RESUMO
Neuronal activity-induced enhancers drive gene activation. We demonstrate that BRG1, the core subunit of SWI/SNF-like BAF ATP-dependent chromatin remodeling complexes, regulates neuronal activity-induced enhancers. Upon stimulation, BRG1 is recruited to enhancers in an H3K27Ac-dependent manner. BRG1 regulates enhancer basal activities and inducibility by affecting cohesin binding, enhancer-promoter looping, RNA polymerase II recruitment, and enhancer RNA expression. We identify a serine phosphorylation site in BRG1 that is induced by neuronal stimulations and is sensitive to CaMKII inhibition. BRG1 phosphorylation affects its interaction with several transcription co-factors, including the NuRD repressor complex and cohesin, possibly modulating BRG1-mediated transcription outcomes. Using mice with knockin mutations, we show that non-phosphorylatable BRG1 fails to efficiently induce activity-dependent genes, whereas phosphomimic BRG1 increases enhancer activity and inducibility. These mutant mice display anxiety-like phenotypes and altered responses to stress. Therefore, we reveal a mechanism connecting neuronal signaling to enhancer activities through BRG1 phosphorylation.
Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos/genética , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Ansiedade/patologia , Comportamento Animal , Encéfalo/patologia , DNA Helicases/química , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Proteínas Nucleares/química , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Estresse Psicológico/complicações , Fatores de Transcrição/química , alfa-Fetoproteínas/metabolismoRESUMO
In Sonic hedgehog (SHH) signaling, GLI family zinc finger (GLI)-mediated diverse gene transcription outcomes are strictly regulated and are important for SHH function in both development and disease. However, how the GLI factors differentially regulate transcription in response to variable SHH activities is incompletely understood. Here, using a newly generated, tagged Gli3 knock-in mouse (Gli3TAP ), we performed proteomic analyses and identified the chromatin-associated SAFB-like transcription modulator (SLTM) as a GLI-interacting protein that context-dependently regulates GLI activities. Using immunoprecipitation and immunoblotting, RT-quantitative PCR, and ChIP assays, we show that SLTM interacts with all three GLI proteins and that its cellular levels are regulated by SHH. We also found that SLTM enhances GLI3 binding to chromatin and increases GLI3 repressor (GLI3R) form protein levels. In a GLI3-dependent manner, SLTM promoted the formation of a repressive chromatin environment and functioned as a GLI3 co-repressor. In the absence of GLI3 or in the presence of low GLI3 levels, SLTM co-activated GLI activator (GLIA)-mediated target gene activation and cell differentiation. Moreover, in vivo Sltm deletion generated through CRISPR/Cas9-mediated gene editing caused perinatal lethality and SHH-related abnormal ventral neural tube phenotypes. We conclude that SLTM regulates GLI factor binding to chromatin and contributes to the transcriptional outcomes of SHH signaling via a novel molecular mechanism.
Assuntos
Proteínas Hedgehog/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Transdução de Sinais , Proteína Gli3 com Dedos de Zinco/metabolismo , Animais , Sistemas CRISPR-Cas , Cromatina , Edição de Genes , Técnicas de Introdução de Genes , Proteínas Hedgehog/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteômica , Proteína Gli3 com Dedos de Zinco/genéticaRESUMO
Synapse development requires normal neuronal activities and the precise expression of synapse-related genes. Dysregulation of synaptic genes results in neurological diseases such as autism spectrum disorders (ASD). Mutations in genes encoding chromatin-remodeling factor Brg1/SmarcA4 and its associated proteins are the genetic causes of several developmental diseases with neurological defects and autistic symptoms. Recent large-scale genomic studies predicted Brg1/SmarcA4 as one of the key nodes of the ASD gene network. We report that Brg1 deletion in early postnatal hippocampal neurons led to reduced dendritic spine density and maturation and impaired synapse activities. In developing mice, neuronal Brg1 deletion caused severe neurological defects. Gene expression analyses indicated that Brg1 regulates a significant number of genes known to be involved in synapse function and implicated in ASD. We found that Brg1 is required for dendritic spine/synapse elimination mediated by the ASD-associated transcription factor myocyte enhancer factor 2 (MEF2) and that Brg1 regulates the activity-induced expression of a specific subset of genes that overlap significantly with the targets of MEF2. Our analyses showed that Brg1 interacts with MEF2 and that MEF2 is required for Brg1 recruitment to target genes in response to neuron activation. Thus, Brg1 plays important roles in both synapse development/maturation and MEF2-mediated synapse remodeling. Our study reveals specific functions of the epigenetic regulator Brg1 in synapse development and provides insights into its role in neurological diseases such as ASD.
Assuntos
DNA Helicases/metabolismo , Fatores de Transcrição MEF2/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Sinapses/metabolismo , Fatores de Transcrição/metabolismo , Animais , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Cromatina/metabolismo , DNA Helicases/genética , Espinhas Dendríticas/genética , Hipocampo/metabolismo , Fatores de Transcrição MEF2/genética , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição/genéticaRESUMO
Insights from embryonic development suggest chromatin remodeling is important in adult neural stem cells (aNSCs) maintenance and self-renewal, but this concept has not been fully explored in the adult brain. To assess the role of chromatin remodeling in adult neurogenesis, we inducibly deleted Brg1--the core subunit of SWI/SNF-like Brg1/Brm-associated factor chromatin remodeling complexes--in nestin-expressing aNSCs and their progeny in vivo and in culture. This resulted in abnormal adult neurogenesis in the hippocampus, which initially reduced hippocampal aNSCs and progenitor maintenance, and later reduced its responsiveness to physiological stimulation. Mechanistically, deletion of Brg1 appeared to impair cell cycle progression, which is partially due to elevated p53 pathway and p21 expression. Knockdown of p53 rescued the neurosphere growth defects caused by Brg1 deletion. Our results show that epigenetic chromatin remodeling (via a Brg1 and p53/p21-dependent process) determines the aNSCs and progenitor maintenance and responsiveness of neurogenesis.
Assuntos
Células-Tronco Adultas/metabolismo , DNA Helicases/metabolismo , Hipocampo/metabolismo , Nestina/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Células-Tronco Adultas/citologia , Animais , DNA Helicases/genética , Regulação da Expressão Gênica , Hipocampo/citologia , Camundongos , Camundongos Transgênicos , Nestina/genética , Células-Tronco Neurais/citologia , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
Molecular and functional studies of genes in neurons in mouse models require neuron-specific Cre lines. The current available neuronal Cre transgenic or knock-in lines either result in expression in a subset of neurons or expression in both neuronal and non-neuronal tissues. Previously we identified BAF53b as a neuron-specific subunit of the chromatin remodeling BAF complexes. Using a bacteria artificial chromosome (BAC) construct containing the BAF53b gene, we generated a Cre transgenic mouse under the control of BAF53b regulatory elements. Like the endogenous BAF53b gene, we showed that BAF53b-Cre is largely neuron-specific. In both central and peripheral nervous systems, it was expressed in all developing neurons examined and was not observed in neural progenitors or glial cells. In addition, BAF53b-Cre functioned in primary cultures in a pan-neuron-specific manner. Thus, BAF53b-Cre mice will be a useful genetic tool to manipulate gene expression in developing neurons for molecular, biochemical, and functional studies.
Assuntos
Integrases/metabolismo , Neurônios/metabolismo , Actinas/metabolismo , Animais , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Camundongos , Camundongos Transgênicos , Modelos Animais , Neurônios/enzimologiaRESUMO
The Sonic hedgehog (Shh) signalling pathway plays important roles during development and in cancer. Here we report a Shh-induced epigenetic switch that cooperates with Gli to control transcription outcomes. Before induction, poised Shh target genes are marked by a bivalent chromatin domain containing a repressive histone H3K27me3 mark and an active H3K4me3 mark. Shh activation induces a local switch of epigenetic cofactors from the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) to an H3K27me3 demethylase Jmjd3/Kdm6b-centred coactivator complex. We also find that non-enzymatic activities of Jmjd3 are important and that Jmjd3 recruits the Set1/MLL H3K4 methyltransferase complexes in a Shh-dependent manner to resolve the bivalent domain. In vivo, changes of the bivalent domain accompanied Shh-activated cerebellar progenitor proliferation. Overall, our results reveal a regulatory mechanism that underlies the activation of Shh target genes and provides insight into the causes of various diseases and cancers exhibiting altered Shh signalling.
Assuntos
Epigênese Genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Animais , Células Cultivadas , Cerebelo/crescimento & desenvolvimento , Feminino , Histona-Lisina N-Metiltransferase/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Meduloblastoma/metabolismo , Camundongos Endogâmicos C57BL , Proteína de Leucina Linfoide-Mieloide/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Gravidez , Proteína GLI1 em Dedos de ZincoRESUMO
Sonic hedgehog (Shh) signaling plays diverse roles during animal development and adult tissue homeostasis through differential regulation of Gli family transcription factors. Dysregulated Shh signaling activities have been linked to birth defects and tumorigenesis. Here we report that Brg, an ATP-dependent chromatin remodeling factor, has dual functions in regulating Shh target gene expression. Using a Brg conditional deletion in Shh-responding neural progenitors and fibroblasts, we demonstrate that Brg is required both for repression of the basal expression and for the activation of signal-induced transcription of Shh target genes. In developing telencephalons deficient for Brg, Shh target genes were derepressed, whereas Brg-deleted cerebellar granule neuron precursors failed to respond to Shh to increase their proliferation. The repressor function of Brg was mediated through Gli3 and both the repressor and activator functions of Brg appeared to be independent of its ATPase activity. Furthermore, Brg facilitates Gli coactivator histone deacetylase (HDAC) binding to the regulatory regions of Shh target genes, providing a possible mechanism for its positive role in Shh signaling. Our results thus reveal that a complex chromatin regulation mechanism underlies the precise transcription outcomes of Shh signaling and its diverse roles during development.
Assuntos
DNA Helicases/metabolismo , Proteínas Hedgehog/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , DNA Helicases/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Proteínas Hedgehog/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Immunoblotting , Hibridização In Situ , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Proteínas Nucleares/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Telencéfalo/citologia , Telencéfalo/embriologia , Telencéfalo/metabolismo , Técnicas de Cultura de Tecidos , Fatores de Transcrição/genética , Proteína Gli3 com Dedos de ZincoRESUMO
Replication stress often induces chromosome instability. In this study, we explore which factors in replication-compromised cells promote abnormal chromosome ploidy. We expressed mutant forms of either polymerase α (Polα) or polymerase δ (Polδ) in normal human fibroblasts to compromise DNA replication. Cells expressing the mutant Polα-protein failed to sustain mitotic arrest and, when propagated progressively, down-regulated Mad2 and BubR1 and accumulated 4N-DNA from the 2N-DNA cells. Significantly, a population of these cells became tetraploids. The Polα mutant expressing cells also exhibited elevated cellular senescence markers, suggesting as a mechanism to limit proliferation of the tetraploids. Expression of the Polδ mutant also caused cells to accumulate 4N-DNA. In contrast to the Polα mutant expressing cells, the Polδ mutant expressing cells expressed sufficient levels of Mad2, BubR1, and cyclin B1 to sustain mitotic arrest, and these cells had normal chromosome ploidy. Together, these results suggest that replication-compromised cells depend on the mitotic checkpoint to prevent mitotic slippage that could result in tetraploidization.
Assuntos
Instabilidade Cromossômica , DNA Polimerase I/metabolismo , DNA Polimerase beta/metabolismo , Fibroblastos/metabolismo , Mitose , Poliploidia , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular , Ciclina B1/biossíntese , Ciclina B1/genética , DNA Polimerase I/genética , DNA Polimerase beta/genética , Humanos , Proteínas Mad2 , Mutação , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genéticaRESUMO
It has been demonstrated that interleukin 24 (IL-24, also called melanoma differentiation associated gene 7) exerts antitumor activity. In this study, we investigated whether oncolytic adenovirus-mediated gene transfer of IL-24 could induce strong antitumor activity. A tumor-selective replicating adenovirus expressing IL-24 (ZD55-IL-24) was constructed by insertion of an IL-24 expression cassette into the ZD55 vector, which is based on deletion of the adenoviral E1B 55-kDa gene. ZD55-IL-24 could express substantially more IL-24 than Ad-IL-24 because of replication of the vector. It has been shown that ZD55-IL-24 exerted a strong cytopathic effect and significant apoptosis in tumor cells with p53 dysfunction. Moreover, no cytotoxic and apoptotic effects could be seen in normal cells infected with ZD55-IL-24. Expression of IL-24 did not interfere with viral replication induced by oncolytic adenovirus. Activation of caspase 3 and caspase 9, and induction of bax gene expression, were involved in tumor cell apoptosis induced by ZD55-IL-24. Treatment of established tumors with ZD55-IL-24 showed much stronger antitumor activity than that induced by ONYX-015 or Ad-IL- 24. These data indicated that oncolytic adenovirus expressing IL-24 could exert potential antitumor activity and offer a novel approach to cancer therapy.
Assuntos
Adenoviridae/genética , Neoplasias Colorretais/terapia , Terapia Genética , Interleucinas/genética , Proteínas E1B de Adenovirus/genética , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular , Neoplasias Colorretais/metabolismo , Feminino , Expressão Gênica , Genes Supressores de Tumor , Vetores Genéticos/genética , Humanos , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Armed oncolytic adenoviruses represent an appealing tumor treatment approach, as they can attack tumors at multiple levels. In this study, considering that angiogenesis plays a central role in tumor growth, we inserted an antiangiogenic gene, sflt-1(1-3) (the first three extracellular domains of FLT1, the hVEGF receptor-1), into an E1B-55-kDa-deleted oncolytic adenovirus (ZD55) to construct ZD55-sflt-1. Although soluble (s) Flt-1 did not affect tumor cell growth, ZD55-sflt-1 could specifically induce a cytopathic effect in tumor cells, like ONYX-015. The secretion of sFlt-1 from ZD55-sflt-1 was much higher than that from replication-deficient Ad-sflt-1 upon infection of SW620 human colon tumor cells, leading to a stronger inhibitory effect on VEGF-induced proliferation and tube formation ability of HUVECs. Moreover, marked reduction of tumor growth and long-term survival rates were observed in ZD55-sflt-1-treated nude mice with subcutaneous SW620 tumor. Its efficacy correlated with a decrease in microvessel density and an increase in apoptotic tumor cells. In addition, ZD55-sflt-1 showed a synergic effect with the chemotherapeutic agent 5-FU. These results indicate that ZD55-sflt-1, combining the advantages of oncolytic adenovirus and antiangiogenic gene therapy, is a powerful agent for human tumor treatment.
Assuntos
Neoplasias do Colo/terapia , Genes Supressores de Tumor , Terapia Genética , Neovascularização Patológica/terapia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Adenoviridae/genética , Proteínas E1B de Adenovirus/genética , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Apoptose , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/tratamento farmacológico , Feminino , Fluoruracila/uso terapêutico , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Células Tumorais Cultivadas , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Replicação Viral/genéticaRESUMO
The naloxone-precipitated withdrawal syndrome in mice and rats after intrathecal injection of recombinant human interleukin-2 protein (rIL-2) or its gene was studied. The results showed that rIL-2 could significantly decrease the number of jumps in mice. In rats, rIL-2 significantly suppressed irritating, diarrhea, weight loss, abnormal posture and salivation. Tendencies towards reductions in teeth chewing and dog-shaking were also observed. Furthermore, pcDNA3-IL-2 (8 microg DNA) had a similar effect as 1x10 IU rIL-2 protein on inhibition of morphine withdrawal syndrome in mice, and the expression of rIL-2 protein in spinal cord could be detected for 6 days. These findings provided further evidence for the neuroregulatory function of an immunological molecule such as IL-2.
Assuntos
Técnicas de Transferência de Genes , Interleucina-2/administração & dosagem , Interleucina-2/genética , Dependência de Morfina/terapia , Síndrome de Abstinência a Substâncias/terapia , Animais , Citomegalovirus/genética , DNA Complementar/administração & dosagem , Vetores Genéticos , Injeções Espinhais , Interleucina-2/biossíntese , Masculino , Camundongos , Dependência de Morfina/genética , Dependência de Morfina/prevenção & controle , Naloxona/farmacologia , Regiões Promotoras Genéticas/genética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Síndrome de Abstinência a Substâncias/genética , Síndrome de Abstinência a Substâncias/prevenção & controleRESUMO
We previously blocked the heat shock transcription factor 1 function with a dominant-negative mutant (mHSF1) in breast cancer cell line Bcap37, and found that mHSF1 sensitizes Bcap37 cells to hyperthermia by promoting the apoptotic process. Here we studied the mechanism of this abolishing process and how thermotolerance develops in Bcap37 cells. The results indicated that mHSF1 abolished acquired or intrinsic thermotolerance in Bcap37 cells by enhancing JNK and caspase-3 pathways, two stress-induced apoptotic pathways, after hyperthermia, and interference with either one of them attenuated hyperthermia-induced apoptosis. Furthermore, epistasis assay of these two pathways suggested that JNK was upstream of the caspase-3 pathway. Conversely, other hyperthermia-induced kinases implicated in cell survival and death, Akt, ERK or p38, did not influence the effect of mHSF1, indicating that these kinases were not implicated in this abolishing process. In addition, we found that the development of acquired thermotolerance of Bcap37 cells was associated with the suppression of JNK activation after mild preheat treatment and was not reduced by Akt, ERK or p38 inhibition. In contrast, the intrinsic thermotolerance of Bcap37 cells was due to the intrinsic high levels of Akt and ERK activities since Akt or ERK inhibition resulted in increased thermosensitivity of Bcap37 cells. Our results suggest that mHSF1 plays a valuable role in the thermotolerance abolishment of Bcap37 cells, which likely contributes to tumor therapy in combination with hyperthermia.
Assuntos
Caspases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Temperatura Alta , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias/metabolismo , Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Caspase 3 , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Feminino , Fatores de Transcrição de Choque Térmico , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Mutação , Proibitinas , Transdução de Sinais/fisiologia , Fatores de TranscriçãoRESUMO
To improve the therapeutic effect of ONYX015, an E1B55kD-deleted replication-competent adenovirus, ZD55 was constructed and armed with the therapeutic gene hTRAIL to form ZD55-hTRAIL, which was used for cancer therapy and which we call Targeting Gene-ViroTherapy. In vitro experiments with SW620, HCT116, and HT29 colorectal carcinoma cell lines demonstrated that they were all sensitive to ZD55-hTRAIL, and especially sensitive to ZD55-hTRAIL plus 5-fluorouracil (5-FU) treatment. In the SW620 xenograft tumor model, various treatment groups showed marked differences at week 11, with the tumor volume for the phosphate-buffered saline (PBS) treatment group >1700 mm3, for 5-FU > 1300 mm3, for ONYX015 1051.3 mm3, for ZD55-hTRAIL 600.05 mm3, and for ZD55-hTRAIL plus 5-FU 230.2 mm3. At the end of week 14, tumor-bearing mice in the other groups almost all died, whereas all the mice in the combined treatment group were alive, with one mouse tumor free. By transmission electron microscopy (TEM) assay, most tumor cells treated with ONYX015 or with ZD55-hTRAIL singly or in combination with 5-FU were lysed due to viral propagation. RT-PCR analysis and immunohistochemistry examination revealed that hTRAIL was expressed in ZD55-hTRAIL-treated SW620 tumor tissue. Furthermore, no detectable hepatoxicity was found by serum enzyme level analysis. These results suggest that ZD55-hTRAIL alone or in combination with 5-FU may have potential clinical implications.
Assuntos
Adenoviridae/genética , Antimetabólitos Antineoplásicos/uso terapêutico , Neoplasias Colorretais/terapia , Fluoruracila/uso terapêutico , Terapia Genética , Adenoviridae/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/virologia , Sinergismo Farmacológico , Fluoruracila/farmacologia , Vetores Genéticos/química , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Vetores Genéticos/farmacologia , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/uso terapêutico , Camundongos , Camundongos Nus , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatocellular carcinoma (HCC) displays a high resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cell death. To increase sensitivity of HCC cells to TRAIL, we have constructed an oncolytic adenoviral vector (ZD55) and used this vector to deliver second mitochondria-derived activator of caspases (Smac) and TRAIL genes (ZD55-Smac and ZD55-TRAIL, respectively) into HCC cells. Our data showed that human HCC cells express high levels of inhibitor of apoptosis proteins (IAPs). Transfected HCC cells expressing exogenous X-linked IAPs (XIAPs) displayed more resistance to TRAIL. The expression of Smac led to rapid and potent activation of apoptosis in HCC cells after infection with ZD55-Smac. The activation of caspases and induction of apoptosis could be enhanced further through coinfection with ZD55-TRAIL. The combined treatment of ZD55-Smac and ZD55-TRAIL resulted in significant reduction of XIAP expression levels. In addition, our in vivo data in mice showed only a partial response in the established tumor treated either by ZD55-Smac or ZD55-TRAIL alone. By contrast, complete tumor regression was observed by combination of ZD55-Smac and ZD55-TRAIL in all treated animals. This strong antitumoral activity achieved by this combination was due to a dramatic induction of tumor cell apoptosis in the treated tumors. In conclusion, our data indicate that Smac antagonizes the IAPs in HCC tumor cells and enhances tumor cell death induced by TRAIL in the oncolytic adenoviral vector. The combination of Smac and TRAIL delivered by way of the oncolytic adenoviral vector would provide a useful strategy for therapy of HCC and might also be applied to other IAPs abundant in cancers.
Assuntos
Adenoviridae/genética , Carcinoma Hepatocelular/terapia , Proteínas de Transporte/genética , Terapia Genética , Neoplasias Hepáticas Experimentais/terapia , Glicoproteínas de Membrana/metabolismo , Proteínas Mitocondriais/genética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Carcinoma Hepatocelular/metabolismo , Caspases/metabolismo , Feminino , Expressão Gênica , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Telomerase is a therapeutic target for cancer. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionaly upregulated exclusively in about 90% of cancer cells. Previous studies have demonstrated that hTERT promoter can control the expression of exogenous genes to the telomerase-positive cancer cells, thus hTERT promoter is an excellent candidate for generating cancer-specific oncolytic adenovirus. In this study, we devised a novel oncolytic adenovirus (Ad.TERT) by replacing the normal E1A regulatory elements with hTERT promoter. Ad.TERT displays cancer-specific E1A expression, virus replication and cytolysis in in vitro experiments. In animal experiments, intratumoral administration of Ad.TERT demonstrates potent antitumoral efficacy at least in two xenograft models (Bcap37 and BEL7404). Ad.TERT was targeted by the telomerase activity in cancer cells and has potent antitumoral efficacy in vivo, and since telomerase activity is a wide-ranged tumor marker, Ad.TERT could be a powerful therapeutic agent for a variety of cancers.
Assuntos
Adenoviridae/genética , Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Antineoplásicos , Neoplasias/enzimologia , Neoplasias/patologia , Telomerase/genética , Telomerase/metabolismo , Animais , Western Blotting , Morte Celular , Proteínas de Ligação a DNA , Feminino , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutagênese Sítio-Dirigida , Transplante de Neoplasias , Neoplasias/genética , Neoplasias/virologia , Proibitinas , Regiões Promotoras Genéticas/genética , Células Tumorais Cultivadas , Replicação ViralRESUMO
Neovascularization is a prerequisite for progressive growth of most tumors and their metastases. Therefore, inhibition of angiogenesis could be one of the most promising strategies that might lead to the development of novel anticancer therapy. New blood vessels forming in tumors can be avoided by interfering the process of angiogenesis through suppressing the proangiogenic signal or augmenting the antiangiogenic factors. Concentrated efforts in this area have lead to the discovery of many proangiogenic factors as well as their inhibitors, and antiangiogenic factors. For the established tumor vasculature, tumor vasculature-targeted delivery of antiangiogenic substances and endothelial cell vaccines has been explored. Although some antiangiogenic drugs are currently in clinical development, for future reason, more efficient therapeutic methods, including antiangiogenic gene therapy strategy, targeted drug delivery system, and the combination of antiangiogenic agents with immunotherapy, chemotherapy or radiotherapy are being explored. With the development of tumor model assessment system, clinical use of the above antiangiogenic tumor therapy methods could be achieved.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores da Angiogênese/genética , Animais , Terapia Genética/métodos , Terapia Genética/tendências , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/terapia , Neovascularização Patológica/prevenção & controleRESUMO
Two tandem cassettes, one containing the telomerase reverse transcriptase gene (hTERT) promoter upstream of a constitutively activated form of heat shock transcription factor 1 (cHSF1) and followed by the other containing the heat shock protein 70B (hsp70B) promoter (HSE) upstream of the cytosine deaminase (CD) gene, could greatly enhance the efficiency of CD gene therapy while retaining tumor specificity in vitro and in vivo. This hTERT-cHSF1/HSE promoter could restrict gene expression in tumor cells and was about 1.5-3-fold more potent than the cytomegalovirus (CMV) promoter. hTERT-cHSF1/HSE-CD transfection led to tumor cells more sensitive to 5-fluorocytosine compared with hTERT-CD and its toxicity was comparable to that of CMV-CD. Besides enhancement of promoter activity, cHSF1 overexpression itself could enhance the bystander effect of CD gene therapy that could be reversed by anti-Fas antibody. This system also led to activation of stress-related genes such as hsp70 in tumor cells, which in the presence of cell killing by the cytotoxic gene is a highly immunostimulatory event. Furthermore, a more potent anti-tumor effect of hTERT-cHSF1/HSE-CD was observed in nude mice inoculated with Bcap37 cells. No obvious activity of the hTERT-cHSF1/HSE promoter was observed in normal tissues after intravenous administration. These results indicate that the hTERT-cHSF1/HSE promoter is highly tumor-specific and strong with potential application in targeted gene therapy, and therefore may be useful for construction of vectors for systemic therapy.