimulated proliferation. Furthermore, the suppression of hTERT by siRNAs caused an enhancement of the antiproliferative capacity of the chemotherapeutics mitomycin C and cisplatin. The results presented herein may support the hypothesis that hTERT promotes the growth of tumor cells by mechanisms independent from telomere lengthening. The detailed clarification of these processes will shed light on the question, whether telomerase inhibitors might constitute suitable anticancer tools.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , RNA Interferente Pequeno/farmacologia , Telomerase/antagonistas & inibidores , Neoplasias da Bexiga Urinária/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Humanos , Análise em Microsséries , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/genética , Telomerase/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Immunotherapies might represent promising alternatives for the treatment of patients with hormone-refractory prostate cancer (HRPC). In a Phase I clinical trial, we evaluated a vaccination with dendritic cells (DCs) loaded with a cocktail consisting of HLA-A*0201-restricted peptides derived from five different prostate cancer-associated antigens [prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), survivin, prostein, transient receptor potential p8 (trp-p8)]. METHODS: Eight HRPC patients received a total of four vaccinations every other week. Clinical and immunological responses were monitored by the determination of the serum PSA levels and by enzyme linked immunospot (ELISPOT) analyses, respectively. RESULTS: Apart from local skin reactions no side effects were noted. One patient displayed a partial response (PR; PSA decrease >50%) and three other patients showed stable PSA values or decelerated PSA increases. In ELISPOT analyses, three of four PSA responders also showed antigen-specific CD8+ T-cell activation against prostein, survivin, and PSMA. CONCLUSIONS: The described protocol represents a safe and feasible concept for the induction of clinical and immunological responses. The application of a peptide cocktail-derived from different antigens as a novel treatment modality is supposed to allow for the genetic and biologic heterogeneity of PCa.
Assuntos
Vacinas Anticâncer/uso terapêutico , Células Dendríticas/imunologia , Antígenos HLA-A/uso terapêutico , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia , Idoso , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Glutamato Carboxipeptidase II/análise , Glutamato Carboxipeptidase II/imunologia , Antígenos HLA-A/química , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Técnicas Imunoenzimáticas , Separação Imunomagnética , Imunoterapia Ativa , Proteínas Inibidoras de Apoptose , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/imunologia , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/imunologia , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/patologia , Survivina , Canais de Cátion TRPM/análise , Canais de Cátion TRPM/imunologiaRESUMO
Survivin is known to be overexpressed in numerous tumor types including human bladder cancer and to cause resistance to radiation and chemotherapy. Therefore, we tested the antisense oligodeoxynucleotide AS-SVV286 and the small interfering RNA si-SVV284 to down-regulate survivin in the BCa cell lines EJ28 and 5637 thereby acting as sensitizers for chemotherapy. Pretreatment with these inhibitors followed by chemotherapy caused an enhanced decrease in cell viability. The observed reduction in cell counts associated with increased rates of apoptosis paralleled the degree of reduction of survivin expression that was achieved more efficiently by the siRNA than by the AS-ODN. Nevertheless, both therapy approaches in combination with all tested chemotherapeutics provoked a remarkable inhibition of viability and may serve as suitable additive tools for chemosensitization of bladder cancer cells.
Assuntos
Antineoplásicos/farmacologia , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Oligodesoxirribonucleotídeos Antissenso/farmacologia , RNA Interferente Pequeno/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Survivina , Neoplasias da Bexiga Urinária/patologiaRESUMO
BACKGROUND: To compare the clinical value of the measurement of complex and total PSA in the discrimination between benign prostatic hyperplasia (BPH) and prostate cancer. METHODS: In serum samples collected from 166 men with histopathologically proven clinically localized prostate cancer and of 97 men with BPH, total prostate-specific antigen (PSA), complexed PSA and the free to total PSA ratio were determined. The statistical analysis was done by the comparison of the receiver operator characteristic (ROC) curves. RESULTS: The areas under the ROC curves were 0.776 for total PSA, 0.799 for complexed PSA (total PSA vs. cPSA: p < 0.0001) and 0.812 for the free to total PSA ratio. With a cut-off of 3.0 ng/ml for complexed PSA, the sensitivity was 90%, the specificity 58%, the positive and the negative predictive values 79 and 78%, respectively. With a cut-off of 4.0 ng/ml for total PSA, the sensitivity was 87%, the specificity 59%, the positive and the negative predictive values were 78 and 72%, respectively. CONCLUSIONS: There was a statistically significant advantage for complexed PSA compared to total PSA in the discrimination between BPH and prostate cancer. The difference was, however, small and its clinical relevance is questionable.
Assuntos
Antígeno Prostático Específico/sangue , Hiperplasia Prostática/sangue , Hiperplasia Prostática/diagnóstico , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Diagnóstico Diferencial , Humanos , MasculinoRESUMO
Recently, we identified the novel protein D-GPCR (Dresden G protein-coupled receptor) which is selectively overexpressed in human prostate cancer (PCa) and belongs to the subfamily of odorant-like orphan GPCRs. Quantification of D-GPCR transcripts in paired malignant and non-malignant prostate tissues of 106 patients with primary PCa by real-time PCR demonstrated a significant up-regulation of this gene in tumor samples. Furthermore, its expression increases with higher tumor stages and grades. The evaluation of D-GPCR expression as a potential molecular tumor marker was performed by receiver-operating characteristic curve (ROC) analysis resulting in an area under the curve (AUC) value of 0.6452. Hence, the evaluation of D-GPCR as possible additive diagnostic tool and putative therapy target appears promising.