RESUMEN
One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options.
RESUMEN
Obesity is associated with increased risk for developing pancreatic cancer, and it is suggested that insulin resistance provides the missing link. Here we demonstrate that under the context of genetic susceptibility, a high fat diet (HFD) predisposes mice with oncogenic K-ras activation to accelerated pancreatic intraepithelial neoplasm (PanIN) development. Tumor promotion is closely associated with increased inflammation and abrogation of TNFR1 signaling significantly blocks this process underlining a central role for TNFalpha in obesity-mediated enhancement of PanIN lesions. Interestingly, however, despite increased TNFalpha levels, mice remain insulin sensitive. We show that, while aggravating tumor promotion, a HFD exerts dramatic changes in energy metabolism through enhancement of pancreatic exocrine insufficiency, metabolic rates, and expression of genes involved in mitochondrial fatty acid (FA) beta-oxidation that collectively contribute to improved glucose tolerance in these mice. While on one hand these findings provide significant evidence that obesity is linked to tumor promotion in the pancreas, on the other it suggests alterations in inflammatory responses and bioenergetic pathways as the potential underlying cause.
Asunto(s)
Ácidos Grasos/metabolismo , Mitocondrias/metabolismo , Obesidad/metabolismo , Neoplasias Pancreáticas/metabolismo , Alimentación Animal , Animales , Progresión de la Enfermedad , Insuficiencia Pancreática Exocrina/inducido químicamente , Insuficiencia Pancreática Exocrina/metabolismo , Insuficiencia Pancreática Exocrina/patología , Ácidos Grasos/farmacología , Inflamación/genética , Inflamación/metabolismo , Resistencia a la Insulina , Ratones , Mitocondrias/efectos de los fármacos , Oxidación-Reducción , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factores de Tiempo , Proteínas ras/metabolismoRESUMEN
To identify functions of the IKKalpha subunit of IkappaB kinase that require catalytic activity, we generated an Ikkalpha(AA) knockin allele containing alanines instead of serines in the activation loop. Ikkalpha(AA/AA) mice are healthy and fertile, but females display a severe lactation defect due to impaired proliferation of mammary epithelial cells. IKKalpha activity is required for NF-kappaB activation in mammary epithelial cells during pregnancy and in response to RANK ligand but not TNFalpha. IKKalpha and NF-kappaB activation are also required for optimal cyclin D1 induction. Defective RANK signaling or cyclin D1 expression results in the same phenotypic effect as the Ikkalpha(AA) mutation, which is completely suppressed by a mammary specific cyclin D1 transgene. Thus, IKKalpha is a critical intermediate in a pathway that controls mammary epithelial proliferation in response to RANK signaling via cyclin D1.
Asunto(s)
Ciclina D1/metabolismo , Células Epiteliales/metabolismo , Glicoproteínas/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caseínas/genética , Caseínas/metabolismo , Células Cultivadas , Ciclina D1/genética , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Glicoproteínas/genética , Humanos , Quinasa I-kappa B , Lactancia/fisiología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/trasplante , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Osteoprotegerina , Embarazo , Proteínas Serina-Treonina Quinasas/genética , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/genética , Receptores del Factor de Necrosis Tumoral , Trasplante de Tejidos , Transgenes , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
In mammals, the canonical nuclear factor kappaB (NF-kappaB) signaling pathway activated in response to infections is based on degradation of IkappaB inhibitors. This pathway depends on the IkappaB kinase (IKK), which contains two catalytic subunits, IKKalpha and IKKbeta. IKKbeta is essential for inducible IkappaB phosphorylation and degradation, whereas IKKalpha is not. Here we show that IKKalpha is required for B cell maturation, formation of secondary lymphoid organs, increased expression of certain NF-kappaB target genes, and processing of the NF-kappaB2 (p100) precursor. IKKalpha preferentially phosphorylates NF-kappaB2, and this activity requires its phosphorylation by upstream kinases, one of which may be NF-kappaB-inducing kinase (NIK). IKKalpha is therefore a pivotal component of a second NF-kappaB activation pathway based on regulated NF-kappaB2 processing rather than IkappaB degradation.
Asunto(s)
Linfocitos B/fisiología , Tejido Linfoide/fisiología , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Linfocitos B/inmunología , Células de la Médula Ósea/metabolismo , Evolución Molecular , Femenino , Regulación de la Expresión Génica , Centro Germinal , Quinasa I-kappa B , Proteínas I-kappa B/metabolismo , Inmunoglobulina D/análisis , Lipopolisacáridos/farmacología , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidad p52 de NF-kappa B , Fosforilación , Procesamiento Proteico-Postraduccional , Quimera por Radiación , Proteínas Recombinantes/metabolismo , Bazo/citología , Bazo/inmunología , Transcripción Genética , Transfección , Quinasa de Factor Nuclear kappa BRESUMEN
We used a murine tumor progression model for the evaluation of potential proliferation markers using positron emission tomography (PET). 5-[(18)F]-2'-deoxyuridine ([(18)F]FdUrd) was synthesized with >98% radiochemical purity and investigated in a pancreatic cancer model, transforming growth factor alpha transgenic mice crossbred to p53 deficient mice. Thymidylate synthase was increased already in premalignant lesions, whereas thymidine kinase 1 mRNA levels were up-regulated 4-fold in the pancreatic cancer specimen of these mice. PET imaging was performed after injection of 1 MBq of [(18)F]FdUrd and 1 MBq of [(18)F]fluoro-deoxyglucose. Animals with pancreatic cancer displayed focal uptake of both tracers. The [(18)F]FdUrd uptake ratio closely correlated with the proliferation index as evaluated in morphometric and fluorescence-activated cell sorter analysis. These results indicate the potential of our tumor model for the evaluation of PET tracers and suggest [(18)F]FdUrd as a tracer for the assessment of proliferation in vivo.
Asunto(s)
Floxuridina , Neoplasias Pancreáticas/diagnóstico por imagen , Radiofármacos , Animales , División Celular/fisiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Floxuridina/síntesis química , Floxuridina/farmacocinética , Fluorodesoxiglucosa F18 , Genes p53/genética , Ratones , Ratones Transgénicos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Cintigrafía , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Timidina Quinasa/biosíntesis , Timidina Quinasa/genética , Timidilato Sintasa/biosíntesis , Timidilato Sintasa/genética , Factor de Crecimiento Transformador alfa/genética , Regulación hacia ArribaRESUMEN
This study describes a tumor progression model for ductal pancreatic cancer in mice overexpressing TGF-alpha. Activation of Ras and Erk causes induction of cyclin D1-Cdk4 without increase of cyclin E or PCNA in ductal lesions. Thus, TGF-alpha is able to promote progression throughout G1, but not S phase. Crossbreeding with p53 null mice accelerates tumor development in TGF-alpha transgenic mice dramatically. In tumors developing in these mice, biallelic deletion of Ink4a/Arf or LOH of the Smad4 locus is found suggesting that loci in addition to p53 are involved in antitumor activities. We conclude that these genetic events are critical for pancreatic tumor formation in mice. This model recapitulates pathomorphological features and genetic alterations of the human disease.
Asunto(s)
Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas , Factor de Crecimiento Transformador alfa/genética , Animales , Proteínas Portadoras/genética , Ciclina D1/genética , Quinasa 4 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Fase G1/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Invasividad Neoplásica , Neoplasias Pancreáticas/patología , Fenotipo , Fase S/fisiología , Factor de Crecimiento Transformador alfa/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Proteínas ras/metabolismoRESUMEN
Pancreatic cancer is a devastating disease with a fatal prognosis due to late diagnosis and resistance to radiation and chemotherapy. The average survival after diagnosis is still 3 to 8 months. In the last few years genetic alterations in cancer-causing genes have been identified in tumors and putative premalignant lesions using microdissection techniques. However, the functional consequence of these genetic alterations for pancreatic growth and differentiation is unknown. TGF alpha overexpressed in the pancreas causes the development of tubular structures and fibrosis. Mice older than one year develop ductal pancreatic cancer. Crossbreeding these mice with p53 knockout mice dramatically accelerated tumor development. Moreover, tumors developing in these mice show frequently biallelic deletion of the Ink4a locus or LOH of SMAD4. These mice represent the first model of pancreatic adenocarcinomas with genetic alterations as well as growth characteristics similar to the human disease.
Asunto(s)
Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Factor de Crecimiento Transformador alfa/genética , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Genes p53/genética , Humanos , Ratones , Ratones Transgénicos , Lesiones Precancerosas/patología , Factor de Crecimiento Transformador alfa/fisiologíaRESUMEN
The molecular evolution of the opioid receptor family has been studied by isolating cDNAs that encode six distinct opioid receptor-like proteins from a lower vertebrate, the teleost fish Catostomus commersoni. One of these, which has been obtained in full-length form, encodes a 383-amino acid protein that exhibits greatest sequence similarity to mammalian mu-opioid receptors; the corresponding gene is expressed predominantly in brain and pituitary. Transfection of the teleost cDNA into HEK 293 cells resulted in the appearance of a receptor having high affinity for the mu-selective agonist [D-Ala2, MePhe4-Gly-ol5]enkephalin (DAMGO) (Kd = 0.63 +/- 0.15 nM) and for the nonselective antagonist naloxone (Kd = 3.1 +/- 1.3 nM). The receptor had negligible affinity for U50488 and [D-Pen2, D-Pen5]enkephalin (DPDPE), which are kappa- and delta-opioid receptor selective agonists, respectively. Stimulation of transfected cells with 1 microM DAMGO lowered forskolin-induced cAMP levels, an effect that could be reversed by naloxone. Experiments in Xenopus oocytes have demonstrated that the fish opioid receptor can, in an agonist-dependent fashion, activate a coexpressed mouse G-protein-gated inward-rectifying potassium channel (GIRK1). The identification of six distinct fish opioid receptor-like proteins suggests that additional mammalian opioid receptors remain to be identified at the molecular level. Furthermore, our data indicate that the mu-opioid receptor arose very early in evolution, perhaps before the appearance of vertebrates, and that the pharmacological and functional properties of this receptor have been conserved over a period of approximately 400 million years implying that it fulfills an important physiological role.