RESUMEN
G-protein coupled receptor 3 (GPR3), GPR6, and GPR12 belong to a family of constitutively active Gs-coupled receptors that activate 3'-5'-cyclic adenosine monophosphate (cAMP) and are highly expressed in the brain. Among these receptors, the endogenous expression of GPR3 in cerebellar granule neurons (CGNs) is increased following development. GPR3 is important for neurite outgrowth and neural maturation; however, the physiological functions of GPR3 remain to be fully elucidated. Here, we investigated the survival and antiapoptotic functions of GPR3 under normal and apoptosis-inducing culture conditions. Under normal culture conditions, CGNs from GPR3-knockout mice demonstrated lower survival than did CGNs from wild-type or GPR3-heterozygous mice. Cerebellar sections from GPR3-/- mice at P7, P14, and P21 revealed more caspase-3-positive neurons in the internal granular layer than in cerebellar sections from wild-type mice. Conversely, in a potassium-deprivation model of apoptosis, increased expression of these three receptors promoted neuronal survival. The antiapoptotic effect of GPR3 was also observed under hypoxic (1% O2/5% CO2) and reactive oxygen species (ROS)-induced apoptotic conditions. We further investigated the signaling pathways involved in the GPR3-mediated antiapoptotic effect. The addition of the PKA inhibitor KT5720, the MAP kinase inhibitor U0126, and the PI3 kinase inhibitor LY294002 abrogated the GPR3-mediated antiapoptotic effect in a potassium-deprivation model of apoptosis, whereas the PKC inhibitor Gö6976 did not affect the antiapoptotic function of GPR3. Furthermore, downregulation of endogenous GPR3 expression in CGNs resulted in a marked reduction in the basal levels of ERK and Akt phosphorylation under normal culture conditions. Finally, we used a transient middle cerebral artery occlusion (tMCAO) model in wild-type and GPR3-knockout mice to determine whether GPR3 expression modulates neuronal survival after brain ischemia. After tMCAO, GPR3-knockout mice exhibited a significantly larger infarct area than did wild-type mice. Collectively, these in vitro and in vivo results suggest that the developmental expression of constitutively active Gs-coupled GPR3 activates the ERK and Akt signaling pathways at the basal level, thereby protecting neurons from apoptosis that is induced by various stimuli.
Asunto(s)
Apoptosis/genética , Cerebelo/citología , Regulación del Desarrollo de la Expresión Génica/genética , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Factores de Edad , Animales , Cardiotónicos/farmacología , Supervivencia Celular/genética , Colforsina/farmacología , Medios de Cultivo Condicionados/farmacología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Isquemia/metabolismo , Isquemia/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
BACKGROUND: During postnatal murine and rodent cerebellar development, cerebellar granule precursors (CGP) gradually stop proliferating as they differentiate after migration to the internal granule layer (IGL). Molecular events that govern this program remain to be fully elucidated. GPR3 belongs to a family of Gs-linked receptors that activate cyclic AMP and are abundantly expressed in the adult brain. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of this orphan receptor in CGP differentiation, we determined that exogenous GPR3 expression in rat cerebellar granule neurons partially antagonized the proliferative effect of Sonic hedgehog (Shh), while endogenous GPR3 inhibition by siRNA stimulated Shh-induced CGP proliferation. In addition, exogenous GPR3 expression in CGPs correlated with increased p27/kip expression, while GPR3 knock-down led to a decrease in p27/kip expression. In wild-type mice, GPR3 expression increased postnatally and its expression was concentrated in the internal granular layer (IGL). In GPR3 -/- mice, the IGL was widened with increased proliferation of CGPs, as measured by bromodeoxyuridine incorporation. Cell cycle kinetics of GPR3-transfected medulloblastoma cells revealed a G0/G1 block, consistent with cell cycle exit. CONCLUSIONS/SIGNIFICANCE: These results thus indicate that GPR3 is a novel antiproliferative mediator of CGPs in the postnatal development of murine cerebellum.
Asunto(s)
Cerebelo/citología , Regulación del Desarrollo de la Expresión Génica , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/fisiología , Animales , Encéfalo/metabolismo , Proliferación Celular , AMP Cíclico/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Hedgehog/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
In the adult cerebellum, basket/stellate cells are scattered throughout the ML, but little is known about the process underlying the cell dispersion. To determine the allocation of stellate/basket cells within the ML, we examined their migration in the early postnatal mouse cerebellum. We found that after entering the ML, basket/stellate cells sequentially exhibit four distinct phases of migration. First, the cells migrated radially from the bottom to the top while exhibiting saltatory movement with a single leading process (Phase I). Second, the cells turned at the top and migrated tangentially in a rostro-caudal direction, with an occasional reversal of the direction of migration (Phase II). Third, the cells turned and migrated radially within the ML at a significantly reduced speed while repeatedly extending and withdrawing the leading processes (Phase III). Fourth, the cells turned at the middle and migrated tangentially at their slowest speed, while extending several dendrite-like processes after having completely withdrawn the leading process (Phase IV). Finally, the cells stopped and completed their migration. These results suggest that the dispersion of basket/stellate cells in the ML is controlled by the orchestrated activity of external guidance cues, cell-cell contact and intrinsic programs in a position- and time-dependent manner.
Asunto(s)
Movimiento Celular/fisiología , Cerebelo , Neuronas , Animales , Animales Recién Nacidos , Células Cultivadas , Cerebelo/citología , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Humanos , Ratones , Neuronas/citología , Neuronas/fisiologíaRESUMEN
The herpes simplex virus (HSV) amplicon vector produces an initial host response that limits transgene expression. In this study, we hypothesized that restoration of the HSV gene infected cell protein (ICP0) into the amplicon could circumvent this host response and thus overcome silencing of encoded transgenes. To test this, we constructed an amplicon vector that encodes the ICP0 under control of its native promoter (ICP0+ amplicon). Expression of ICP0 was transient and, at a multiplicity of infection (MOI) of 1, did not significantly alter interferon (IFN)-based responses against the vector or cell kinetics/apoptosis of infected cells. Chromatin immunoprecipitation (ChIP) PCR analysis revealed that conventional amplicon DNA became associated with histone deacetylase 1 (HDAC1) immediately after infection, whereas ICP0+ amplicon DNA remained relatively unbound by HDAC1 for at least 72 hours after infection. Mice administered systemic ICP0+ amplicon exhibited significantly greater and more sustained transgene expression in their livers than did those receiving conventional amplicon, likely due to increased transcriptional or post-transcriptional activity rather than increased copy numbers of vector DNA. These findings indicate that restoration of ICP0 expression may be employed within HSV amplicon constructs to decrease transgene silencing in vitro and in vivo.
Asunto(s)
Proteínas Inmediatas-Precoces/fisiología , Simplexvirus/fisiología , Transgenes , Ubiquitina-Proteína Ligasas/fisiología , Animales , Secuencia de Bases , Inmunoprecipitación de Cromatina , Cartilla de ADN , ADN Viral/genética , Histona Desacetilasa 1 , Histona Desacetilasas/metabolismo , Ratones , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simplexvirus/genéticaRESUMEN
BACKGROUND: Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers. METHODOLOGY/PRINCIPAL FINDINGS: Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice. CONCLUSIONS/SIGNIFICANCE: These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Células Madre Neoplásicas/citología , Neuroblastoma/metabolismo , Virus Oncolíticos/metabolismo , Antígeno AC133 , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Antígenos CD/biosíntesis , Línea Celular Tumoral , Linaje de la Célula , Chlorocebus aethiops , Glicoproteínas/biosíntesis , Humanos , Proteínas de Filamentos Intermediarios/biosíntesis , Proteínas de Neoplasias/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Nestina , Péptidos , Células Madre/metabolismo , Transcripción Genética , Células VeroRESUMEN
Therapies targeting glioma cells that diffusely infiltrate normal brain are highly sought after. Our aim was to identify novel approaches to this problem using glioma spheroid migration assays. Lithium, a currently approved drug for the treatment of bipolar illnesses, has not been previously examined in the context of glioma migration. We found that lithium treatment potently blocked glioma cell migration in spheroid, wound-healing, and brain slice assays. The effects observed were dose dependent and reversible, and worked using every glioma cell line tested. In addition, there was little effect on cell viability at lithium concentrations that inhibit migration, showing that this is a specific effect. Lithium treatment was associated with a marked change in cell morphology, with cells retracting the long extensions at their leading edge. Examination of known targets of lithium showed that inositol monophosphatase inhibition had no effect on glioma migration, whereas inhibition of glycogen synthase kinase-3 (GSK-3) did. This suggested that the effects of lithium on glioma cell migration could possibly be mediated through GSK-3. Specific pharmacologic GSK-3 inhibitors and siRNA knockdown of GSK-3alpha or GSK-3beta isoforms both reduced cell motility. These data outline previously unidentified pathways and inhibitors that may be useful for the development of novel anti-invasive therapeutics for the treatment of brain tumors.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/enzimología , Movimiento Celular/efectos de los fármacos , Glioma/enzimología , Glucógeno Sintasa Quinasa 3/metabolismo , Cloruro de Litio/farmacología , Animales , Western Blotting , Neoplasias Encefálicas/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Glioma/patología , Glucógeno Sintasa Quinasa 3/efectos de los fármacos , Humanos , Ratones , Invasividad Neoplásica/fisiopatología , Neuronas/efectos de los fármacos , Técnicas de Cultivo de ÓrganosRESUMEN
The herpes simplex virus (HSV) amplicon is a plasmid-based, infectious gene delivery system that carries up to 150 kilobase (kb) of exogenous DNA. We previously characterized early host responses and stability of transgene expression in mice systemically injected with HSV amplicon vectors. Transgene expression was readily detected primarily in the liver but rapidly declined to undetectable levels within 2 weeks. Molecular analyses revealed induction of type I interferons (IFN) as the primary response, and early transcriptional silencing of the vector followed IFN's activation of signal transducers and activators of transcription 1 (STAT1). In this study, we investigate vector administration by stereotactic injection into the striatum. In the brain, induction of type I IFN was rather modest, and transgene expression lasted more than 1 year despite dose-dependent inflammation and infiltration of immune cells around injection sites. Further analyses revealed dose-dependent upregulation of immunosuppressive cytokines and molecular markers specific to regulatory T cells in the injected brain regions, which supported the immune-privileged properties of the brain parenchyma. Overall, our findings indicate that the spectrum of host responses can differ significantly depending on target organs and administrative routes, and that HSV amplicon vectors hold great potential for gene therapy of chronic neurological disorders.
Asunto(s)
Encéfalo/metabolismo , Expresión Génica , Vectores Genéticos , Simplexvirus/genética , Transgenes , Animales , Secuencia de Bases , Cuerpo Estriado/inmunología , Cuerpo Estriado/virología , Cartilla de ADN , Ratones , Ratones Endogámicos C57BLRESUMEN
Replication-conditional (oncolytic) mutants of herpes simplex virus (HSV), are considered promising therapeutic alternatives for human malignancies, and chemotherapeutic adjuvants are increasingly sought to augment their efficacy. Histone deacetylase (HDAC) inhibitors are a new class of antineoplastic agents because of their potent activity in growth arrest, differentiation, and apoptotic death of cancer cells. The ability of the HDAC inhibitors to upregulate exogenous transgene expression and inhibit interferon (IFN) responses prompted our exploration of their use in improving the antitumor efficacy of oncolytic HSV. We discovered that the yield of viral progeny increased significantly when cultured glioma cells were treated with HDAC inhibitors before viral infection. Valproic acid (VPA), a commonly used antiepileptic agent with HDAC inhibitory activity, proved most effective when used to treat glioma cells before viral infection, but not concomitantly with viral infection. Pretreatment with VPA inhibited the induction of several IFN-responsive antiviral genes, augmented the transcriptional level of viral genes, and improved viral propagation, even in the presence of type I IFNs. Moreover, VPA pretreatment improved the propagation and therapeutic efficacy of oncolytic HSV in a human glioma xenograft model in vivo. These findings indicate that HDAC inhibitors can improve the efficacy of tumor virotherapies.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Glioma/tratamiento farmacológico , Herpesvirus Humano 1/genética , Inhibidores de Histona Desacetilasas , Interferón beta/uso terapéutico , Virus Oncolíticos , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/virología , Butiratos/uso terapéutico , Sinergismo Farmacológico , Quimioterapia Combinada , Terapia Genética , Vectores Genéticos/uso terapéutico , Glioma/genética , Glioma/virología , Infecciones por Herpesviridae/tratamiento farmacológico , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/virología , Herpesvirus Humano 1/metabolismo , Humanos , Interferón beta-1a , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/virología , Ratones , Ratones Desnudos , Proteínas Recombinantes/uso terapéutico , Células Tumorales Cultivadas , Ácido Valproico/uso terapéutico , Replicación Viral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Malignant solid tumors remain a significant clinical challenge, necessitating innovative therapeutic approaches. Oncolytic viral therapy is a nonmutagenic, biological anticancer therapeutic shown to be effective against human cancer in early studies. Because matrix metalloproteinases (MMP) play important roles in the pathogenesis and progression of cancer, we sought to determine if "arming" an oncolytic herpes simplex virus (oHSV) with an MMP-antagonizing transgene would increase virus-mediated antitumor efficacy. We generated oHSVs that express human tissue inhibitor of metalloproteinases 3 (TIMP3) or firefly luciferase and designated them rQT3 and rQLuc, respectively. We evaluated the antitumor efficacy of these viruses against neuroblastoma and malignant peripheral nerve sheath tumor (MPNST) xenografts. Relative to rQLuc, rQT3-infected primary human MPNST and neuroblastoma cells exhibited equivalent virus replication but increased cytotoxicity and reduced MMP activity. In vivo, rQT3-treated tumors showed delayed tumor growth, increased peak levels of infectious virus, immature collagen extracellular matrix, and reduced tumor vascular density. Remarkably, rQT3 treatment reduced circulating endothelial progenitors, suggesting virus-mediated antivasculogenesis. We conclude that rQT3 enhanced antitumor efficacy through multiple mechanisms, including direct cytotoxicity, elevated virus titer, and reduced tumor neovascularization. These findings support the further development of combined TIMP-3 and oncolytic virotherapy for cancer.
Asunto(s)
Terapia Genética/métodos , Neoplasias de la Vaina del Nervio/terapia , Neuroblastoma/terapia , Viroterapia Oncolítica/métodos , Simplexvirus/fisiología , Inhibidor Tisular de Metaloproteinasa-3/genética , Animales , Chlorocebus aethiops , Terapia Combinada , Femenino , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/virología , Humanos , Luciferasas de Luciérnaga/genética , Ratones , Ratones Desnudos , Neoplasias de la Vaina del Nervio/sangre , Neoplasias de la Vaina del Nervio/genética , Neoplasias de la Vaina del Nervio/virología , Neuroblastoma/sangre , Neuroblastoma/genética , Neuroblastoma/virología , Simplexvirus/genética , Células Vero , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Replication-conditional (oncolytic) mutants of herpes simplex virus (HSV), are considered promising therapeutic alternatives for human malignancies, and chemotherapeutic adjuvants are increasingly sought to augment their efficacy. Histone deacetylase (HDAC) inhibitors are a new class of antineoplastic agents because of their potent activity in growth arrest, differentiation, and apoptotic death of cancer cells. The ability of the HDAC inhibitors to upregulate exogenous transgene expression and inhibit interferon (IFN) responses prompted our exploration of their use in improving the antitumor efficacy of oncolytic HSV. We discovered that the yield of viral progeny increased significantly when cultured glioma cells were treated with HDAC inhibitors before viral infection. Valproic acid (VPA), a commonly used antiepileptic agent with HDAC inhibitory activity, proved most effective when used to treat glioma cells before viral infection, but not concomitantly with viral infection. Pretreatment with VPA inhibited the induction of several IFN-responsive antiviral genes, augmented the transcriptional level of viral genes, and improved viral propagation, even in the presence of type I IFNs. Moreover, VPA pretreatment improved the propagation and therapeutic efficacy of oncolytic HSV in a human glioma xenograft model in vivo. These findings indicate that HDAC inhibitors can improve the efficacy of tumor virotherapies.
RESUMEN
The herpes simplex virus (HSV) amplicon vector is a powerful gene delivery vehicle that can accommodate up to 150 kilobase of exogenous DNA. However, amplicon-mediated transgene expression is often transient outside the nervous system. In order to define the role of host immune responses in silencing amplicon-encoded transgenes, we evaluated the kinetics of cytokine-/chemokine-expression after tail-vein injection of a luciferase-encoding amplicon into mice. Type I interferons (IFNs) were induced earliest, within an hour after injection, and several other cytokines/chemokines were subsequently upregulated in the livers of wild-type (WT) mice. When the same experiment was performed in signal transducers and activators of transcription 1 (STAT1)-knockout (KO) mice, the levels of type I IFN expression were significantly lower and chemokine induction was almost non-existent. Importantly, STAT1-KO mice exhibited significantly higher and more sustained luciferase activity than did the WT mice, which is attributable to increased transcriptional activity rather than increased copy numbers of luciferase-encoding vector DNA. Further studies using primary cultured fibroblasts derived from WT and STAT1-KO mice revealed the significance of STAT1 signaling in transcriptional silencing of the amplicon-encoded transgene in vitro. Our results indicate that type I IFNs induced by systemic delivery of HSV amplicon vectors initiate a cascade of immune responses and suppress transgene expression at the transcriptional level by activation of STAT1.
Asunto(s)
Vectores Genéticos/genética , Factor de Transcripción STAT1/metabolismo , Simplexvirus/genética , Transcripción Genética/genética , Transgenes/genética , Animales , Antígeno CD11b/metabolismo , Chlorocebus aethiops , Cromatina/genética , Citocinas/biosíntesis , ADN Viral/genética , Fibroblastos , Expresión Génica , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción STAT1/deficiencia , Factor de Transcripción STAT1/genética , Factores de Tiempo , Células VeroRESUMEN
Cyclic AMP regulates multiple neuronal functions, including neurite outgrowth and axonal regeneration. GPR3, GPR6, and GPR12 make up a family of constitutively active G protein-coupled receptors (GPCRs) that share greater than 50% identity and 65% similarity at the amino acid level. They are highly expressed in the central nervous system, and their expression in various cell lines results in constitutive stimulation of cAMP production. When the constitutively active GPCRs were overexpressed in rat cerebellar granule neurons in culture, the transfected neurons exhibited significantly enhanced neurite outgrowth and overcame growth inhibition caused by myelin-associated glycoprotein. GPR12-mediated neurite outgrowth was the most prominent and was shown to depend on G(s) and cAMP-dependent protein kinase. Moreover, the GPR12-mediated rescue from myelin-associated glycoprotein inhibition was attributable to cAMP-dependent protein kinase-mediated inhibition of the small GTPase, RhoA. Among the three receptors, GPR3 was revealed to be enriched in the developing rat cerebellar granule neurons. When the endogenous GPR3 was knocked down, significant reduction of neurite growth was observed, which was reversed by expression of either GPR3 or GPR12. Taken together, our results indicate that expression of the constitutively active GPCRs up-regulates cAMP production in neurons, stimulates neurite outgrowth, and counteracts myelin inhibition. Further characterization of the GPCRs in developing and injured mammalian neurons should provide insights into how basal cAMP levels are regulated in neurons and could establish a firm scientific foundation for applying receptor biology to treatment of various neurological disorders.
Asunto(s)
Cerebelo/metabolismo , AMP Cíclico/biosíntesis , Neuritas/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Regulación hacia Arriba , Animales , Línea Celular , Cerebelo/citología , Eliminación de Gen , Humanos , Vaina de Mielina/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Transfección , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Approaches to improve the oncolytic potency of replication-competent adenoviruses include the insertion of therapeutic transgenes into the viral genome. Little is known about the levels and duration of in vivo transgene expression by cells infected with such "armed" viruses. Using a tumor-selective adenovirus encoding firefly luciferase (AdDelta24CMV-Luc) we investigated these questions in an intracranial mouse model for malignant glioma. Luciferase expression was detected by bioluminescence imaging, and the effect of the immunosuppressive agent cyclophosphamide (CPA) on transgene expression was assessed. Intratumoral AdDelta24CMV-Luc injection led to a localized dose-dependent expression of luciferase. Surprisingly, this expression decreased rapidly during the course of 14 days. In contrast, mice injected with nonreplicating Ad.CMV-Luc demonstrated stable transgene expression. Treatment of mice with CPA in combination with AdDelta24CMV-Luc retarded the loss of transgene expression. Staining of mouse brains for inflammatory cells demonstrated decreased tumor infiltration by immune cells in CPA-treated mice. Moreover, in immunodeficient NOD/SCID mice loss of transgene expression was less rapid and not prevented by CPA treatment. Together, our data demonstrate that transgene expression and viral replication decrease rapidly after intratumoral injection of oncolytic adenovirus in mouse brains and that treatment with the immunomodulator CPA prolongs viral-mediated gene expression.
Asunto(s)
Adenoviridae/genética , Ciclofosfamida/farmacología , Glioma/genética , Mediciones Luminiscentes/métodos , Transgenes/genética , Animales , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Antineoplásicos Alquilantes/administración & dosificación , Antineoplásicos Alquilantes/farmacología , Línea Celular , Línea Celular Tumoral , Ciclofosfamida/administración & dosificación , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Glioma/metabolismo , Glioma/patología , Humanos , Inmunohistoquímica , Antígenos Comunes de Leucocito/análisis , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Virus Oncolíticos/genética , Trasplante HeterólogoRESUMEN
The herpes simplex virus (HSV) amplicon vector is a versatile plasmid-based gene delivery vehicle with a large transgene capacity (up to 150 kb) and the ability to infect a broad range of cell types. The vector system was originally developed by Frenkel and her colleagues in 1980. Ever since, a great deal of effort by various investigators has been directed at minimizing the toxicity associated with the inevitable contamination by helper virus. In 1996, Fraefel and his colleagues successfully devised a cosmid-based packaging system that was free of contamination by helper virus (so-called helper virus-free packaging), which utilized as helper a set of 5 overlapping cosmid clones that covered the entire HSV genome, which lacked the DNA packaging/cleavage signals. With the helper virus-free system, broader applications of the vector became possible. Cloning of the entire HSV genome in bacteria artificial chromosome (BAC) plasmids enabled stable maintenance and propagation of the helper HSV genome in bacteria. It also allowed for the development of BAC-based helper virus-free packaging systems. In this article, we review various versions of DNA-based methods to prepare HSV amplicon vectors free of helper virus contamination. We also examine recent advances in vector design, including methods of vector construction, hybrid amplicon vectors, and the infectious BAC system. Future directions in improving packaging systems and vector designs are discussed.
Asunto(s)
ADN , Vectores Genéticos , Plásmidos , Simplexvirus/genética , Cromosomas Artificiales BacterianosRESUMEN
The herpes simplex virus (HSV)-based amplicon vector, a bacterial-viral-mammalian cell shuttle system, holds promise as a versatile gene delivery vehicle because of its large transgene capacity. However, amplicon-mediated transgene expression is often transient. We hypothesized that the presence of prokaryotic DNA sequences within the packaged vector genome can trigger transcriptional silencing of the entire vector sequence. To test this, we constructed a novel amplicon vector devoid of bacterial sequences (minicircle [MC] amplicon). Although the same dose of the minicircle amplicon vector in normal human fibroblasts resulted in an expression of luciferase approximately 20 times higher than that caused by the conventional amplicon vector, no significant difference was observed in copy numbers of luciferase DNA between MC amplicon- and control-transduced cells. Quantitative analyses of levels of luciferase mRNA revealed that differential expression of luciferase was controlled at the transcriptional level. Chromatin immunoprecipitation PCR analyses of several regions of vector genomes revealed that the bacterial sequences found in the conventional amplicon DNA were associated with an inactive form of chromatin immediately after infection. The presence of bacterial sequences also affected the remaining vector sequences in the conventional amplicon vector. Finally, nude mice injected with the MC amplicon exhibited higher and more sustained expression of luciferase than those injected with the conventional amplicon, confirming the usefulness of the MC amplicon devoid of bacterial sequences. Although additional improvements are absolutely required, these findings are a significant first step toward developing a novel HSV amplicon vector that can achieve enhanced long-term transgene expression.
Asunto(s)
Cromatina/genética , Dependovirus/genética , Silenciador del Gen , Vectores Genéticos , Herpesvirus Humano 1/genética , Plásmidos , Transgenes , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Dependovirus/metabolismo , Fibroblastos/metabolismo , Amplificación de Genes , Técnicas de Transferencia de Gen , Genes Reporteros , Genes Virales , Herpesvirus Humano 1/metabolismo , Humanos , Cinética , Luciferasas/metabolismo , Ratones , Ratones Desnudos , ARN Mensajero/análisisAsunto(s)
Sistema Nervioso Central/enzimología , Técnicas de Transferencia de Gen , Simplexvirus/genética , Transducción Genética , Transposasas/genética , Virión/genética , Animales , Regulación de la Expresión Génica , Terapia Genética/tendencias , Vectores Genéticos/administración & dosificación , Vectores Genéticos/uso terapéutico , Humanos , Ratones , Transposasas/biosíntesisRESUMEN
Conditionally replicating herpes simplex virus-1 (HSV-1) vectors are promising therapeutic agents for cancer. Insertion of therapeutic transgenes into the viral genome should confer desired anticancer functions in addition to oncolytic activities. Herein, using bacterial artificial chromosome and two recombinase-mediated recombinations, we simultaneously created four "armed" oncolytic HSV-1, designated vHsv-B7.1-Ig, vHsv-interleukin (IL)-12, vHsv-IL-18, and vHsv-null, which express murine soluble B7.1 (B7.1-Ig), murine IL-12, murine IL-18, and no transgene, respectively. These vHsv vectors possess deletions in the gamma34.5 genes and contain the green fluorescent protein gene as a histochemical marker and the immunostimulatory transgene inserted in the deleted ICP6 locus. The vHsv showed similar replicative capabilities in vitro. The in vivo efficacy was tested in A/J mice harboring s.c. tumors of syngeneic and poorly immunogenic Neuro2a neuroblastoma. The triple combination of vHsv-B7.1-Ig, vHsv-IL-12, and vHsv-IL-18 exhibited the highest efficacy among all single vHsv or combinations of two viruses. Combining 1 x 10(5) plaque-forming units each of the three armed viruses showed stronger antitumor activities than any single armed virus at 3 x 10(5) plaque-forming units in inoculated tumors as well as in noninoculated remote tumors. Studies using athymic mice indicated that this enhancement of antitumor efficacy was likely mediated by T-cell immune responses. The combined use of multiple oncolytic HSV-1 armed with different immunostimulatory genes may be a useful strategy for cancer therapy.
Asunto(s)
Antígeno B7-1 , Vectores Genéticos/uso terapéutico , Herpesvirus Humano 1/genética , Interleucina-12 , Interleucina-18 , Neuroblastoma/terapia , Viroterapia Oncolítica , Animales , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Antígeno B7-1/metabolismo , Chlorocebus aethiops , Cromosomas Artificiales Bacterianos , Quimioterapia Combinada , Vectores Genéticos/genética , Humanos , Interleucina-12/genética , Interleucina-12/inmunología , Interleucina-12/metabolismo , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-18/metabolismo , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos BALB C , Ratones Desnudos , Neuroblastoma/genética , Neuroblastoma/inmunología , Recombinasas/genética , Linfocitos T/inmunología , Transfección , Transgenes/fisiología , Células Tumorales Cultivadas , Células VeroRESUMEN
Volumetric detection and accurate quantification of fluorescent proteins in entire animals would greatly enhance our ability to monitor biological processes in vivo. Here we present a quantitative tomographic technique for visualization of superficial and deep-seated (>2-3 mm) fluorescent protein activity in vivo. We demonstrate noninvasive imaging of lung tumor progression in a murine model, as well as imaging of gene delivery using a herpes virus vector. This technology can significantly improve imaging capacity over the current state of the art and should find wide in vivo imaging applications in drug discovery, immunology, and cancer research.
Asunto(s)
Proteínas Luminiscentes/análisis , Tomografía/métodos , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Vectores Genéticos/genética , Humanos , Proteínas Luminiscentes/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Ratas , Simplexvirus/genéticaRESUMEN
The success of cancer virotherapy depends on its efficacy versus toxicity profile in human clinical trials. Progress towards clinical trials can be hampered by the relatively elevated doses of oncolytic viruses administered in animal models to achieve an anticancer effect and by the even higher doses required in humans to approximate an animal bioequivalent dose. Such elevated doses of injected viral proteins may also lead to undesirable toxicities and are also very difficult to produce in a biotechnological setting. We report that a relatively potent herpes simplex virus type 1 oncolytic virus (rQNestin34.5) produces 45% survivors at a dose of 3 x 10(4) plaque-forming units (pfu) in a 9-day-old mouse model of human glioma. Unlike our previous findings with less potent oncolytic viruses, though, the preadministration of cyclophosphamide did not enhance this survival or affect oncolytic virus tumor distribution and tumor volume. However, when oncolytic virus doses were reduced (3 x 10(3) and 3 x 10(2) pfu), cyclophosphamide significantly enhanced both animal survival and oncolytic virus tumor distribution and also reduced tumor volumes. These findings thus show that cyclophosphamide allows for dose reduction of doses of a relatively potent oncolytic virus, a finding with implications for the development of clinical trials.
Asunto(s)
Antineoplásicos Alquilantes/uso terapéutico , Neoplasias Encefálicas/terapia , Ciclofosfamida/uso terapéutico , Terapia Genética , Glioma/terapia , Herpesvirus Humano 1/fisiología , Virus Oncolíticos/genética , Animales , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/virología , Terapia Combinada , Vectores Genéticos/administración & dosificación , Glioma/patología , Glioma/virología , Humanos , Ratones , Tasa de Supervivencia , Células Tumorales Cultivadas , Replicación ViralRESUMEN
The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein G(s) and an orphan member of the G protein-coupled receptor family, GPR3. To determine whether GPR3 activates G(s), the localization of Galpha(s) in follicle-enclosed oocytes from Gpr3(+/+) and Gpr3(-/-) mice was compared by using immunofluorescence and Galpha(s)GFP. GPR3 decreased the ratio of Galpha(s) in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Galpha(s) in the oocyte. Both of these properties indicate that GPR3 activates G(s). The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent G(s) activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity.