RESUMO
BACKGROUND: Glioblastoma (GB) is the most common and most aggressive malignant brain tumor. In understanding its resistance to conventional treatments, iron metabolism and related pathways may represent a novel avenue. As for many cancer cells, GB cell growth is dependent on iron, which is tightly involved in red-ox reactions related to radiotherapy effectiveness. From new observations indicating an impact of RX radiations on the expression of ceruloplasmin (CP), an important regulator of iron metabolism, the aim of the present work was to study the functional effects of constitutive expression of CP within GB lines in response to beam radiation depending on the oxygen status (21% O2 versus 3% O2). METHODS AND RESULTS: After analysis of radiation responses (Hoechst staining, LDH release, Caspase 3 activation) in U251-MG and U87-MG human GB cell lines, described as radiosensitive and radioresistant respectively, the expression of 9 iron partners (TFR1, DMT1, FTH1, FTL, MFRN1, MFRN2, FXN, FPN1, CP) were tested by RTqPCR and western blots at 3 and 8 days following 4 Gy irradiation. Among those, only CP was significantly downregulated, both at transcript and protein levels in the two lines, with however, a weaker effect in the U87-MG, observable at 3% O2. To investigate specific role of CP in GB radioresistance, U251-MG and U87-MG cells were modified genetically to obtain CP depleted and overexpressing cells, respectively. Manipulation of CP expression in GB lines demonstrated impact both on cell survival and on activation of DNA repair/damage machinery (γH2AX); specifically high levels of CP led to increased production of reactive oxygen species, as shown by elevated levels of superoxide anion, SOD1 synthesis and cellular Fe2 + . CONCLUSIONS: Taken together, these in vitro results indicate for the first time that CP plays a positive role in the efficiency of radiotherapy on GB cells.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Ceruloplasmina/farmacologia , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Humanos , Ferro/farmacologia , Oxigênio/metabolismo , Tolerância a Radiação/genéticaRESUMO
A role for glucose-dependent insulinotropic polypeptide (GIP) in controlling bone resorption has been suspected. However uncertainty remains to identify whether GIP act directly on osteoclasts. The aim of the present study were (i) to identify in different osteoclast differentiation models (human peripheral blood mononuclear cells-PBMC, murine bone marrow macrophage-BMM and murine Raw 264.7 cells) whether GIP was capable of reducing osteoclast formation and resorption; (ii) ascertain whether the highly potent GIP analogue N-AcGIP was capable of inducing a response at lower concentrations and (iii) to decipher the molecular mechanisms responsible for such effects. [d-Ala(2)]-GIP dose-dependently reduced osteoclast formation at concentration as low as 1nM in human PBMC and 10nM in murine BMM cultures. Furthermore, [d-Ala(2)]-GIP also reduced the extent of osteoclast resorption at concentration as low as 1nM in human PBMC and murine BMM cultures. The mechanism of action of [d-Ala(2)]-GIP appeared to be mediated by reduction in intracellular calcium concentration and oscillation that subsequently inhibited calcineurin activity and NFATc1 nuclear translocation. The potency of the highly potent N-AcGIP was determined and highlighted an effect on osteoclast formation and resorption at concentration ten times lower than observed with [d-Ala(2)]-GIP in vitro. Furthermore, N-AcGIP was also capable of reducing the number of osteoclast in ovariectomized mice as well as the circulating level of type I collagen C-telopeptide. Pharmacological concentrations required for reducing osteoclast formation and resorption provide the impetus to design and exploit enzymatically stable GIP analogues for the treatment of bone resorption disorders in humans.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Polipeptídeo Inibidor Gástrico/farmacologia , Polipeptídeo Inibidor Gástrico/uso terapêutico , Osteoclastos/patologia , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Osso Esponjoso/diagnóstico por imagem , Osso Esponjoso/efeitos dos fármacos , Osso Esponjoso/patologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Osso Cortical/diagnóstico por imagem , Osso Cortical/efeitos dos fármacos , Osso Cortical/patologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Polipeptídeo Inibidor Gástrico/química , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Osteoclastos/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Transporte Proteico/efeitos dos fármacos , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
Precise control of transgene expression in a tissue-specific and temporally regulated manner is desirable for many basic and applied investigations gene therapy applications. This is important to regulate dose of transgene products and minimize unwanted effects. Previously described methods have employed tissue specific promoters, miRNA-based transgene silencing or tetR-KRAB-mediated suppression of transgene promoters. To improve on versatility of transgene expression control, we have developed expression systems that use combinations of a tetR-KRAB artificial transgene-repressor, endogenous miRNA silencing machinery and tissue specific promoters. Precise control of transgene expression was demonstrated in liver-, macrophage- and muscle-derived cells. Efficiency was also demonstrated in vivo in murine muscle. This multicomponent and modular regulatory system provides a robust and easily adaptable method for achieving regulated transgene expression in different tissue types. The improved precision of regulation will be useful for many gene therapy applications requiring specific spatiotemporal transgene regulation.
Assuntos
Vetores Genéticos/genética , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/genética , Proteínas Repressoras/genética , Transcrição Gênica , Transgenes , Animais , Linhagem Celular , Linhagem Celular Tumoral , Inativação Gênica , Células HEK293 , Humanos , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células Musculares/metabolismo , Regiões Promotoras GenéticasRESUMO
Lentiviral vectors are promising tools for liver disease gene therapy, because they can achieve protracted expression of transgenes in hepatocytes. However, the question as to whether cell division is required for optimal hepatocyte transduction has still not been completely answered. Liver gene-transfer efficiency after in vivo administration of recombinant lentiviral vectors carrying a green fluorescent protein reporter gene under the control of a liver-specific promoter in mice that were either hepatectomized or treated with cholic acid or phenobarbital was compared. Phenobarbital is known as a weak inducer of hepatocyte proliferation, whereas cholic acid has no direct effect on the cell cycle. This study shows that cholic acid is able to prime hepatocytes without mitosis induction. Both phenobarbital and cholic acid significantly increased hepatocyte transduction six- to ninefold, although cholic acid did not modify the mitotic index or cell-cycle entry. However, the effect of either compound was weaker than that observed after partial hepatectomy. In no cases was there a correlation between the expression of cell-cycle marker and transduction efficiency. We conclude that priming of hepatocytes should be considered a clinically applicable strategy to enhance in vivo liver gene therapy with lentiviral vectors.
Assuntos
Ácido Cólico/farmacologia , Terapia Genética/métodos , Vetores Genéticos/genética , Hepatócitos/efeitos dos fármacos , Fígado/metabolismo , Transdução Genética/métodos , Animais , Proliferação de Células/efeitos dos fármacos , Primers do DNA/genética , Proteínas de Fluorescência Verde/genética , Hematócrito , Imuno-Histoquímica , Lentivirus/genética , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenobarbital/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não ParamétricasRESUMO
BACKGROUND & AIMS: Crigler-Najjar type 1 (CN-I) is an inherited liver disease caused by an absence of bilirubin-uridine 5'-diphosphate-glucuronosyltransferase (UGT1A1) activity. It results in life-threatening levels of unconjugated bilirubin, and therapeutic options are limited. We used adult Gunn rats (an animal model of the disease) to evaluate the efficiency of lentiviral-based gene therapy to express UGT1A1 in liver. METHODS: Gunn rats were given intraportal injections of VSVG-pseudotyped lentiviral vectors that encode UGT1A1 under the control of a liver-specific transthyretin promoter (mTTR.hUGT1A1); this vector does not contain target sequences for miR-142, a microRNA that is expressed specifically in hematopoietic cells. Rats were also injected with the vector mTTR.hUGT1A1.142T, which contains 4 copies of the miR-142 target sequences; its messenger RNA should be degraded in antigen-presenting cells. Bilirubinemia was monitored, and the presence of transduced hepatocytes was analyzed by quantitative polymerase chain reaction. Vector expression was tested in vitro in rat hematopoietic cells. RESULTS: In Gunn rats, bilirubin levels normalized 2 weeks after administration of mTTR.hUGT1A1. However, hyperbilirubinemia resumed 8 weeks after vector administration, concomitant with the induction of an immune response. In contrast, in rats injected with mTTR-UGT1A1.142T, bilirubin levels normalized for up to 6 months and transduced cells were not eliminated. CONCLUSIONS: Lentiviral vectors that express UGT1A1 reduce hyperbilirubinemia in immunocompetent Gunn rats for at least 6 months. The immune response against virally expressed UGT1A1 can be circumvented by inclusion of miR-142 target sequences, which reduce vector expression in antigen-presenting cells. This lentiviral-based gene therapy approach might be developed to treat patients with CN-I.
Assuntos
Síndrome de Crigler-Najjar/terapia , Terapia Genética/métodos , Vetores Genéticos , Glucuronosiltransferase/genética , Lentivirus/genética , Fígado/enzimologia , MicroRNAs/metabolismo , Animais , Anticorpos/sangue , Células Apresentadoras de Antígenos/imunologia , Bilirrubina/sangue , Biomarcadores/sangue , Síndrome de Crigler-Najjar/enzimologia , Síndrome de Crigler-Najjar/genética , Síndrome de Crigler-Najjar/imunologia , Modelos Animais de Doenças , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/imunologia , Células HeLa , Humanos , Masculino , Pré-Albumina/genética , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ratos , Ratos Gunn , Fatores de Tempo , Transdução GenéticaRESUMO
Lentiviral vectors can stably transduce hepatocytes and are promising tools for gene therapy of hepatic diseases. Although hepatocytes are accessible to blood-borne viral vectors through fenestrations of the hepatic endothelium, improved liver transduction after delivery of vectors to the blood stream is needed. As the normal endothelial fenestration and lentiviral vectors are similar in size (150 nm), we hypothesized that a transient increase in hepatic blood pressure may enhance in vivo gene transfer to hepatocytes. We designed a simple surgical procedure, by which the liver is temporarily excluded from blood flow. Lentiviral vectors were injected in a large volume to increase intrahepatic pressure. We demonstrated that in the Gunn rat, a model of Crigler-Najjar disease, the administration of low vector doses (corresponding to a multiplicity of infection of 0.2) by this procedure resulted in therapeutic correction of hyperbilirubinemia, without toxicity. The correction was sustained for 10 months (end of study). The same vector amounts yielded only partial correction after intraportal delivery. We believe that this new and clinically applicable strategy may broaden the range of genetic liver diseases accessible to gene therapy.
Assuntos
Síndrome de Crigler-Najjar/terapia , Terapia Genética/métodos , Vetores Genéticos , Hiperbilirrubinemia/terapia , Lentivirus/genética , Fígado , Transdução Genética , Animais , Bilirrubina/sangue , Biomarcadores/sangue , Pressão Sanguínea , Síndrome de Crigler-Najjar/genética , Modelos Animais de Doenças , Hepatócitos , Fígado/irrigação sanguínea , Fígado/cirurgia , Testes de Função Hepática , Reação em Cadeia da Polimerase , Pressão , Ratos , Ratos GunnRESUMO
The enteric nervous system (ENS) and its major component, enteric glial cells (EGCs), have recently been identified as a major regulator of intestinal epithelial barrier functions. Indeed, EGCs inhibit intestinal epithelial cell (IEC) proliferation and increase barrier resistance and IEC adhesion via the release of EGC-derived soluble factors. Interestingly, EGC regulation of intestinal epithelial barrier functions is reminiscent of previously reported peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent functional effects. In this context, the present study aimed at identifying whether EGC could synthesize and release the main PPARgamma ligand, 15-deoxy-(12,14)-prostaglandin J2 (15dPGJ2), and regulate IEC functions such as proliferation and differentiation via a PPARgamma dependent pathway. First, we demonstrated that the lipocalin but not the haematopoetic form for prostaglandin D synthase (PGDS), the enzyme responsible of 15dPGJ2 synthesis, was expressed in EGCs of the human submucosal plexus and of the subepithelium, as well as in rat primary culture of ENS and EGC lines. Next, 15dPGJ2 was identified in EGC supernatants of various EGC lines. 15dPGJ2 reproduced EGC inhibitory effects upon IEC proliferation, and inhibition of lipocalin PGDS expression by shRNA abrogated these effects. Furthermore, EGCs induced nuclear translocation of PPARgamma in IEC, and both EGC and 15dPGJ2 effects upon IEC proliferation were prevented by the PPARgamma antagonist GW9662. Finally, EGC induced differentiation-related gene expression in IEC through a PPARgamma-dependent pathway. Our results identified 15dPGJ2 as a novel glial-derived mediator involved in the control of IEC proliferation/differentiation through activation of PPARgamma. They also suggest that alterations of glial PGDS expression may modify intestinal epithelial barrier functions and be involved in the development of pathologies such as cancer or inflammatory bowel diseases.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Sistema Nervoso Entérico/fisiologia , Mucosa Intestinal/fisiologia , Neuroglia/fisiologia , Prostaglandina D2/análogos & derivados , Animais , Células Cultivadas , Humanos , Mucosa Intestinal/inervação , Oxirredutases Intramoleculares/análise , Oxirredutases Intramoleculares/metabolismo , Oxirredutases Intramoleculares/fisiologia , Lipocalinas/análise , Lipocalinas/metabolismo , Lipocalinas/fisiologia , PPAR gama/análise , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , PPAR gama/fisiologia , Prostaglandina D2/biossíntese , Prostaglandina D2/metabolismo , Prostaglandina D2/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Plasma hepatitis C virus (HCV) originates from hepatocytes. However, in certain subjects, B cells may harbour both plasma strains and occult HCV strains tha t are not detected in the plasma. The internal ribosome entry site (IRES) of these latter strains is mutated, suggesting that the efficiency of viral translation could drive the cellular tropism of HCV. AIMS: To determine if the translational efficiency of IRES variants in cultured hepatocytes or B cells is correlated with their cellular tropism in vivo. METHODS: The efficiency of IRES of 10 B cell-specific variants and nine plasma variants, isolated from six patients with compartmentalised variants in B cells, was estimated by bicistronic dual luciferase expression in hepatocyte cell types (Huh7), in primary cultured human hepatocytes (PCHs) and in two B cell lines (Raji and Daudi). RESULTS: For each of the six subjects, the plasma IRESes were significantly and repeatedly more efficient than B cell IRESes in Huh7 (1.7+/-0.3 vs 0.7+/-0.2; p<0.01) and PCH cells. In B cell lines, B cell and plasma IRES had similar low efficiencies (0.8+/-0.1 vs 0.9+/-0.1; NS). For three subjects, two IRES variants from the same compartment could be analysed, and had the same efficiency in each cell type. Silencing the lupus antigen, a known IRES trans-acting factor, inhibited plasma IRES variants to a greater extent than B cell-specific IRESes. CONCLUSIONS: B cells can harbour occult variants that have a poor translational efficiency in hepatocytes, strongly suggesting their extra-hepatic origin and raising the hypothesis that competition between HCV variants with different IRESes is driven at a translational level in hepatic, as well as in extra-hepatic, sites.
Assuntos
Linfócitos B/virologia , Hepacivirus/genética , Hepatite C Crônica/virologia , Hepatócitos/virologia , Adulto , Sequência de Bases , Células Cultivadas , Feminino , Hepacivirus/isolamento & purificação , Hepacivirus/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Filogenia , Biossíntese de Proteínas , Alinhamento de Sequência , Células Tumorais Cultivadas , Carga Viral , Tropismo Viral/genética , Replicação Viral/genéticaRESUMO
This report describes the development, optimization and implementation of a persistent cell-based system to test inhibitors of hepatitis C (HCV) translation. The assay is based on a heterologous human immunodeficiency virus-1/simian immunodeficiency virus (HIV-1/SIV) lentiviral vector expressing the bicistronic cassette containing the firefly and renilla luciferase genes, respectively, as reporters, and the HCV internal ribosome entry site (IRES) inserted in between, under the control of the cytomegalovirus (CMV) promoter. The drug target in this assay is the HCV IRES, the activity of which leads to modulation of the renilla luciferase gene expression under its control, which is monitored by luminometry. The system has been validated using interferon (IFN), which is still the only consensual antiviral agent against HCV infection, associated with ribavirin. This bicistronic vector, extended to other viral IRESs and assayed in different cell lines, exhibited weak cell tropism, allowing its broad use in gene therapy, which frequently needs a multicistronic transfer vector to follow the expression of a gene of interest inside the target cells with the aid of a reporter, a drug selection marker, or a suicide gene, expressed from the same transcript.
Assuntos
Antivirais/farmacologia , Vetores Genéticos , Hepacivirus/efeitos dos fármacos , Lentivirus/genética , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , HIV-1/genética , Hepacivirus/genética , Humanos , Interferons/farmacologia , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Vírus da Imunodeficiência Símia/genéticaRESUMO
Translation initiation of hepatitis C virus (HCV) RNA occurs through an internal ribosome entry site (IRES) located at its 5' end. As a positive-stranded virus, HCV uses the genomic RNA template for translation and replication, but the transition between these two processes remains poorly understood. HCV core protein (HCV-C) has been proposed as a good candidate to modulate such a regulation. However, current data are still the subject of controversy in attributing any potential role in HCV translation to the HCV core protein. Here we demonstrate that HCV-C displays binding activities toward both HCV IRES and the 40 S ribosomal subunit by using centrifugation on sucrose gradients. To gain further insight into these interactions, we investigated the effect of exogenous addition of purified HCV-C on HCV IRES activity by using an in vitro reporter assay. We found that HCV IRES-mediated translation was specifically modulated by HCV-C provided in trans, in a dose-dependent manner, with up to a 5-fold stimulation of the IRES efficiency upon addition of low amounts of HCV-C, followed by a decrease at high doses. Interestingly, mutations within some domains of the IRES as well as the presence of an upstream reporter gene both lead to changes in the expected effects, consistent with the high dependence of HCV IRES function on its overall structure. Collectively, these results indicate that the HCV core protein is involved in a tight modulation of HCV translation initiation, depending on its concentration, and they suggest an important biological role of this protein in viral gene expression.