RESUMEN
BACKGROUND: Monogenetic forms of amyotrophic lateral sclerosis (ALS) offer an opportunity for unraveling the molecular mechanisms underlying this devastating neurodegenerative disorder. In order to identify a link between ALS-related metabolic changes and neurodegeneration, we investigated whether ALS-causing mutations interfere with the peripheral and brain-specific expression and signaling of the metabolic master regulator PGC (PPAR gamma coactivator)-1α (PGC-1α). METHODS: We analyzed the expression of PGC-1α isoforms and target genes in two mouse models of familial ALS and validated the stimulated PGC-1α signaling in primary adipocytes and neurons of these animal models and in iPS derived motoneurons of two ALS patients harboring two different frame-shift FUS/TLS mutations. RESULTS: Mutations in SOD1 and FUS/TLS decrease Ppargc1a levels in the CNS whereas in muscle and brown adipose tissue Ppargc1a mRNA levels were increased. Probing the underlying mechanism in neurons, we identified the monocarboxylate lactate as a previously unrecognized potent and selective inducer of the CNS-specific PGC-1α isoforms. Lactate also induced genes like brain-derived neurotrophic factor, transcription factor EB and superoxide dismutase 3 that are down-regulated in PGC-1α deficient neurons. The lactate-induced CNS-specific PGC-1α signaling system is completely silenced in motoneurons derived from induced pluripotent stem cells obtained from two ALS patients harboring two different frame-shift FUS/TLS mutations. CONCLUSION: ALS mutations increase the canonical PGC-1α system in the periphery while inhibiting the CNS-specific isoforms. We identify lactate as an inducer of the neuronal PGC-1α system directly linking brain metabolism and neuroprotection. Changes in the PGC-1α system might be involved in the ALS accompanied metabolic changes and in neurodegeneration.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Encéfalo/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Proteína FUS de Unión a ARN/genética , Superóxido Dismutasa-1/genética , Tejido Adiposo Pardo/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Línea Celular , Modelos Animales de Enfermedad , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Esquelético/metabolismo , Mutación , Neuronas/metabolismo , Isoformas de Proteínas , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Ratas , Superóxido Dismutasa-1/metabolismoRESUMEN
Aging is associated with a rising incidence of cutaneous squamous cell carcinoma (cSCC), an aggressive skin cancer with the potential for local invasion and metastasis. Acquisition of a senescence-associated secretory phenotype (SASP) in dermal fibroblasts has been postulated to promote skin cancer progression in elderly individuals. The underlying molecular mechanisms are largely unexplored. We show that Chemerin, a previously unreported SASP factor released from senescent human dermal fibroblasts, promotes cSCC cell migration, a key feature driving tumor progression. Whereas the Chemerin abundance is downregulated in malignant cSCC cells, increased Chemerin transcripts and protein concentrations are detected in replicative senescent fibroblasts in vitro and in the fibroblast of skin sections from old donors, indicating that a Chemerin gradient is built up in the dermis of elderly. Using Transwell® migration assays, we show that Chemerin enhances the chemotaxis of different cSCC cell lines. Notably, the Chemerin receptor CCRL2 is remarkably upregulated in cSCC cell lines and human patient biopsies. Silencing Chemerin in senescent fibroblasts or the CCRL2 and GPR1 receptors in the SCL-1 cSCC cell line abrogates the Chemerin-mediated chemotaxis. Chemerin triggers the MAPK cascade via JNK and ERK1 activation, whereby the inhibition impairs the SASP- or Chemerin-mediated cSCC cell migration.Taken together, we uncover a key role for Chemerin, as a major factor in the secretome of senescent fibroblasts, promoting cSCC cell migration and possibly progression, relaying its signals through CCRL2 and GPR1 receptors with subsequent MAPK activation. These findings might have implications for targeted therapeutic interventions in elderly patients.
Asunto(s)
Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma de Células Escamosas/metabolismo , Senescencia Celular , Quimiocinas/metabolismo , Quimiotaxis , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Comunicación Paracrina , Neoplasias Cutáneas/metabolismo , Anciano de 80 o más Años , Fibroblastos Asociados al Cáncer/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Proliferación Celular , Quimiocinas/genética , Técnicas de Cocultivo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Invasividad Neoplásica , Interferencia de ARN , Receptores CCR/genética , Receptores CCR/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , TransfecciónRESUMEN
Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFß receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety.
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Adenoviridae/genética , Proteínas de la Cápside/genética , Terapia Genética , Vectores Genéticos/genética , Virus Oncolíticos/genética , Neoplasias Pancreáticas/patología , Ingeniería de Proteínas , Adenoviridae/fisiología , Secuencia de Aminoácidos , Animales , Transporte Biológico , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Factor X/metabolismo , Vectores Genéticos/metabolismo , Hepatocitos/patología , Hepatocitos/virología , Humanos , Inmunoglobulina M/metabolismo , Macrófagos/metabolismo , Ratones , Datos de Secuencia Molecular , Virus Oncolíticos/fisiología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/virología , Células Estrelladas Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Células del Estroma/virología , Transducción Genética , Tropismo Viral , Replicación ViralRESUMEN
Recombinant adenovirus vectors (AdVs) have been used for the development of vaccines, as gene therapy vectors and for protein production. Currently, the production of clinical grade batches of recombinant E1-deleted adenovirus type 5 vectors is performed using human-derived HEK293 or PER.C6(®) cell lines. In this work we describe the generation of a new human amniocyte-derived cell line named 1G3 and show that it can be used as a very promising cell host for AdV production in serum-free conditions, allowing for production in high cell density cultures and avoiding the typical cell density effect observed for HEK293. By design, this cell line makes the generation of replication-competent adenovirus during production of E1-deleted AdVs very unlikely. The impact of the culture system (static versus agitated) and AdV infection parameters such as multiplicity of infection, time of harvesting and cell concentration at infection were evaluated and compared with HEK293. Using stirred tanks bioreactors, it was possible to grow 1G3 cells to cell densities of up to 9 × 10(6) cells/mL using serum-free media. Moreover, without a medium exchange step at infection, a three-fold increase in AdV volumetric titers was obtained, as no cell density effect was observed at CCI 3. Overall, our results clearly demonstrate the potential of the human amniocyte-derived newly established cell line 1G3 for AdV production in a serum-free scalable process, paving the way for further process improvements based on fed-batch or perfusion strategies.
Asunto(s)
Adenovirus Humanos/crecimiento & desarrollo , Líquido Amniótico/citología , Técnicas de Cultivo Celular por Lotes/métodos , Medio de Cultivo Libre de Suero/metabolismo , Adenovirus Humanos/genética , Reactores Biológicos , Recuento de Células , Línea Celular , Femenino , Vectores Genéticos , Células HEK293 , Humanos , Embarazo , Carga Viral , Cultivo de Virus/métodosRESUMEN
Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington's disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on "gutless" adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Línea Celular , Dicroismo Circular , Doxiciclina/farmacología , Humanos , Proteína Huntingtina , Mutación , Proteínas del Tejido Nervioso/química , Fosforilación , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The evolutionarily conserved IGF-1 signalling pathway is associated with longevity, metabolism, tissue homeostasis, and cancer progression. Its regulation relies on the delicate balance between activating kinases and suppressing phosphatases and is still not very well understood. We report here that IGF-1 signalling in vitro and in a murine ageing model in vivo is suppressed in response to accumulation of superoxide anions (O2â-) in mitochondria, either by chemical inhibition of complex I or by genetic silencing of O2â--dismutating mitochondrial Sod2. The O2â--dependent suppression of IGF-1 signalling resulted in decreased proliferation of murine dermal fibroblasts, affected translation initiation factors and suppressed the expression of α1(I), α1(III), and α2(I) collagen, the hallmarks of skin ageing. Enhanced O2â- led to activation of the phosphatases PTP1B and PTEN, which via dephosphorylation of the IGF-1 receptor and phosphatidylinositol 3,4,5-triphosphate dampened IGF-1 signalling. Genetic and pharmacologic inhibition of PTP1B and PTEN abrogated O2â--induced IGF-1 resistance and rescued the ageing skin phenotype. We thus identify previously unreported signature events with O2â-, PTP1B, and PTEN as promising targets for drug development to prevent IGF-1 resistance-related pathologies.
Asunto(s)
Envejecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Superóxidos/metabolismo , Envejecimiento/genética , Animales , Humanos , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Fosfohidrolasa PTEN/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Transducción de Señal , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Replication deficient adenovirus vectors are frequently used tools for the delivery of transgenes in vitro and in vivo. In addition, several therapeutic products based on adenovirus are under clinical development. This review outlines adenovirus vector production discussing different vector types, available production cell lines and state of the art of production process development and purification.