RESUMO
PURPOSE: Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity. METHODS: Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72. RESULTS: The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37 °C after heat treatment at 44 °C for 15 min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3 h of incubation at 37 °C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3 h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment. CONCLUSIONS: These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.
Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico/metabolismo , Autoantígeno Ku/metabolismo , Animais , Humanos , CamundongosRESUMO
The accumulation of abnormal proteins is a common characteristic of neurodegenerative diseases. This accumulation reflects a severe disturbance of cellular homeostasis in pathogenic protein clearance. Here, we demonstrated that the activation of the two major proteolytic machineries, the molecular chaperone-ubiquitin proteasome system (UPS) and the autophagy system, were simultaneously enhanced by paeoniflorin (PF), a major component of Paeonia plants, and exerted therapeutic effects in models of spinal and bulbar muscular atrophy (SBMA). PF significantly increased the expression of nuclear factor-YA (NF-YA), which strongly upregulated the molecules involved in the proteolytic machinery [molecular chaperones, carboxyl terminus of Hsc70-interacting protein and transcription factor EB], which thus mitigated the behavioral and pathological impairments in an SBMA mouse model through the upregulation of pathogenic androgen receptor protein clearance in motor neurons and muscles. These findings demonstrated that PF is able to enhance both the UPS and autophagy systems by upregulating the expression of NF-YA, which promotes therapeutic effects in an SBMA model.
Assuntos
Glucosídeos/uso terapêutico , Monoterpenos/uso terapêutico , Receptores Androgênicos/genética , Animais , Linhagem Celular , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Imuno-Histoquímica , Camundongos , Atrofia Muscular Espinal , Proteólise/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
PURPOSE: Heat stress induces complex cellular responses, and its detailed molecular mechanisms still remain to be clarified. The objective of this study was to investigate the molecular mechanisms underlying cellular responses to mild hyperthermia (MHT) in normal human fibroblastic (NHF) cells. MATERIALS AND METHODS: Cells were treated with MHT (41°C, 30 min) and then cultured at 37°C. Gene expression was determined by the GeneChip® system and bioinformatics tools. RESULTS: Treatment of the NHF cell lines, Hs68 and OUMS-36, with MHT did not affect the cell viability or cell cycle. In contrast, many probe sets were differentially expressed by >1.5-fold in both cell lines after MHT treatment. Of the 1,196 commonly and differentially expressed probe sets analysed by k-means clustering, three gene clusters, Up-I, Down-I and Down-II, were observed. Interestingly, two gene networks were obtained from the up-regulated genes in cluster Up-I. The gene network E contained DDIT3 and HSPA5 and was mainly associated with the biological process of endoplasmic reticulum stress, while the network S contained HBEGF and LIF and was associated with the biological process of cell survival. Eighteen genes were validated by quantitative real-time polymerase chain reaction, consistent with the microarray data, in four kinds of NHF cells. CONCLUSIONS: Common genes that were differentially expressed and/or acted within a gene network in response to MHT in NHF cells were identified. These findings provide the molecular basis for a further understanding of the mechanisms of the MHT response in NHF cells.
Assuntos
Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Hipertermia Induzida , Ciclo Celular , Linhagem Celular , Sobrevivência Celular , Chaperona BiP do Retículo Endoplasmático , Fibroblastos/citologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , TemperaturaRESUMO
To define the molecular mechanisms that mediate hyperthermia-induced apoptosis, we performed microarray and computational gene expression analyses. U937 cells, a human myelomonocytic lymphoma cell line, were treated with hyperthermia at 42 degrees C for 90 min and cultured at 37 degrees C. Apoptotic cells ( approximately 15%) were seen 6 h after hyperthermic treatment, and elevated expression of heat shock proteins (HSPs) including Hsp27, Hsp40, and Hsp70 was detected, following the activation of heat shock factor-1. Of the 54,675 probe sets analyzed, 1334 were upregulated and 4214 were downregulated by >2.0-fold in the cells treated with hyperthermia. A non-hierarchical gene clustering algorithm, K-means clustering, demonstrated 10 gene clusters. The gene network U1 or U2 that was obtained from up-regulated genes in cluster I or IX contained HSPA1B, DNAJB1, HSPH1, and TXN or PML, LYN, and DUSP1, and were mainly associated with cellular compromise, and cellular function and maintenance or death, and cancer, respectively. In the decreased gene cluster II, the gene network D1 including CCNE1 and CEBPE was associated with the cell cycle and cellular growth and proliferation. These findings will provide a basis for understanding the detailed molecular mechanisms of apoptosis induced by hyperthermia at 42 degrees C in cells.
Assuntos
Apoptose/genética , Redes Reguladoras de Genes/fisiologia , Proteínas de Choque Térmico/genética , Linfoma/genética , Análise por Conglomerados , Regulação Leucêmica da Expressão Gênica , Temperatura Alta , Humanos , Hipertermia Induzida , Linfoma/patologia , Linfoma/terapia , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Células U937RESUMO
In this study, to better understand the molecular mechanism underlying cellular responses to mild hyperthermia, we investigated gene expression patterns and genetic networks in human myelomonocytic lymphoma U937 cells using high-density oligonucleotide microarrays and computational gene expression analysis tools. The cells were incubated at 41 degrees C for 30 min (mild hyperthermia treatment) and then at 37 degrees C for 0-6 h. Although the mild hyperthermia treatment of the cells did not induce apoptosis, significant increases in the protein expression levels of heat shock proteins (HSPs), namely, Hsp27, Hsp40 and Hsp70, were observed following the activation of heat shock factor-1. Of the 22,283 probe sets analyzed, 423 probe sets were up-regulated and 515 probe sets were down-regulated by >1.5-fold in the cells 3 h post-treatment. Computational gene network analysis demonstrated that the significant genetic network A that contained many HSPs such as DNAJB1, HSPA1A, and HSPA1B was associated with cellular function and maintenance, post-transcriptional modification, or protein folding. Moreover, the significant genetic network B whose core contained v-myc myelocytomatosis viral oncogene homolog (MYC) was associated with cell morphology, cell cycle, and cellular development. The expression levels of nine selected genes were comparable to those determined by microarray analysis with real-time quantitative PCR assay. The present results indicate that mild hyperthermia affects the expression of a large number of genes and provides additional novel insights into the molecular basis of mild hyperthermia in cells.
Assuntos
Febre/fisiopatologia , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Linfoma/genética , Células U937/fisiologia , Perfilação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The effects of heat shock protein 70 (Hsp70), a molecular chaperone, on the degradation and functional alterations of a mutant large T antigen induced by a nonpermissive temperature were examined. In this study, mouse tracheal epithelial TM02-3 cells harboring temperature-sensitive simian virus 40 large T antigen and stable TM02-3 cells overexpressing human Hsp70 and/or Hsp40 were used. Although the temperature shift from 33 degrees C (permissive temperature) to 39 degrees C (nonpermissive temperature) induced increases in the endogenous chaperones including Hsp70 and Hsp40, degradation of the T antigen, activation of the p53-p21(waf1) pathway, and an arrest of cell growth were observed in the mock cells. In contrast, these changes induced by the temperature shift were partially but significantly prevented in stable cells overexpressing human Hsp70 and/or Hsp40. A combination of Hsp70 and Hsp40 was the most effective, suggesting that Hsp40 may cooperate with Hsp70. Moreover, immunocytochemical observation indicated that human Hsp70 was expressed in the cytoplasm at 33 degrees C, but it colocalized with T antigen in the nucleus at 39 degrees C. These results suggest that overexpressed Hsp70 translocates from the cytoplasm to nucleus, and significantly restores the structural stability and functional defects of mutant large T antigen in the cells.
Assuntos
Antígenos Virais de Tumores/genética , Proteínas de Choque Térmico HSP70/metabolismo , Mutação , Vírus 40 dos Símios/imunologia , Temperatura , Animais , Contagem de Células , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Conformação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traqueia/citologia , Traqueia/metabolismoRESUMO
Heat shock proteins (Hsp) 70 and Hsp 40 are stress proteins that cooperate as chaperones in mammalian cells. We determined the expression of Hsp 70 and Hsp 40 in 81 gastric cancers. Immunoreactivities to Hsp 70 and Hsp 40 were detected in 67.9 and 22.2% of tumors, respectively. Immunohistochemical analysis showed enhanced Hsp 70 and Hsp 40 expression in gastric tumor tissue, relative to the surrounding normal tissue. Overexpression of Hsp 70 and Hsp 40 was also confirmed by immunoblotting. Among various clinicopathological parameters, low histopathological differentiation was associated with reduced expression of both proteins.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Neoplasias Gástricas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Proteínas de Choque Térmico HSP40 , Humanos , Immunoblotting , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Gástricas/cirurgiaRESUMO
Heat shock proteins (HSPs) are induced by various physical, chemical, and biological stresses. HSPs are known to function as molecular chaperones, and they not only regulate various processes of protein biogenesis but also function as lifeguards against proteotoxic stresses. Because it is very useful to discover nontoxic chaperone-inducing compounds, we searched for them in herbal medicines. Some herbal medicines had positive effects on the induction of HSPs (Hsp70, Hsp40, and Hsp27) in cultured mammalian cells. We next examined 2 major constituents of these herbal medicines, glycyrrhizin and paeoniflorin, with previously defined chemical structures. Glycyrrhizin had an enhancing effect on the HSP induction by heat shock but could not induce HSPs by itself. In contrast, paeoniflorin had not only an enhancing effect but also an inducing effect by itself on HSP expression. Thus, paeoniflorin might be termed a chaperone inducer and glycyrrhizin a chaperone coinducer. Treatment of cells with paeoniflorin but not glycyrrhizin resulted in enhanced phosphorylation and acquisition of the deoxyribonucleic acid-binding ability of heat shock transcription factor 1 (HSF1), as well as the formation of characteristic HSF1 granules in the nucleus, suggesting that the induction of HSPs by paeoniflorin is mediated by the activation of HSF1. Also, thermotolerance was induced by treatment with paeoniflorin but not glycyrrhizin. Paeoniflorin had no toxic effect at concentrations as high as 80 microg/ mL (166.4 microM). To our knowledge, this is the first report on the induction of HSPs by herbal medicines.
Assuntos
Benzoatos/metabolismo , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Glucosídeos/metabolismo , Proteínas de Choque Térmico/metabolismo , Paeonia/metabolismo , Animais , Anti-Infecciosos/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Glucosídeos/genética , Ácido Glicirrízico/farmacologia , Células HeLa , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/efeitos dos fármacos , Medicina Herbária , Temperatura Alta , Humanos , Monoterpenos , Fitoterapia , Ratos , Fatores de TranscriçãoRESUMO
Mutations of the superoxide dismutase 1 (SOD1) gene cause familial amyotrophic lateral sclerosis (FALS). Intracytoplasmic aggregate formation consisting of mutant SOD1 is the histological hallmark of FALS. Since a previous report revealed that Hsp70 reduced aggregate formation and cell death in a cell model of FALS, here we examined the combined effects of Hsp70 and its cofactor, Hsp40, on a cell model of FALS. The combination of Hsp70 and Hsp40 reduced intracytoplasmic aggregates and markedly improved neurite outgrowth. They also prevented cell death to a relatively lesser extent. Neurite outgrowth was recognized almost exclusively in the cells without intracytoplasmic aggregates. Hsp70 and Hsp40 were upregulated in cells expressing mutant SOD1, and were colocalized with intracytoplasmic aggregates of mutant SOD1. These findings suggest that heat shock proteins (HSPs) promote neurite outgrowth by suppressing intracytoplasmic aggregate formation and restoring cellular dysfunctions. This is the first demonstration that overexpression of HSPs improved neurite outgrowth as it suppressed intracytoplasmic aggregate formation and cell death in a cultured neuronal cell model of FALS. These findings may provide a basis for the utilization of HSPs in developing a treatment for FALS.
Assuntos
Citoplasma/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Mutação , Neuritos , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/metabolismo , Baculoviridae , Western Blotting , Técnicas de Cultura de Células , Morte Celular/genética , Citoplasma/ultraestrutura , Imunofluorescência , Regulação Enzimológica da Expressão Gênica , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico/biossíntese , Humanos , Microscopia Confocal , Neuritos/metabolismo , Superóxido Dismutase-1 , Transfecção , Regulação para CimaRESUMO
Heat shock protein (Hsp) 70 and Hsp 40 are stress proteins that cooperate as chaperones in mammalian cells. The present study was designed to determine the expression levels of Hsp 70 and Hsp 40 in colorectal cancer by immunohistochemistry and Western blot analysis. Among 50 colorectal cancer tissues studied, 80% and 14% of tumors showed specific immunoreactivity to Hsp 70 and Hsp 40, respectively. Hsp 70 and Hsp 40 were overexpressed in cancer tissue samples compared with normal tissues, on both analytic sets. However, there were no significant correlations between their expression and various clinicopathological parameters of colorectal cancer. Hsp 70 and Hsp 40 may be tumor markers for colorectal cancer.
Assuntos
Neoplasias Colorretais/metabolismo , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/biossíntese , Western Blotting , Feminino , Proteínas de Choque Térmico HSP40 , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-IdadeRESUMO
Local hyperthermia (HT) for various types of malignant tumors has shown promising antitumor effects. To confirm the detailed molecular mechanism underlying cell death induced by HT, gene expression patterns and gene networks in human oral squamous cell carcinoma (OSCC) cells were examined using a combination of DNA microarray and bioinformatics tools. OSCC HSC-3 cells were treated with HT at 44ËC for 90 min or mild hyperthermia (MHT) at 42ËC for 90 min, followed by culturing at 37ËC for 0-24 h. Treatment of cells with HT prevented cell proliferation (62%) and induced cell death (17%), whereas these alterations were not observed in cells treated with MHT. Microarray analysis revealed substantial differences with respect to gene expression patterns and biological function for the two different hyperthermic treatments. Moreover, we identified the temperature-specific gene networks D and H that were obtained from significantly up-regulated genes in the HT and MHT conditions, respectively, using Ingenuity pathway analysis tools. Gene network D, which contains 14 genes such as ATF3, DUSP1 and JUN, was associated with relevant biological functions including cell death and cellular movement. Gene network H, which contains 13 genes such as BAG3, DNAJB1 and HSPA1B, was associated with cellular function and maintenance and cellular assembly and organization. These findings provide a basis for understanding the detailed molecular mechanisms of cell death elicited by HT in human OSCC cells.
Assuntos
Carcinoma de Células Escamosas/genética , Redes Reguladoras de Genes , Temperatura Alta , Neoplasias Bucais/genética , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores de TempoRESUMO
AIMS: We previously found that paeoniflorin, a major constituent of Paeonia lactiflora Pall, could induce heat shock proteins (HSPs) in cultured mammalian cells without apparent toxicity (Yan et al. 2004). We here investigated the induction of HSPs by paeoniflorin in mouse stomach and the effect of paeoniflorin on the HCl- and ethanol-triggered gastric mucosal injury in mouse. MAIN METHODS: Paeoniflorin and quercetin were intraperitoneally administered in mouse and Hsp70 and other proteins in mouse tissues were detected by western blotting. KEY FINDINGS: The intraperitoneal administration of paeoniflorin clearly induced Hsp70 in mouse stomach, and paeoniflorin had a protective effect on the HCl- and ethanol-triggered gastric mucosal injury. When quercetin was injected before paeoniflorin administration, the induction of Hsp70 was suppressed and the protective effect of paeoniflorin was also diminished. Thus, the expression level of Hsp70 was well correlated with the extent of protection against irritant-induced gastric mucosal injury. Oral injection of HCl activated nuclear factor kappa B (NF-κB) and elicited the expression of cyclooxygenase-2 (COX-2) in gastric mucosa. Prior administration of paeoniflorin, however, suppressed these effects. No apparent systemic side effect of paeoniflorin has been observed so far. Hsp70 was also induced in the liver, heart, and brain by paeoniflorin. SIGNIFICANCE: From these results, it is suggested that paeoniflorin and paeoniflorin-containing herbal medicines might be used clinically as HSP inducers for the prevention and treatment of diseases associated with protein conformation and of various other pathological states, such as stress ulcers and irritant- or ischemia-induced injuries.
Assuntos
Benzoatos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Glucosídeos/farmacologia , Proteínas de Choque Térmico HSP70/agonistas , Animais , Benzoatos/antagonistas & inibidores , Western Blotting , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Hidrocarbonetos Aromáticos com Pontes/antagonistas & inibidores , Ciclo-Oxigenase 2/biossíntese , Etanol/farmacologia , Mucosa Gástrica/lesões , Mucosa Gástrica/metabolismo , Glucosídeos/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/biossíntese , Coração/efeitos dos fármacos , Ácido Clorídrico/farmacologia , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Monoterpenos , Miocárdio/metabolismo , NF-kappa B/biossíntese , Quercetina/farmacologiaRESUMO
Although cancer cells exposed to temperatures >42.5°C undergo cell death as the temperature rises, exposure of up to 42.5°C induces slight or no cytotoxicity. The temperature of 42.5°C is, therefore, well known to be the inflection point of hyperthermia. To better understand the molecular mechanisms underlying cellular responses to heat stress at temperatures higher and lower than the inflection point, we carried out global scale microarray and computational gene expression analyses. Human leukemia U937 cells were incubated at 42°C or 44°C for 15 min and cultured at 37°C for 0-6 h. Apoptosis accompanied by the activation of caspase-3 and DNA fragmentation was only observed in cells treated with heat stress at 44°C, but not at 42°C. Although a large number of genes were differentially expressed by a factor of 2.0 or greater, we found substantial differences with respect to the biological functions and gene networks of the genes differentially expressed at the two temperatures examined. Interestingly, we identified temperature-specific gene networks that were considered to be mainly associated with cell death or cellular compromise and cellular function and maintenance at 44°C or 42°C, respectively, by using the Ingenuity pathway analysis tools. These findings provide the molecular basis for a further understanding of the mechanisms of the biological changes that are responsive to heat stress in human lymphoma cells.
Assuntos
Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/genética , Temperatura Alta , Linfoma/genética , Linfoma/metabolismo , Estresse Fisiológico , Apoptose/genética , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Humanos , Estresse Fisiológico/genética , Fatores de Transcrição/genética , Células U937RESUMO
Carbenoxolone (CBX) is a semisynthetic derivative of the licorice root substance glycyrrhizinic acid and has been previously reported to induce only heat shock protein 70 [Hsp70, HSPA1A (the systematic name of heat shock protein is given in the parenthesis after each HSP, according to the recent nomenclature guidelines, Kampinga et al., Cell Stress Chaperones, 14:105-111, 2008) but not other heat shock proteins (HSPs) (Nagayama et al., Life Sci. 69:2867-2873, 2001). In this study, we reinvestigated the effect of CBX on the induction of HSPs in HeLa and human neuroblastoma (A-172) cells. CBX clearly induced not only Hsp70 but also Hsp90 (HSPC1), Hsp40 (DNAJB1), and Hsp27 (HSPB1) at concentrations of 10 to 800 microM for 16 h incubation. At higher concentrations (more than 400 microM), however, CBX appeared to be toxic. Treatment of cells with CBX resulted in enhanced phosphorylation and acquisition of DNA-binding ability of heat shock transcription factor 1 (HSF1). Furthermore, characteristic HSF1 granules were formed in the nucleus, suggesting that the induction of HSPs by CBX is mediated by the activation of HSF1. Furthermore, thermotolerance was induced by CBX treatment, as determined by clonogenic survival. Although the precise target of CBX is not known at present, these results indicate that CBX is one of the molecular chaperone inducers and suggest that some pharmacological activities of CBX might be ascribable in part to its molecular chaperone-inducing property.
Assuntos
Carbenoxolona/farmacologia , Proteínas de Choque Térmico/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Fosforilação , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismoRESUMO
PURPOSE AND BACKGROUND: Paeoniflorin (PF) isolated from peony root (Paeoniae radix) has been used as a herbal medicine in East Asia for its anti-allergic, anti-inflammatory, and immunoregulatory effects. PF is known to cause apoptosis and to be a chemical heat shock protein (HSP) inducer. With this information, the effects on the gene expression in human leukemia U937 cells treated with PF were investigated. METHODS: U937 cells, a human myelomonocytic cell line, were treated with PF at different concentrations (0-640 microg/ml). Expression level of Hsp70 was monitored by Western blotting. Gene expression was evaluated using high-density oligonucleotide microarrays and computational gene expression analysis tools and the results were verified by real-time quantitative PCR. RESULTS: Although cell viability was not affected after PF treatment at a high concentration of 640 microg/ml, PF treatment (80-640 microg/ml) significantly elevated Hsp70 expression in a concentration-dependent manner. When the cells were treated with PF (160 microg/ml; 30 min), 35 up-regulated and 29 down-regulated genes were identified. Among the differentially expressed genes, a significant genetic network containing CDC2, FOSL1 and EGR1 was associated with biological functions such as cell death, gene expression or cellular growth and proliferation. CONCLUSION: The present results indicate that PF affects the expression of many genes including Hsp70 and will provide a better understanding on the molecular mechanism of action of this compound in inducing HSPs in cells.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Benzoatos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosídeos/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Apoptose/efeitos dos fármacos , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Humanos , Monoterpenos , Análise de Sequência com Séries de Oligonucleotídeos , Células U937 , Regulação para CimaRESUMO
Heat shock proteins (HSPs) have molecular chaperone functions in protein biogenesis as well as cytoprotective functions against deleterious environmental stresses, and they work mainly inside of the cells. HSPs are usually induced in living cells that have been exposed to mild stresses or have recovered from severe stresses. Here, we show the enhanced synthesis of HSPs in gradually and necrotically dying cells that were treated with a high concentration of acrylamide (10 mM). This treatment caused irreversible cell death. The synthesis of HSPs, which was enhanced before cell death, was mediated by the activation of heat shock transcription factor 1 (HSF1); that is, the treatment led to the phosphorylation of HSF1, formation of characteristic HSF1 granules in the nucleus, and acquisition of DNA binding ability of HSF1. The induction of HSPs by acrylamide treatment was dependent on the consensus sequence of heat shock element (HSE) as demonstrated by a reporter assay. Also, several HSPs (Hsp90, Hsc70, Hsp70, Hsp60, Hsp47, Hsp40, and Hsp27) were detected outside of the cells after the treatment with acrylamide, indicating that these HSPs are released from necrotically dead cells. These results suggest that when cells are slowly and irreversibly dying, they augment the expression of HSPs and release them outside of the cells as a danger signal or dying messages.
Assuntos
Acrilamida/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Necrose , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Humanos , Fatores de Tempo , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
DNA fragmentation is a hallmark of cells undergoing apoptosis and is mediated mainly by the caspase-activated DNase (CAD or DNA-fragmentation factor 40 [DFF40]), which is activated when released from its inhibitor protein (ICAD or DFF45) upon apoptosis signals. Here we analyzed the effect of heat shock protein 70 (Hsp70) on CAD activity in T-cell receptor (TCR)-induced apoptosis using a T-cell line (TAg-Jurkat). Overexpression of Hsp70 significantly augmented the apoptotic cell death as well as DNA fragmentation in CD3/CD28- or staurosporine-stimulated cells. Following stimulation of cells with CD3/CD28 or staurosporine, Hsp70 was coprecipitated with free CAD, but not with CAD associated with ICAD. Furthermore, the purified Hsp70 dose-dependently augmented DNA-fragmentation activity of caspase-3-activated CAD in a cell-free system. Peptide-binding domain-deleted Hsp70 could neither bind nor augment its activity, while adenosine triphosphate (ATP)-binding domain-deleted Hsp70 or the peptide-binding domain itself bound CAD and augmented its activity. These results indicate that the the binding of Hsp70 to the activated CAD via the peptide-binding domain augments its activity. Although CAD lost its activity in an hour after being released from ICAD in vitro, its activity was retained after an hour of incubation in the presence of Hsp70, suggesting that Hsp70 may be involved in stabilization of CAD activity. Finally, CAD that had been coprecipitated with Hsp70 from the cell lysate of staurosporine-activated 293T cells induced chromatin DNA fragmentation and its activity was not inhibited by ICAD. These results suggest that Hsp70 binds free CAD in TCR-stimulated T cells to stabilize and augment its activity.
Assuntos
Desoxirribonucleases/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/enzimologia , Caspases/metabolismo , Sistema Livre de Células , Fragmentação do DNA/imunologia , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/farmacologia , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/farmacologia , Humanos , Técnicas In Vitro , Células Jurkat , Estrutura Terciária de Proteína , Proteínas Recombinantes/farmacologia , Estaurosporina/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
The HSV-2 UL14 gene encodes a 32 kDa protein that is a minor component of the viral tegument. The protein relocates other viral proteins such as VP26 and UL33 protein into the nuclei of transiently coexpressing cells (Yamauchi et al., 2001). We found that the protein shared some characteristics of heat shock proteins (HSPs) or molecular chaperones, such as nuclear translocation upon heat shock, ATP deprivation and osmotic shock. Interestingly, a significant homology over a stretch of 15 amino acids was found between an N-terminal region of HSV UL14 protein and the substrate-binding domain of Hsp70 family proteins. Two arginine residues in this region were important for nuclear translocation of VP26. In addition, overexpression of UL14 protein increased the activity of coexpressed firefly luciferase, which suggested that the protein functioned in the folding of newly synthesized luciferase. We thus conclude that UL14 protein can act as a chaperone-like protein in a singly expressed state.
Assuntos
Proteínas de Choque Térmico/metabolismo , Herpes Simples/metabolismo , Herpesvirus Humano 2/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos/fisiologia , Animais , Arginina/metabolismo , Compartimento Celular/fisiologia , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Chlorocebus aethiops , Regulação Viral da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Herpes Simples/fisiopatologia , Humanos , Luciferases/genética , Luciferases/metabolismo , Chaperonas Moleculares/genética , Oligonucleotídeos Antissenso , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Células Vero , Proteínas Virais/genéticaRESUMO
Herpes simplex virus (HSV) is a large, enveloped DNA virus that replicates in the nucleus and is assembled in the cytoplasm to the mature infectious virion. In this study, we present evidence that, in HSV-2-infected cells, some tegument proteins (UL46 and VP16) and newly synthesized nucleocapsids accumulate in a juxtanuclear domain sharing characteristics with aggresomes, cellular structures formed in response to misfolded proteins [J. Cell Biol. 146 (1999) 1239, J. Cell Biol. 143 (1998) 2010]. The juxtanuclear domains (aggresome-like structures) induced by HSV-2 infection localize to the microtubule organizing center (MTOC) where the clustering mitochondria, Golgi-derived vesicles, and cellular chaperones including heat shock protein (Hsp)40 and Hsp70 were recruited. Formation of aggresome-like structures was blocked by the presence of microtubule-disassembling drug nocodazole, indicating that microtubule-dependent transport may be involved in the accumulation of viral and cellular proteins at these sites in HSV-2-infected cells. These features are similar to those governing the formation of aggresomes. In contrast to aggresomes, however, the vimentin cage surrounding the MTOC was not observed with the aggresome-like structures in HSV-2-infected cells, and the maintenance of these structures required an intact microtubular network. Disruption of the aggresome-like structures by nocodazole treatment led to a low but consistent effect (10-fold decrease) on the production of intracellular infectious particles. These results suggest that aggresome-like structures do not play a critical but augmentary role in HSV-2 replication.