RESUMO
Cellular cholesterol plays fundamental and diverse roles in many biological processes and affects the pathology of various diseases. Comprehensive and detailed understanding of the cellular functions and characteristics of cholesterol requires visualization of its subcellular distribution, which can be achieved by fluorescence microscopy. Many attempts have been made to develop fluorescent cholesterol reporters, but so far, none of them seems to be ideal for studying all aspects of cholesterol management. To meet the requirements for the right probe remains a great challenge, and progress in this field continues. The main objective of this review is to not only present the current state of the art, but also critically evaluate the applicability of individual probes and for what purpose they can be used to obtain relevant data. Hence, the data obtained with different probes might provide complementary information to build an integrated picture about the cellular cholesterol.
Assuntos
Colesterol/metabolismo , Corantes Fluorescentes , Animais , Transporte Biológico/fisiologia , HumanosRESUMO
A novel pentamethinium salt was synthesized with an unforeseen expanded conjugated quinoxaline unit directly incorporated into a pentamethinium chain. The compound exhibited high fluorescence intensity, selective mitochondrial localization, high cytotoxicity, and selectivity toward malignant cell lines, and resulted in remarkable in vivo suppression of tumor growth in mice.
Assuntos
Antineoplásicos/química , Hexametônio/química , Neoplasias/tratamento farmacológico , Quinoxalinas/química , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ciclização , Hexametônio/uso terapêutico , Camundongos , Neoplasias/patologia , Quinoxalinas/uso terapêuticoRESUMO
BACKGROUND: Malignant melanoma is considered to be highly resistant to chemotherapy, radiotherapy, hormonotherapy and standard immunotherapy (interleukin 2, interferon alpha). Radical surgery in the early stages of the disease is still the most efficient method. Since the development of immunotherapy and targeted therapy, the role of palliative chemotherapy for advanced disease may be changing. CASE: A case report regarding 44-year-old woman with extensive tumor of the pectoral wall with contralateral axillary lymphadenopathy is presented. On the basis of imaging methods, histology and immunohistochemistry, the tumor was defined as a sarcoma. Due to PAX7-FKHR fusion gene positivity, rhabdomyosarcoma was the most probable classification. The patient was treated with radical chemotherapy including iphosphamide, vincristine, actinomycin D and doxorubicin with the effect of partial regression of the tumor. This enabled radical surgery of the chest wall tumor. Pathology proved 70% necrosis of the tumor. A contralateral axillary dissection was performed with a finding of two lymph nodes infiltrated with melanoma. The immunohistochemistry markers S100, HMB-45 and Melan A were positive. This resulted in a reclassification of the chest wall tumor to malignant melanoma. The following PET/CT scan was negative. A massive progression of the disease occurred after 5 months. B-RAF mutation leads to a plan of targeted therapy with vemurafenib. CONCLUSION: The case demonstrates the limits of the sensitivity and specificity of immunohistochemical markers of melanoma and the ability of this tumor to imitate various tumors including soft tissue sarcomas. A rare -PAX7-FKHR fusion gene positivity considered specific for rhabdomyosarcoma was found. An extraordinary response to radical chemotherapy with surgical resection led to an improvement of the quality of life and to a prolonged survival comparable with the effect of new targeted treatment for malignant melanoma.
Assuntos
Melanoma/diagnóstico , Rabdomiossarcoma/diagnóstico , Sarcoma/diagnóstico , Neoplasias Cutâneas/diagnóstico , Neoplasias Torácicas/diagnóstico , Parede Torácica , Adulto , Terapia Combinada , Diagnóstico Diferencial , Feminino , Humanos , Melanoma/tratamento farmacológico , Rabdomiossarcoma/cirurgia , Sarcoma/cirurgia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Torácicas/cirurgiaRESUMO
The aim was to study the antioxidant properties of four wine polyphenols (flavonoids catechin, epicatechin, and quercetin, and hydroxystilbene resveratrol). All three flavonoids exerted significant and dose-dependent scavenging effects against peroxyl radical and nitric oxide in chemical systems. The scavenging effect of resveratrol was significantly lower. All polyphenols decreased production of reactive oxygen species (ROS) by RAW264.7 macrophages. Only quercetin quenched ROS produced by lipopolysaccharide-stimulated RAW264.7 macrophages incubated for 24 h with polyphenols. Quercetin and resveratrol decreased the release of nitric oxide by these cells in a dose-dependent manner which corresponded to a decrease in iNOS expression in the case of quercetin. In conclusion, the higher number of hydroxyl substituents is an important structural feature of flavonoids in respect to their scavenging activity against ROS and nitric oxide, while C-2,3 double bond (present in quercetin and resveratrol) might be important for inhibition of ROS and nitric oxide production by RAW 264.7 macrophages.
Assuntos
Flavonoides/farmacologia , Sequestradores de Radicais Livres/farmacologia , Macrófagos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fenóis/farmacologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vinho , Animais , Catequina/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Flavonoides/química , Sequestradores de Radicais Livres/química , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Fenóis/química , Polifenóis , Quercetina/farmacologia , Resveratrol , Estilbenos/farmacologia , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Pluripotential embryonal carcinoma cells such as those of the P19 line differentiate when exposed to retinoic acid (RA). The RAC65 cell line is a mutant clone of P19 cells selected to be RA nonresponsive. RAC65 cells carry a rearrangement affecting one of the genes encoding a nuclear retinoic acid receptor (RAR alpha). The mutant gene encodes a protein, RAR alpha', that has lost its 70 C-terminal amino acids, thus truncating the RA-binding domain. The RAR alpha' was found to be a dominant repressor of transcription from an RA-responsive target gene; however, expression of RAR alpha' was insufficient to confer RA nonresponsiveness, suggesting that RAC65 cells carry an additional mutation(s) affecting RA-induced genes.
Assuntos
Proteínas de Transporte/genética , Genes Dominantes , Mutação , Teratoma/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Diferenciação Celular , Linhagem Celular , DNA de Neoplasias/genética , Resistência a Medicamentos , Biblioteca Gênica , Genes , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , Receptores do Ácido Retinoico , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
v-rel is the oncogenic member of the Rel/NF-kappaB family of transcription factors. The mechanism by which v-Rel induces transformation of avian lymphoid cells and fibroblasts is not precisely known. However, most models propose that v-rel disrupts the normal transcriptional regulatory network. In this study we evaluated the role of AP-1 family members in v-Rel-mediated transformation. The overexpression of v-Rel, c-Rel, and c-Rel delta resulted in a prolonged elevation of c-fos and c-jun expression and in a sustained repression of fra-2 at both the mRNA and protein levels in fibroblasts and lymphoid cells. Moreover, the transforming abilities of these Rel proteins correlated with their ability to alter the expression of these AP-1 factors. v-Rel exhibited the most pronounced effect, whereas c-Rel, with poor transforming ability, elicited only moderate changes in AP-1 levels. Furthermore, c-Rel delta, which exhibits enhanced transforming potential relative to c-Rel, induced intermediate changes in AP-1 expression. To directly evaluate the role of AP-1 family members in the v-Rel transformation process, a supjun-1 transdominant mutant was used. The supjun-1 mutant functions as a general inhibitor of AP-1 activity by inhibiting AP-1-mediated transactivation and by reducing AP-1 DNA-binding activity. Coinfection or sequential infection of fibroblasts or lymphoid cells with viruses carrying rel oncogenes and supjun-1 resulted in a reduction of the transformation efficiency of the Rel proteins. The expression of supjun-1 inhibited the ability of v-Rel transformed lymphoid cells and fibroblasts to form colonies in soft agar by over 70%. Furthermore, the expression of supjun-1 strongly interfered with the ability of v-Rel to morphologically transform avian fibroblasts. This is the first report showing that v-Rel might execute its oncogenic potential through modulating the activity of early response genes.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Proteínas de Ligação a DNA/genética , Fibroblastos/citologia , Genes Reporter , Modelos Genéticos , Proteínas Oncogênicas v-rel , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-rel , Supressão Genética , Fator de Transcrição AP-1/genética , Ativação TranscricionalRESUMO
A new generator of two successive shock waves focused to a common focal point has been developed. Cylindrical pressure waves created by multichannel electrical discharges on two cylindrical composite anodes are focused by a metallic parabolic reflector - cathode, and near the focus they are transformed to strong shock waves. Schlieren photos of the focal region have demonstrated that mutual interaction of the two waves results in generation of a large number of secondary short-wavelength shocks. Interaction of the focused shockwaves with liver tissues and cancer cell suspensions was investigated. Localized injury of rabbit liver induced by the shock waves was demonstrated by magnetic resonance imaging. Histological analysis of liver samples taken from the injured region revealed that the transition between the injured and the healthy tissues is sharp. Suspension of melanoma B16 cells was exposed and the number of the surviving cells rapidly decreased with increasing number of shocks and only 8 % of cells survived 350 shocks. Photographs of cells demonstrate that even small number of shocks results in perforation of cell membranes.
Assuntos
Membrana Celular/patologia , Fígado/patologia , Melanoma Experimental/patologia , Ultrassom/efeitos adversos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Desenho de Equipamento , Fígado/lesões , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Necrose , Coelhos , Fatores de TempoRESUMO
Two hundred-sixty-one (261) cases of cerebral malaria within last 8 years from 3 tropical clinics in South Sudan were analyzed. Coma was present at 79.8% and convulsions at 25.6%. However 90.5% of children were cured. Commonest antimalarial drugs used were intravenous quinine, clindamycin, artesunate and artemeter.
Assuntos
Antimaláricos/uso terapêutico , Coma/epidemiologia , Malária Cerebral/epidemiologia , Artemeter , Artemisininas/uso terapêutico , Pré-Escolar , Clindamicina/uso terapêutico , Comorbidade , Quimioterapia Combinada , Febre/epidemiologia , Humanos , Lactente , Malária Cerebral/tratamento farmacológico , Malária Cerebral/mortalidade , Quinina/uso terapêutico , Estudos Retrospectivos , Convulsões/epidemiologia , Sudão/epidemiologiaRESUMO
The aim of this study was to assess mortality and sequellae within cases from Nationwide survey of community acquired meningitis and identify risk factors for inferior outcome. Risk factors such as underlying disease (diabetes mellitus, cancer, trauma, neonatal age, splenectomy, alcoholism, sepsis, other infections), etiology, clinical symptoms and outcome (death, improvement and cured after modifications of ATB therapy, cured without change of therapy, cured with neurologic sequellae) were recorded and analysed with univariate analysis (chi2 or t test for trends, CDC Atlanta 2004). Analysing risk factors for inferior outcome (death or cured with neurologic sequellae), we compared patients who died or survived with neurologic sequellae to all patients with community acquired bacterial meningitis. Univariate analysis showed that trauma (p<0.05), alcohol abuse (p<0.05), diabetes, S. aureus (p<0.05) and gram-negative etiology (A. baumannii, Ps. aeruginosa or Enterobacteriaceae) (36% vs. 11,9%, p<0.05) were predicting inferior outcome. Analysing risk factors for treatment failure (death or failed but cured after change of antibiotic treatment) prior sepsis (34.1% vs. 13.9%, p<0.01) and gram-negative etiology (25% vs. 11.9%, p<0.02) were statistically significant predictors of treatment failure. Neisseria meningitis had less failures (p<0.05). Concerning infection associated mortality again diabetes mellitus (p<0.05), alcoholism (p<0.05) staphylococcal and gram-negative etiology (p<0.05) were significant predictors of death. N. meningitis had surprisingly less treatment failures (appropriate and rapid initial therapy). Neurologic sequellae were more common in patients with alcohol abuse (p<0.05), craniocerbral trauma (p<0.05) and less common in meningitis with pneumococcal etiology (p<0.05).
Assuntos
Alcoolismo/complicações , Dano Encefálico Crônico/etiologia , Lesões Encefálicas/complicações , Infecções por Bactérias Gram-Negativas/complicações , Meningites Bacterianas/terapia , Alcoolismo/mortalidade , Lesões Encefálicas/mortalidade , Distribuição de Qui-Quadrado , Infecções Comunitárias Adquiridas/complicações , Infecções Comunitárias Adquiridas/mortalidade , Infecções Comunitárias Adquiridas/terapia , Diabetes Mellitus , Infecções por Bactérias Gram-Negativas/mortalidade , Infecções por Bactérias Gram-Negativas/terapia , Humanos , Meningites Bacterianas/complicações , Meningites Bacterianas/mortalidade , Fatores de Risco , Eslováquia , Falha de TratamentoRESUMO
This review summarizes recent developments in the area of porphyrin chemistry in the direction of biological applications. Novel synthetic methodologies are reviewed for porphyrin synthesis, porphyrin analog synthesis, stable porphyrinogens -- calixpyrroles, expanded porphyrins. Unique biological properties of those compounds are desribed with focus on photodynamic therapy (PDT) and molecular recognition properties. Special attentions given to metalloporphyrins with potential to affect heme degradation and CO formation.
Assuntos
Fotoquimioterapia , Porfirinas/química , Monóxido de Carbono/metabolismo , Heme/metabolismo , Modelos Biológicos , Porfirinas/uso terapêuticoRESUMO
Rel/NF-kappa B transcription factors and I Kappa B alpha function in an autoregulatory network. Avian I kappa B alpha transcription is increased in response to both c-Rel and v-Rel. This study shows that I kappa B alpha transcription is synergistically stimulated by Rel and AP-1 factors (c-Fos and c-Jun). However, the response to v-Rel and the AP-1 factors was not as vigorous as that of c-Rel and AP-1. A 386 bp region of the I kappa B alpha promoter (containing two NF-kappa B and one AP-1 binding sites) was shown to be both necessary and sufficient for response to both Rel factors alone or Rel factors in conjunction with the AP-1 proteins. In addition, an imperfect NF-kappa B binding site was found to overlap the AP-1 binding site. Mutation of either of the NF-kappa B binding sites or the AP-1 binding site dramatically decreased the response of the I kappa B alpha promoter to Rel proteins alone or Rel and AP-1 factors. Overexpression of c-Rel or v-Rel resulted in the formation of DNA binding complexes associated with the imperfect NF-kappa B binding site which overlaps the AP-1 site. v-Rel associated with the imperfect NF-kappa B site stronger than c-Rel, and overexpression of v-Rel also resulted in the formation of a v-Rel containing complex bound to a consensus AP-1 site. These studies address the difference in c-Rel and v-Rel's ability to synergistically stimulate I kappa B alpha expression in conjunction with the AP-1 factors.
Assuntos
Proteínas de Ligação a DNA/genética , Genes fos , Genes jun , Proteínas I-kappa B , Proteínas Oncogênicas de Retroviridae/genética , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Embrião de Galinha , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Luciferases/genética , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Oncogênicas v-rel , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-rel , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Previous studies have shown that treatment of avian reticuloendotheliosis virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells with 10 microg/ml (18.8 microM) of RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) for 3 days induced approximately 50% of the cells to undergo apoptosis. Elevated and prolonged expression of c-jun mRNA and protein was temporally correlated with VES-induced cell death. Data presented in this paper show that the elevated and prolonged expression of c-jun message and protein are not accounted for by enhanced stability, and show the involvement of c-Jun in VES-induced apoptosis in this lymphoblastoid cell type. C4-1 cells infected with a virus carrying a dominant, negatively acting mutant form of c-Jun, supjun-1, exhibited: (i) 71% reduction in VES-induced apoptosis, (ii) a 2.0-2.5-fold decrease in wildtype, endogenous c-Jun expression, and (iii) a 2.4-2.6-fold reduction in AP-1 binding activity. Additionally, cells co-treated with VES plus RRR-alpha-tocopherol, exhibited a 70% reduction in apoptosis, a marked reduction in c-Jun expression and a 1.6-fold reduction in AP-1 binding activity. These studies suggest that c-Jun plays a crucial role in VES-induced apoptosis in C4-1 cells, and add to our understanding of mechanisms of action involved in VES-mediated tumor cell growth inhibition.
Assuntos
Apoptose/efeitos dos fármacos , Transformação Celular Neoplásica , Proteínas Proto-Oncogênicas c-jun/fisiologia , Vírus da Reticuloendoteliose/genética , Vitamina E/análogos & derivados , Animais , Linhagem Celular , Galinhas , Linfócitos/efeitos dos fármacos , RNA Mensageiro/análise , Tocoferóis , Vitamina E/farmacologiaRESUMO
Mouse P19 embryonal carcinoma (EC) cells express on their surfaces a Thy-1 glycoprotein. The expression of Thy-1 at the mRNA and protein levels is down-regulated during differentiation induced by retinoic acid (RA). Thy-1 is also expressed in human NTERA-2 EC cells, but its expression is not down-regulated during RA-induced differentiation. As a first step towards understanding differential regulation of the mouse and human Thy-1 gene in EC cells, we have introduced genomic DNA fragments encompassing the mouse or human Thy-1 gene into NTERA-2 and P19-derived cells and analyzed surface properties of the transfectants. In the transient transfection assay, both mouse and human Thy-1 genes were expressed on cell surfaces at comparable levels. P19-derived stable transfectants exhibited great clonal variations in the expressions of the transfected Thy-1 gene products, which in part reflected copy numbers. There was no simple correlation between the expression of the transfected Thy-1 gene and two stem cell surface markers, TEC-1 and TEC-4. In the course of differentiation induced by RA several clones with a surface phenotype of EC cells exhibited a significant decrease in the expression of the transfected mouse Thy-1, whereas expression of the human Thy-1 was less efficiently down-regulated. The results suggest the presence of multiple cis- and trans-acting elements controlling expression of the mouse and human Thy-1 genes in P19 EC cells and their differentiated derivatives.
Assuntos
Antígenos de Superfície/análise , Glicoproteínas de Membrana/análise , Células-Tronco Neoplásicas/metabolismo , Animais , Antígenos de Superfície/biossíntese , Diferenciação Celular/genética , Células-Tronco de Carcinoma Embrionário , Regulação Neoplásica da Expressão Gênica , Humanos , Glicoproteínas de Membrana/biossíntese , Camundongos , Antígenos Thy-1 , Transfecção , TretinoínaRESUMO
Post-translational modification of nuclear proteins by poly(ADP-ribose) polymerase 1 (PARP-1) is involved in the regulation of DNA repair, cell death, and maintenance of genomic stability. Recently, several PARP-1 homologues have been identified constituting a family of poly(ADP-ribosyl)ating proteins. We cloned and sequenced the cDNAs of the mouse PARP-3 (Adprt3) gene encoding poly(ADP-ribose) polymerase 3 and of the closely linked U3-55k gene coding for the U3 small nucleolar ribonucleoprotein complex-associated 55-kilodalton protein. The two genes are located in a head-to-head orientation on mouse chromosome 9 and are linked by an approximately 1.5-kb putative bi-directional promoter region. This gene arrangement is conserved between mouse and human orthologues. Three alternative non-coding 5'-end exons were found in the mouse PARP-3 mRNA. The expression patterns of PARP-3, U3-55k, PARP-2, and PARP-1 genes were determined using Northern blot with mRNA from various adult mouse tissues and organs. PARP-3 expression was found to be regulated in a tissue-specific manner. The highest expression of PARP-3 was detected in the skeletal muscle, high to moderate levels were found in the lung, liver, kidney, ovary, spleen and heart, while thymus, small intestine and colon contained lower levels of the PARP-3 transcripts. Notably, PARP-3 expression was barely detectable in the whole brain and testis mRNA. In contrast to PARP-3, the other three genes showed ubiquitous expression with less variable mRNA levels. Interestingly, the mouse and human PARP-2 gene has recently been shown to be connected via a bi-directional promoter with the gene for the RNase P RNA subunit (Amé et al., J. Biol. Chem. 276: 11092-11099, 2001). As both the U3-55k protein and the RNase P RNA are involved in the processing of precursor RNAs of the protein-synthesizing machinery (pre-rRNA and pre-tRNA, respectively), it is tempting to hypothesize that expression of some members of the two groups of genes (i.e. PARP vs. protein-synthesizing machinery RNA-processing genes) may be coordinately regulated under certain physiological or pathological conditions and/or in some cell types.
Assuntos
Cromossomos , Poli(ADP-Ribose) Polimerases/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Processamento Alternativo , Animais , Sequência de Bases , Clonagem Molecular , Éxons , Feminino , Regulação da Expressão Gênica , Ordem dos Genes , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Músculo Esquelético/fisiologia , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosAssuntos
Meningite Meningocócica/epidemiologia , Vigilância da População , Refugiados , Antibacterianos/uso terapêutico , Ceftriaxona/uso terapêutico , Criança , Pré-Escolar , Cloranfenicol/uso terapêutico , Notificação de Doenças , Humanos , Meningite Meningocócica/tratamento farmacológico , Sudão/epidemiologia , Resultado do TratamentoRESUMO
v-Rel is the acutely oncogenic member of the NF-κB family of transcription factors. Infection with retroviruses expressing v-Rel rapidly induces fatal lymphomas in birds and transforms primary lymphocytes and fibroblasts in vitro. We have previously shown that AP-1 transcriptional activity contributes to v-Rel-mediated transformation. Although v-Rel increases the expression of these factors, their activity may also be induced through phosphorylation by the mitogen-activated protein kinases (MAPKs). The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways. This induction is critical for the v-Rel-transformed phenotype, as suppression of MAPK activity with chemical inhibitors or small interfering RNA severely impairs colony formation of v-Rel-transformed lymphoid cell lines. However, signaling must be maintained within an optimal range in these cells, as strong additional activation of either pathway beyond the levels induced by v-Rel through the expression of constitutively active MAPK proteins attenuates the transformed phenotype. MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified. We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.
Assuntos
Transformação Celular Viral/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes rel/genética , Genes rel/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/deficiência , MAP Quinases Reguladas por Sinal Extracelular/genética , Técnicas de Silenciamento de Genes , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/deficiência , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fenótipo , RNA Interferente Pequeno/genéticaRESUMO
Cell transformation by the v-rel oncogene is mediated by the aberrant expression of genes that are normally tightly regulated by other Rel/NF-kappaB family members. Although a number of genes inappropriately activated or suppressed by v-Rel have been identified, their contributions to the v-Rel transformation process have been poorly characterized. Here, we examine the role of individual AP-1 proteins in v-Rel-mediated transformation. v-Rel-transformed cells exhibit elevated RNA and protein expression of c-Fos, c-Jun and ATF2 and sustained repression of Fra-2. c-Fos and c-Jun are essential in both the initiation and maintenance of v-Rel-mediated transformation, whereas Fra-2 is dispensable. By employing a c-Jun dimerization mutant, we further identified Fos/Jun heterodimers as major contributors to the v-Rel transformation process. The inability of c-Rel to induce the expression of c-Fos and c-Jun contributes to its weaker oncogenic potential relative to v-Rel. Our studies also demonstrate that v-Rel may induce AP-1 members by directly upregulating gene expression (c-fos and ATF2) and by activating pathways that stimulate AP-1 activity. Although elevated expression of ATF2 is also required for v-Rel-mediated transformation, its ectopic overexpression is inhibitory. Investigating the mode of ATF2 regulation revealed a positive feedback mechanism whereby ATF2 induces p38 MAPK phosphorylation to further induce its own activity. In addition, these studies identified Ha-Ras as an effector of v-Rel-mediated transformation and reveal a novel role for ATF2 in the inhibition of the Ras-Raf-MEK-ERK signaling pathway. Overall, these studies reveal distinct and complex roles of AP-1 proteins in Rel/NF-kappaB oncogenesis.
Assuntos
Transformação Celular Neoplásica , NF-kappa B/metabolismo , Proteínas Oncogênicas v-rel/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator 2 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Embrião de Galinha , DNA/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Oncogênicas v-rel/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases raf/metabolismoAssuntos
Antígenos de Histocompatibilidade , Pele/imunologia , Timo/imunologia , Animais , Feminino , Masculino , Camundongos , Pele/citologia , Timo/citologiaRESUMO
The interaction of adrenergic agonists and/or antagonists with the adrenergic receptors expressed on immunologically active cells including macrophages plays an important role in regulation of inflammatory responses. Our study investigated the effects of carvedilol, a unique vasodilating beta-adrenergic antagonist, and endogenous adrenergic agonists (adrenalin, noradrenalin, and dopamine) and/or antagonists (prazosin, atenolol) on lipopolysaccharide-stimulated nitric oxide (NO) production from murine macrophage cell line RAW 264.7. The production of NO was determined as the concentration of nitrites in cell supernatants (Griess reaction) and inducible nitric oxide synthase (iNOS) protein expression (Western blot analysis). Scavenging properties against NO were measured electrochemically. Carvedilol in a concentration range of 1, 5, 10 and 25 microM inhibited iNOS protein expression and decreased the nitrite concentration in cell supernatants. Adrenalin, noradrenalin or dopamine also inhibited the iNOS protein expression and the nitrite accumulation. Prazosine and atenolol prevented the effect of both carvedilol and adrenergic agonists on nitrite accumulation and iNOS expression in lipopolysaccharide-stimulated cells. These results, together with the absence of scavenging properties of carvedilol against NO, imply that both carvedilol and adrenergic agonists suppress the lipopolysaccharide-evoked NO production by macrophages through the activation and modulation of signaling pathways connected with adrenergic receptors.
Assuntos
Agonistas Adrenérgicos/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Carbazóis/farmacologia , Propanolaminas/farmacologia , Receptores Adrenérgicos/efeitos dos fármacos , Antagonistas Adrenérgicos alfa/farmacologia , Antagonistas Adrenérgicos beta/administração & dosagem , Animais , Carbazóis/administração & dosagem , Carvedilol , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Propanolaminas/administração & dosagem , Receptores Adrenérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
In this study, we provide evidence that photostimulation of various cancer cells preloaded with a new photosensitizing compound, tetrakis-meso-(4-ethyleneglycol-2,3,5,6-tetrafluorophenyl) porphyrin (PORF-TEG), results in rapid activation of the cell death machinery. PORF-TEG, although primarily localized in lysosomes, induces mitochondria-driven apoptosis. The induction of apoptosis is accompanied by immediate and sustained activation of p38 mitogen-activated protein kinase (MAPK) and transient activation of c-Jun N-terminal kinase (JNK). Conversely, the inhibition of p38 by PD 169316 or SB202190 and by the p38alpha dominant-negative mutant as well as the deletion of the p38alpha gene (MEFs-KO) protected cells from apoptosis, whereas inhibition of JNK did not. Activation of the p38 signaling pathway occurs upstream of caspase activation. In addition, preincubation of cells with scavengers of reactive oxygen species attenuated p38 and caspase activation and increased cell survival, thus connecting reactive oxygen species formation with the activation of the p38 pathway. Later events included degradation of Bcl-2, activation of tBid, and cleavage of Bad and Mcl-1. The data suggest a key role for p38 MAPK in PORF-TEG-photoinduced apoptosis.