RESUMO
BACKGROUND: Immune checkpoint blockade with Programmed cell death 1 (PD-1)/PD-L1 inhibitors has been effective in various malignancies and is considered as a standard treatment modality for patients with non-small-cell lung cancer (NSCLC). However, emerging evidence show that PD-1/PD-L1 blockade can lead to hyperprogressive disease (HPD), a flair-up of tumor growth linked to dismal prognosis. This study aimed to evaluate the incidence of HPD and identify the determinants associated with HPD in patients with NSCLC treated with PD-1/PD-L1 blockade. PATIENTS AND METHODS: We enrolled patients with recurrent and/or metastatic NSCLC treated with PD-1/PD-L1 inhibitors between April 2014 and November 2018. Clinicopathologic variables, dynamics of tumor growth, and treatment outcomes were analyzed in patients with NSCLC who received PD-1/PD-L1 blockade. HPD was defined according to tumor growth kinetics (TGK), tumor growth rate (TGR), and time to treatment failure (TTF). Immunophenotyping of peripheral blood CD8+ T lymphocytes was conducted to explore the potential predictive biomarkers of HPD. RESULTS: A total of 263 patients were analyzed. HPD was observed in 55 (20.9%), 54 (20.5%), and 98 (37.3%) patients according to the TGK, TGR, and TTF. HPD meeting both TGK and TGR criteria was associated with worse progression-free survival [hazard ratio (HR) 4.619; 95% confidence interval (CI) 2.868-7.440] and overall survival (HR, 5.079; 95% CI, 3.136-8.226) than progressive disease without HPD. There were no clinicopathologic variables specific for HPD. In the exploratory biomarker analysis with peripheral blood CD8+ T lymphocytes, a lower frequency of effector/memory subsets (CCR7-CD45RA- T cells among the total CD8+ T cells) and a higher frequency of severely exhausted populations (TIGIT+ T cells among PD-1+CD8+ T cells) were associated with HPD and inferior survival rate. CONCLUSION: HPD is common in NSCLC patients treated with PD-1/PD-L1 inhibitors. Biomarkers derived from rationally designed analysis may successfully predict HPD and worse outcomes, meriting further investigation of HPD.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Progressão da Doença , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/imunologia , Metástase Linfática , Linfócitos do Interstício Tumoral/imunologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Prognóstico , Taxa de Sobrevida , Carga TumoralRESUMO
Background: Tumor-specific mutations form novel immunogenic peptides called neoantigens. Neoantigens can be used as a biomarker predicting patient response to cancer immunotherapy. Although a predicted binding affinity (IC50) between peptide and major histocompatibility complex class I is currently used for neoantigen prediction, large number of false-positives exist. Materials and methods: We developed Neopepsee, a machine-learning-based neoantigen prediction program for next-generation sequencing data. With raw RNA-seq data and a list of somatic mutations, Neopepsee automatically extracts mutated peptide sequences and gene expression levels. We tested 14 immunogenicity features to construct a machine-learning classifier and compared with the conventional methods based on IC50 regarding sensitivity and specificity. We tested Neopepsee on independent datasets from melanoma, leukemia, and stomach cancer. Results: Nine of the 14 immunogenicity features that are informative and inter-independent were used to construct the machine-learning classifiers. Neopepsee provides a rich annotation of candidate peptides with 87 immunogenicity-related values, including IC50, expression levels of neopeptides and immune regulatory genes (e.g. PD1, PD-L1), matched epitope sequences, and a three-level (high, medium, and low) call for neoantigen probability. Compared with the conventional methods, the performance was improved in sensitivity and especially two- to threefold in the specificity. Tests with validated datasets and independently proven neoantigens confirmed the improved performance in melanoma and chronic lymphocytic leukemia. Additionally, we found sequence similarity in proteins to known pathogenic epitopes to be a novel feature in classification. Application of Neopepsee to 224 public stomach adenocarcinoma datasets predicted â¼7 neoantigens per patient, the burden of which was correlated with patient prognosis. Conclusions: Neopepsee can detect neoantigen candidates with less false positives and be used to determine the prognosis of the patient. We expect that retrieval of neoantigen sequences with Neopepsee will help advance research on next-generation cancer immunotherapies, predictive biomarkers, and personalized cancer vaccines.
Assuntos
Antígenos/genética , Genoma , Aprendizado de Máquina , Aminoácidos/imunologia , Conjuntos de Dados como Assunto , HumanosRESUMO
In the present study, we established a novel murine model of vitiligo by sequential prime/boost immunizations into the hind footpad and tail dermis with tyrosinase-related protein 2 (TRP2)-180 (SVYDFFVWL) peptide, lipopolysaccharides and cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides. Immunized mice developed epidermal depigmentation in the tail skin without hair depigmentation, thereby differentiating this approach from established models of vitiligo. Following intradermal tail immunization, activated CD8(+) interferon (IFN)-γ(+) T cells were recruited locally to the tail skin. In-vivo cytotoxicity assays demonstrated specific lysis of TRP2-180-presenting cells in immunized mice. Furthermore, the extent of skin depigmentation correlated with the frequency of TRP2-180-specific splenic CD8(+) T cells, as determined by IFN-γ and tumour necrosis factor (TNF)-α production, and cytotoxic degranulation evidenced by CD107a staining. These findings suggest a correlation between the presence of TRP2-180-specific CD8(+) effector T cells and the development of depigmented skin lesions in our vitiligo model. This new model of vitiligo, characterized by skin depigmentation without hair depigmentation, is more similar to human disease than previous murine models. Therefore, this model is well suited to future studies on the pathogenesis of vitiligo and the development of novel therapeutics for vitiligo.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Epiderme/imunologia , Melanócitos/imunologia , Transtornos da Pigmentação/imunologia , Transtornos da Pigmentação/patologia , Vitiligo/imunologia , Vitiligo/patologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Epiderme/metabolismo , Epiderme/patologia , Humanos , Masculino , Melanócitos/patologia , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Transtornos da Pigmentação/etiologia , Vacinação/métodos , Vitiligo/etiologia , Vitiligo/metabolismoRESUMO
BACKGROUND: Since the recent establishment of a murine model of eosinophilic chronic rhinosinusitis with nasal polyps (CRSwNP), both the development of new drugs for treatment or prevention of eosinophilic CRSwNP and elucidation of their pathogenesis have been feasible. We investigated the therapeutic effects of resveratrol on CRSwNP and its mechanism of action using a murine model. METHODS: After induction of eosinophilic CRSwNP, the therapeutic effects of resveratrol were tested and compared with those of triamcinolone acetonide. Histopathologic changes were evaluated using hematoxylin and eosin for overall inflammation, Sirius red for eosinophils, and Masson's trichrome stain for collagen. The expression levels of the interleukin (IL)-4, IL-5, prostaglandin D synthase, and leukotriene C4 synthase genes were assessed by quantitative real-time PCR. Cyclooxygense-2 and 5-lipoxygense levels were evaluated by immunohistochemical staining and Western blot analysis. RESULTS: The degree of eosinophilic infiltration and subepithelial fibrosis was significantly decreased by administration of high-dose resveratrol, the potency of which was similar to that of triamcinolone acetonide. The expression levels of the IL-4, IL-5, prostaglandin D synthase, and leukotriene C4 synthase genes were significantly decreased by administration of low- or high-dose resveratrol. The production of 5-lipoxygenase was strongly inhibited by high-dose resveratrol. CONCLUSIONS: Resveratrol may be useful for the prevention of eosinophilic CRSwNP. A key mechanism of its action is believed to be its anti-inflammatory effect, particularly on eosinophils, by inhibiting the lipoxygenase pathway.
Assuntos
Eosinofilia/tratamento farmacológico , Pólipos Nasais/tratamento farmacológico , Rinite/tratamento farmacológico , Sinusite/tratamento farmacológico , Estilbenos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Biópsia por Agulha , Western Blotting , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Eosinofilia/complicações , Eosinofilia/imunologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Pólipos Nasais/complicações , Pólipos Nasais/patologia , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Resveratrol , Rinite/complicações , Rinite/imunologia , Medição de Risco , Sinusite/complicações , Sinusite/imunologia , Estatísticas não Paramétricas , Resultado do TratamentoRESUMO
Some autoimmune complications such as postinfectious encephalomyelitis are associated with immunologic abnormalities induced by measles virus infection. To address the superantigenic stimulation in measles which might be related with autoimmune complications, T-cells bearing the TCRBV5S2 or TCRBV8 chains and the expression of activation markers were analyzed by monoclonal antibodies. To estimate clonal expansions, the CDR3 length profile in T-cells bearing the TCRBV5S2 or TCRBV8 chains was analyzed by two-stage PCR. Results showed that the expression of DR molecules in CD3+ cells was increased significantly in measles patients (19.6 +/- 20.7%) compared to healthy children (2.9 +/- 1.4%). The mean percentage (7.1 +/- 4.4%) of T-cells bearing the TCRBV8 chain was increased in measles patients compared to healthy children (5.6 +/- 3.1%). The percentage of T-cells bearing the TCRBV5S2 chain in measles patients (3.0 +/- 1.2%) was similar to that in healthy children (2.7 +/- 0.6%). By analysis of the CDR3 length we found that there was no evidence of clonal expansions in T-cells bearing the TCRBV8 chain and that there were clonal expansions in T-cells bearing the TCRBV5S2 chain. These data suggest a conventional antigenic stimulation with T-cells bearing the TCRBV5S2 chain and a superantigenic stimulation with T-cells bearing the TCRBV8 chain may occur in the acute stage of measles infection.
Assuntos
Sarampo/imunologia , Linfócitos T/imunologia , Anticorpos Monoclonais/imunologia , Doenças Autoimunes/complicações , Complexo CD3/análise , Complexo CD3/genética , Relação CD4-CD8 , Pré-Escolar , Células Clonais , Feminino , Citometria de Fluxo , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Antígenos HLA-DR/análise , Humanos , Masculino , Sarampo/complicações , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Interleucina/análise , Superantígenos/imunologiaRESUMO
Hepatocellular carcinoma (HCC) is one of the tumors known to be resistant to Fas-mediated apoptosis. To elucidate the possible mechanisms of this resistance, we examined the mRNA transcripts of genes related to Fas in 10 hepatitis B virus (HBV)-associated HCC cell lines and six HBV-associated HCC tissues. Most of the HBV-associated HCC cell lines showed markedly decreased Fas expression. Some cell lines expressed FAP, a possible inhibitor of apoptosis, or showed downregulated expression of FADD or FLICE, molecules essential to Fas-mediated apoptosis. All six HBV-associated HCC tissues also showed decreased Fas expression compared with their non-malignant counterparts. Our data demonstrate that HBV-associated HCC showed a common defect in the expression of Fas, upregulation of FAP and downregulation of downstream molecules such as FADD and FLICE.
Assuntos
Apoptose/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas/genética , Carcinoma Hepatocelular/virologia , Regulação para Baixo , Citometria de Fluxo , Imunofluorescência , Hepatite B/complicações , Humanos , Neoplasias Hepáticas/virologia , RNA Mensageiro/análise , RNA Neoplásico/análise , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Alpha-synuclein, a presynaptic protein of the central nervous system, has been implicated in the synaptic events such as neuronal plasticity during development and learning, and neuronal degeneration under pathological conditions. As an effort to understand the biological function of alpha-synuclein, we examined the expression patterns of alpha-synuclein in various human hematopoietic cells, and in Drosophila at different developmental stages. The alpha-synuclein was ubiquitously expressed in all the tested hematopoietic cells including T cells, B cells, NK cells, and monocytes, as well as in the lymphoma cell lines, Jurkat and K562. A potential alpha-synuclein homologue was also expressed in Drosophila, and its expression appeared to be temporally and spatially regulated during development. Our data suggest that alpha-synuclein may function in invertebrates as well as in vertebrates and its function may not be restricted to the neuron.
Assuntos
Drosophila/genética , Leucócitos Mononucleares/metabolismo , Proteínas do Tecido Nervoso/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Western Blotting , Drosophila/embriologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células Jurkat , Células K562 , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/citologia , Monócitos/citologia , Monócitos/metabolismo , RNA/genética , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinucleínas , Linfócitos T/citologia , Linfócitos T/metabolismo , alfa-SinucleínaRESUMO
Human natural killer (NK) cells have receptors that bind to HLA class I molecules. Among these receptors, p58 and p50 bind to HLA-C. P58 belongs to the killer inhibitory receptor (KIR) and p50 belongs to the killer activatory receptor (KAR). Previously, we obtained three recombinant p58 KIR or p50 KAR proteins, KAR-K1 (KIR2DS4), KIR-K6 (KIR2DL1), and KIR-K7 (KIR2DL3). In this study, we produced and characterized seven monoclonal antibodies (MAbs) to the p58 KIR and p50 KAR proteins. The MAbs were classified in three groups according to their antigen-binding specificity: 5IB103 and 26ID707 were KAR-K1-specific; A809, 190IIC311, and 197IIC611 were KIR-K7-specific; while A210 and A803g bound to all three recombinant proteins. The MAbs reactive to KIR-K7 bound to the gamma3 domain among two immunoglobulin (Ig) domains of KIR-K7. Immunofluorescence staining and flow cytometric analysis with A803g showed reactivity to about 10% of peripheral blood mononuclear cells and 35% of purified natural killer cells. Double immunofluorescence staining with A803g and anti-CD56 Ab showed that CD56 and p58 or p50 were expressed on NK cells in a mutually exclusive way. We also investigated T-cell markers in A803g+ cells. A803g+ T cells were almost CD8+ cells. MAbs produced in this study can be utilized practically in the investigation of biological characteristics of p58 KIR and p50 KAR.
Assuntos
Anticorpos Monoclonais/imunologia , Células Matadoras Naturais/imunologia , Receptores Imunológicos/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Western Blotting , Antígenos CD4/análise , Antígeno CD56/análise , Antígenos CD8/análise , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos , Hibridomas/imunologia , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores de IgG/análise , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores KIR , Receptores KIR2DL1 , Receptores KIR2DL3 , Receptores de Células Matadoras Naturais , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Subpopulações de Linfócitos T/imunologiaRESUMO
Sporadic occurrence of transformed tumor cells is under the surveillance of the host immune system and such cells are effectively eliminated by immune-mediated cell death. During tumor progression, the antitumor effects of the tumor microenvironment are suppressed by diverse immunosuppressive mechanisms. In this research, we suggest novel immune evasion strategy of tumor cells through a transforming growth factor (TGF)-ß1- and hypoxia-dependent mechanism. Experimental results showed that TGF-ß1 and hypoxia induced mitogen-activated protein kinase phosphatase (MKP)-1 expression within 1 h, resulting in attenuation of c-Jun N-terminal kinase (JNK) phosphorylation and subsequent death receptor-mediated cell death. In addition, analysis of microarray data and immunostaining of MKP-1 in hepatocellular carcinoma (HCC) patient samples revealed that expression of MKP-1 is notably higher in tumors than in normal tissues, implying that MKP-1-dependent suppression of immune-mediated cell death takes place only in the tumor. To prove that MKP-1 can act as a mediator of immune escape by tumors, we determined whether chemo-resistance against several anticancer drugs could be overcome by knockdown of MKP-1. Cytotoxic assays showed that chemotherapy with siRNA targeting MKP-1 was significantly more effective than chemotherapy in the presence of MKP-1. Thus, we conclude that TGF-ß1 and hypoxia ensure tumor cell survival and growth through expression of MKP-1.
Assuntos
Apoptose/efeitos dos fármacos , Hipóxia Celular , Fosfatase 1 de Especificidade Dupla/metabolismo , Receptores de Morte Celular/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Bases de Dados Factuais , Fosfatase 1 de Especificidade Dupla/antagonistas & inibidores , Fosfatase 1 de Especificidade Dupla/genética , Células Hep G2 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Transgênicos , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/antagonistas & inibidores , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
INTRODUCTION: Cytomegalovirus (CMV) can reactivate causing serious clinical problems during immunosuppression. CMV-specific CD8(+) T cells play an important role in the control of CMV reactivation. Using pentameric major histocompatibility complex (MHC) peptide complexes, we investigated cellular immune responses to CMV among healthy individuals and kidney transplantation recipients in Korea, which is an endemic area of CMV infection. MATERIALS AND METHODS: Analysis of CMV-specific T cells was performed on 28 healthy individuals and 40 recipients who bore human leukocyte antigen (HLA)-A2 or -A24. CMV pp65 pentamer-binding cells incubated with various monoclonal antibodies were measured by four-color flow cytometry. RESULTS: Detectable levels of pentamer(+) CD8(+) T cells were present in 109/139 samples (78.4%) that stained with the A*02NLV-pentamer, while 15/67 samples (22.4%) stained with the A*24QYD-pentamer (P < .01). Among patients with HLA-A2, 22/24 (91.7%) samples showing positive CMV antigenemia revealed detectable pentamer(+) CD8(+) T cells, while 87/115 (75.7%) displaying negative CMV antigenemia had detectable pentamer(+) CD8(+) T cells (P = .04). There was no significant difference in percentages of pentamer(+) CD8(+) T cells between patients who did versus who did not experience episodes of CMV infection. The subpopulation of CMV-specific CD8(+) T cells in transplantation recipients was evaluated using phenotypic markers; memory cells comprised the majority of the CMV-specific CD8(+) T-cell population. CONCLUSION: The A*02NLV-pentamer complex was useful to monitor CMV-specific T cells. However, MHC pentamer-based techniques did not provide a clear distinction between patients who are or are not at risk for CMV infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/imunologia , Transplante de Rim , Complexo Principal de Histocompatibilidade , Adulto , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Monitorização FisiológicaRESUMO
It has been postulated that tumor cells expressing Fas ligand (FasL) can evade immune surveillance by inducing apoptosis in T cells expressing Fas. In this study, we investigated FasL expression in 13 human hepatoma cell lines. Strong FasL expression was detected by reverse transcription-polymerase chain reaction or immunofluorescence in Hep G2.2.15, in which the hepatitis-B-virus (HBV) genome was transfected, and in SNU-354, which showed HBx transcripts. To determine the biological activity of FasL, Hep G2.2. 15 was co-cultured with MOLT-4, T-cell-leukemia cells. Hep G2.2.15 induced apoptosis in MOLT-4 and this was inhibited by the antagonistic anti-Fas antibody, ZB4. For further analysis of the role of HBx in the induction of FasL, PLC/PRF/5 cells were transfected transiently with the HBV genome, or HBx, or the frameshift mutant of HBx. In PLC/PRF/5 cells transfected with the HBV genome or HBx but not in cells transfected with the frameshift mutant of HBx, FasL expression was detected. Our data suggest that HBx plays a role in the induction of FasL in hepatoma cells and in the escape from immune surveillance.
Assuntos
Apoptose/fisiologia , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Glicoproteínas de Membrana/fisiologia , Transativadores/fisiologia , Carcinoma Hepatocelular/virologia , Proteína Ligante Fas , Vírus da Hepatite B/genética , Humanos , Neoplasias Hepáticas/virologia , Glicoproteínas de Membrana/metabolismo , Transativadores/genética , Transfecção , Células Tumorais Cultivadas , Evasão Tumoral , Proteínas Virais Reguladoras e AcessóriasRESUMO
Hepatitis B virus (HBV) X protein (HBx) plays an essential role in development of HBV-associated hepatocellular carcinoma (HCC). Recently, we reported that HBx induces Fas Ligand (FasL) expression, which may help HCC cells to evade host-immune surveillance. The aim of this study was to investigate the role of HBx in expression of Nur77, an orphan nuclear receptor implicated in the upregulation of FasL. When Chang X-34 expressing HBx under the control of a doxycycline-inducible promoter was examined, induction of Nur77 was observed following HBx expression. Blocking of Nur77 function by introduction of an antisense or a dominant negative mutant Nur77 significantly inhibited the induction of FasL, indicating that Nur77 plays critical roles in FasL expression. Further, a high-level expression of transcripts and DNA binding of Nur77 were observed in the HBV-integrated cell lines established from HCC patients that express HBx. These results suggested that Nur77 may contribute to leading the HBx-induced Fas/FasL signaling pathway which eliminates invading Fas-expressing lymphocytes.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/biossíntese , Hepatite B/metabolismo , Neoplasias Hepáticas/metabolismo , Transativadores/farmacologia , Fatores de Transcrição/biossíntese , Ativação Transcricional , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Proteína Ligante Fas , Hepatite B/genética , Hepatite B/virologia , Vírus da Hepatite B/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Mutação , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Oligonucleotídeos Antissenso/farmacologia , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Transativadores/biossíntese , Transativadores/genética , Transativadores/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transfecção , Células Tumorais Cultivadas , Proteínas Virais Reguladoras e Acessórias , Integração ViralRESUMO
Many tumors, including hepatocellular carcinomas (HCCs), resist Fas-mediated cell death, which is one of the effector mechanisms in the host's anti-tumor response; however, this resistance can be abolished by interferon-gamma (IFN-gamma). IFN-gamma may sensitize Fas-mediated cell death in several ways, but the exact mechanism in HCCs is uncertain. In this study, we thoroughly investigated the effect of IFN-gamma on the susceptibility of one human normal liver cell line and 12 HCC cell lines to Fas-mediated cell death. We also investigated the effect of IFN-gamma on the expression of various apoptosis-related genes such as the Fas/TNF-related genes, the bcl-2 family, and the caspase family of genes. Although most cell lines showed considerable constitutive expression of Fas, all tested cell lines resisted Fas-mediated cell death without IFN-gamma. When cells were pretreated with IFN-gamma, only three cell lines were made significantly susceptible to Fas-mediated cell death (SNU-354, SNU-387 and SNU-423); the other 10 cell lines were not affected. IFN-gamma increased the mRNA expression of Fas, TRAIL and caspase-1, and surface Fas was also increased. The strongly sensitized cell lines (SNU-354, SNU-387 and SNU-423) showed a particularly potent increment in surface Fas after IFN-gamma treatment (increase in surface Fas > 1.7-fold). This result enabled us to conclude that a potent increment of surface Fas expression is a major sensitizing mechanism of IFN-gamma. We conclude that IFN-gamma cannot play a sensitizing role in most HCC cell lines and that IFN-gamma makes HCC cells susceptible to Fas-mediated cell death through a marked up-regulation of surface Fas in some HCC cells.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Interferon gama/farmacologia , Neoplasias Hepáticas/patologia , Receptor fas/fisiologia , Caspase 1/genética , Genes bcl-2 , Humanos , RNA Mensageiro/análise , Células Tumorais Cultivadas , Receptor fas/genéticaRESUMO
Many chemotherapeutic drugs have been found to exert their mode of action via induction of apoptosis in cancer cells. The mechanisms involved in this process are not clear. Recent studies have shown that the Fas/Fas ligand (FasL) system is a key factor controlling apoptotic cell death. In the present study, the involvement of Fas in chemotherapeutic drug-induced apoptosis in hepatoma cell lines was investigated. Five different human hepatoma cell lines, Hep G2, Hep G2.2.15, Hep 3B, SK-Hep-1, and PLC/PRF/5, were used. It was found that they expressed different levels of Fas. However, all five cell lines were susceptible to apoptosis when treated with chemotherapeutic drugs such as 5-fluorouracil (5-FU) or cisplatin. In Hep G2 that constitutively expressed Fas, 5-FU or cisplatin treatment caused an increase in the expression of Fas before the formation of oligonucleosomal DNA fragments, a typical feature of apoptosis. However, in Hep 3B, where Fas is undetectable, apoptosis could also be induced by 5-FU or cisplatin without induction of Fas. The agonistic anti-Fas antibody (CH-11) was capable of inducing apoptosis by itself and promoted drug-induced apoptosis in Hep G2 but not in Hep 3B. The antagonistic anti-Fas antibody (ZB4) inhibited drug-induced apoptosis in Hep G2. Our results suggest that apoptosis can be induced in hepatoma cell lines via both Fas-dependent and Fas-independent pathways.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Receptor fas/fisiologia , Anticorpos , Apoptose/imunologia , Divisão Celular/efeitos dos fármacos , Cisplatino/farmacologia , Citometria de Fluxo , Fluoruracila/farmacologia , Humanos , Marcação In Situ das Extremidades Cortadas , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Receptor fas/biossíntese , Receptor fas/imunologiaRESUMO
We demonstrated the induction of cell death in a hepatoma cell line by IFN-gamma and its possible mechanism. Among the 2 hepatitis B virus (HBV)-associated hepatoma cell lines, SNU-354 and SNU-368, IFN-gamma induced cell death and increased caspase-3 activity in SNU-368 but not in SNU-354. IFN-gamma induced several changes in the mRNA expression level of apoptosis-regulating genes, e.g., increased expression of Fas, caspase-1 and TNF-related apoptosis-inducing ligand (TRAIL). In particular, IFN-gamma potently increased the mRNA expression of TRAIL in both cell lines. However, it did not change the mRNA expression level of death-mediating TRAIL receptors, e.g., DR4 and DR5, which were constitutively expressed in both cell lines. In contrast, the decoy receptor DcR1 was expressed in SNU-354 but not in SNU-368, and its expression level in SNU-354 was increased by IFN-gamma. Another decoy receptor, DcR2, was constitutively expressed in both cell lines; however, its expression level in SNU-368 was decreased by IFN-gamma. In addition, exogenous recombinant TRAIL reduced viability in SNU-368, but not in SNU-354, cells. From these findings, we speculated that TRAIL up-regulation and the subsequent TRAIL-mediated apoptosis serve as a mechanism of IFN-gamma-induced cell death in SNU-368. To confirm this hypothesis, we demonstrated that soluble DR4-Fc fusion protein, a TRAIL pathway inhibitor, inhibited IFN-gamma-induced cell death in SNU-368. Our results demonstrated that IFN-gamma acts as an inducer of cell death through TRAIL-mediated apoptosis.
Assuntos
Apoptose , Interferon gama/farmacologia , Glicoproteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Reguladoras de Apoptose , Carcinoma Hepatocelular/patologia , Caspase 1/genética , Caspase 1/metabolismo , Caspase 3 , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Proteína Ligante Fas , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Neoplasias Hepáticas/patologia , Glicoproteínas de Membrana/genética , RNA Mensageiro/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/genética , Receptor fas/genética , Receptor fas/metabolismoRESUMO
BACKGROUND: The p16(INK4A) gene encodes 2 cell cycle regulator proteins, p16 and p14(ARF), by alternative splicing. This genetic locus also contains another cell cycle regulator gene, p15(INK4B), which encodes p15. The inactivation of the p16 protein has been demonstrated in some hepatocellular carcinomas (HCCs); however, the inactivation of the other 2 cell regulator proteins and their inactivation patterns are not well characterized. METHODS: To characterize the role of the above 3 cell cycle regulator proteins in HCCs, the authors examined the genomic status of the p16(INK4A) and p15(INK4B) genes and their RNA products in 20 HCC tissues and 7 human HCC cell lines. Homozygous deletions in each exon of p16(INK4A) and p15(INK4B) were evaluated by comparative multiplex polymerase chain reaction (PCR), and the methylation status of the p16(INK4A) and p15(INK4B) promoter region was analyzed by methylation specific PCR. RESULTS: Homozygous deletions were found in 6 of 20 HCCs (30%) and 2 of 7 HCC cell lines (29%). In 20 HCCs, the frequency of homozygous deletions was 20% in exon 1 of p15(INK4B), 20% in exon 2 of p15(INK4B), 10% in exon 1beta of p16(INK4A), 25% in exon 1alpha of p16(INK4A), 15% in exon 2 of p16(INK4A), and 15% in exon 3 of p16(INK4A). The authors found hypermethylation of the p16(INK4A) promoter region in 7 HCCs (35%) and 3 HCC cell lines (43%). The overall frequency of p16 alterations in HCCs, including hypermethylation and homozygous deletions, was 60% (12 of 20 cases). According to reverse transcriptase-PCR analysis, the absence of RNA expression was most frequent in p16 (11 of 20 cases, 55%) and less frequent in p15 (7 of 20 cases, 35%) and p14(ARF) (5 of 20 cases, 25%). CONCLUSIONS: Among the 3 cell cycle regulator proteins encoded at the 9p21 genetic locus, inactivation of p16 is the most frequent event in HCCs in which promoter hypermethylation and homozygous deletions are the common mechanisms.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular , Genes p16/genética , Neoplasias Hepáticas/genética , Regiões Promotoras Genéticas/genética , Proteínas Supressoras de Tumor , Transformação Celular Neoplásica , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina/genética , DNA de Neoplasias/genética , Deleção de Genes , Humanos , Metilação , Proteínas/genética , RNA/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p14ARFRESUMO
Nur77 (NGFI-B) is an orphan nuclear receptor that has been implicated in activation-induced T-cell apoptosis. Retinoids, potent immune modulators, were shown to inhibit the activation-induced apoptosis of immature thymocytes and T-cell hybridomas. To illustrate the mechanism of the inhibition, we examined the effects of retinoic acid (RA) on the expression and transactivation functions of Nur77 in the human peripheral blood mononuclear cells and the human T-cell leukemia, Jurkat. All-trans-RA remarkably repressed the DNA binding and transcriptional induction of Nur77. Among the two potential trans-acting factors that activate Nur77 gene promoter, i.e., AP-1 and related serum response factor (RSRF), all-trans-RA repressed DNA binding and reporter gene activity of AP-1 but not that of RSRF, suggesting that the inhibition may be mediated through AP-1. We also demonstrated a posttranscriptional regulation of Nur77 function by retinoid receptors by showing that transactivation activity of Nur77 was significantly inhibited by cotransfection of RARalpha or RXRalpha. Nur77 bound RARalpha or RXRalpha in both yeast and mammalian two-hybrid tests, suggesting that direct protein-protein interaction between these receptors may mediate the inhibition. Taken all together, we demonstrated that RA repressed Nur77 function through multiple mechanisms that may provide the basis for RA inhibition on the apoptosis of activated T-lymphocytes.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/fisiologia , Linfócitos T/citologia , Fatores de Transcrição/metabolismo , Tretinoína/fisiologia , Motivos de Aminoácidos , DNA/metabolismo , Humanos , Células Jurkat , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Ligação Proteica , Receptores do Ácido Retinoico/metabolismo , Receptores de Esteroides , Transfecção , Tretinoína/metabolismo , Tretinoína/farmacologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Epidemiological studies have demonstrated that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of and mortality from colon cancer. In addition, NSAIDs reduce the number and the size of polyps in patients with familial adenomatous polyposis. The mechanisms responsible for the antineoplastic effect of NSAIDs are not yet completely understood, but one of the possible mechanisms is an induction of apoptosis. We explored the role of caspase-3, a major apoptosis-executing enzyme, in NSAID-induced apoptosis of colon cancer cell line HT-29. Treatment of HT-29 cells with indomethacin induced a dramatic increase in caspase-3-like protease activity measured by a cleavage of the fluorogenic substrate Ac-DEVD-AMC. Western blot analysis showed that indomethacin treatment led both to decrease in procaspase-3 and to cleavage of its substrate poly(ADP-ribose) polymerase (PARP). Furthermore, the caspase-3-like protease inhibitor Ac-DEVD-CHO attenuated indomethacin-induced DNA fragmentation dose dependently. However, mRNA expression of CASP genes was not affected by the addition of indomethacin, highlighting the importance of posttranslational modification of this enzyme for the activation. These results suggest that NSAIDs, including indomethacin, induce apoptosis in colon cancer cells through a caspase-3 dependent mechanism which may contribute to the chemopreventive functions of these agents.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/efeitos dos fármacos , Caspases/efeitos dos fármacos , Precursores Enzimáticos/efeitos dos fármacos , Células HT29/efeitos dos fármacos , Indometacina/farmacologia , Apoptose/fisiologia , Caspase 3 , Caspases/genética , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HT29/enzimologia , Células HT29/patologia , Humanos , Oligopeptídeos/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacosRESUMO
AIMS: To isolate the cellulase gene from Rhizobium leguminosarum bv. trifolii 1536. METHODS AND RESULTS: By the shot-gun method a clone (cel8A) harbouring 3.1 kb genomic DNA fragment from R. leguminosarum bv. trifolii 1536 was obtained. The cel8A gene coded 348 amino acids and it belongs to the glycosyl hydrolase family 8. The molecular mass of Cel8A protein induced from Escherichia coli DH5alpha, appeared to be 35 kDa. The optimum pH and optimum temperature was 7.0, and about 30 degrees C for its enzymatic activity respectively. CONCLUSIONS: R. leguminosarum bv. trifolii 1536 had cel8A gene having an open reading frame of 1047 bp coded for the activity of hydrolyzation of carboxymethyl cellulose. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of celluloytic enzyme by R. leguminosarum bv. trifolii was confirmed, which would play specific roles in rhizobia. Future study should focus on its role in the infection and nodulation phenomena.
Assuntos
Celulase/genética , Celulase/metabolismo , Rhizobium leguminosarum/enzimologia , Rhizobium leguminosarum/genética , Celulase/química , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
AIMS: To examine the diversity of protozoa in the rumen contents of cow. METHODS AND RESULTS: Protozoa that inhabit the rumen were detected by PCR using protozoan-specific primers. Libraries of protozoan rDNA sequences were constructed from rumen fluid, solid tissues and epithelium. Twenty-three clones isolated from rumen fluid fell into two genera identified as Entodinium (69.6% of clones) and Epidinium (31.4% of clones). Of the clones isolated from rumen fluid, a moderate number were unidentifiable (30.4%). CONCLUSIONS: The predominant protozoan genus identified in the whole rumen belonged to the Entodinium group (81.1%). Protozoa were not detected in the rumen epithelium. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that rumen fluid and solid tissues contain different protozoan populations that may play specific roles in rumen function. Quantitative PCR techniques and a more specific set of phylogenetic probes that distinguish between protozoan species are needed to determine the significance of newly identified groups and to determine the distribution of identified protozoan clusters in rumen microbial communities.