RESUMEN
Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 (SMN1) causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The SMN1 protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear. We used an antisense oligonucleotide (ASO)-based inducible mouse model of SMA to investigate the SMN-specific transcriptome changes associated with neurodegeneration. We found evidence of widespread intron retention, particularly of minor U12 introns, in the spinal cord of mice 30 d after SMA induction, which was then rescued by a therapeutic ASO. Intron retention was concomitant with a strong induction of the p53 pathway and DNA damage response, manifesting as γ-H2A.X positivity in neurons of the spinal cord and brain. Widespread intron retention and markers of the DNA damage response were also observed with SMN depletion in human SH-SY5Y neuroblastoma cells and human induced pluripotent stem cell-derived motor neurons. We also found that retained introns, high in GC content, served as substrates for the formation of transcriptional R-loops. We propose that defects in intron removal in SMA promote DNA damage in part through the formation of RNA:DNA hybrid structures, leading to motor neuron death.
Asunto(s)
Daño del ADN , Intrones , Atrofia Muscular Espinal/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Empalme del ARNRESUMEN
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by disruption of the survival motor neuron 1 (SMN1) gene, partly compensated for by the paralogous gene SMN2. Exon 7 inclusion is critical for full-length SMN protein production and occurs at a much lower frequency for SMN2 than for SMN1. Antisense oligonucleotide (ASO)-mediated blockade of an intron 7 splicing silencer was previously shown to promote inclusion of SMN2 exon 7 in SMA mouse models and mediate phenotypic rescue. However, downstream molecular consequences of this ASO therapy have not been defined. Here we characterize the gene-expression changes that occur in an induced model of SMA and show substantial rescue of those changes in central nervous system tissue upon intracerebroventricular administration of an ASO that promotes inclusion of exon 7, with earlier administration promoting greater rescue. This study offers a robust reference set of preclinical pharmacodynamic gene expression effects for comparison of other investigational therapies for SMA.
Asunto(s)
Exones , Expresión Génica , Atrofia Muscular Espinal/genética , Oligonucleótidos Antisentido/farmacología , Animales , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Ratones , Atrofia Muscular Espinal/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/efectos de los fármacos , Proteína 2 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Tumor growth and metastasis require concomitant growth of new blood vessels, which are stimulated by angiogenic factors, including vascular endothelial growth factor (VEGF), secreted by most tumors. Whereas the angiogenic property and molecular mechanisms of VEGF have been well studied, the biological function of its related homolog, placenta growth factor (PlGF), is poorly understood. Here we demonstrate that PlGF-1, an alternatively spliced isoform of the PlGF gene, antagonizes VEGF-induced angiogenesis when both factors are coexpressed in murine fibrosarcoma cells. Overexpression of PlGF-1 in VEGF-producing tumor cells results in the formation of PlGF-1/VEGF heterodimers and depletion of the majority of mouse VEGF homodimers. The heterodimeric form of PlGF-1/VEGF lacks the ability to induce angiogenesis in vitro and in vivo. Similarly, PlGF-1/VEGF fails to activate the VEGFR-2-mediated signaling pathways. Further, PlGF-1 inhibits the growth of a murine fibrosarcoma by approximately 90% when PlGF-1-expressing tumor cells are implanted in syngeneic mice. In contrast, overexpression of human VEGF in murine tumor cells causes accelerated and exponential growth of primary fibrosarcomas and early hepatic metastases. Our data demonstrate that PlGF-1, a member of the VEGF family, acts as a natural antagonist of VEGF when both factors are synthesized in the same population of cells. The underlying mechanism is due to the formation of functionally inactive heterodimers.
Asunto(s)
Neovascularización de la Córnea/prevención & control , Factores de Crecimiento Endotelial/fisiología , Fibrosarcoma/prevención & control , Linfocinas/fisiología , Proteínas Gestacionales/fisiología , Neoplasias Cutáneas/prevención & control , Inductores de la Angiogénesis/fisiología , Animales , Quimiotaxis , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Ensayo de Inmunoadsorción Enzimática , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Expresión Génica/fisiología , Humanos , Técnicas para Inmunoenzimas , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Factor de Crecimiento Placentario , Plásmidos , Isoformas de Proteínas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Breast cancer development is a complex pathobiological process involving sequential genetic alterations in normal epithelial cells that results in uncontrolled growth in a permissive microenvironment. Accordingly, physiologically relevant models of human breast cancer that recapitulate these events are needed to study cancer biology and evaluate therapeutic agents. Here, we report the generation and utilization of the human breast cancer in mouse (HIM) model, which is composed of genetically engineered primary human breast epithelial organoids and activated human breast stromal cells. By using this approach, we have defined key genetic events required to drive the development of human preneoplastic lesions as well as invasive adenocarcinomas that are histologically similar to those in patients. Tumor development in the HIM model proceeds through defined histological stages of hyperplasia, DCIS to invasive carcinoma. Moreover, HIM tumors display characteristic responses to targeted therapies, such as HER2 inhibitors, further validating the utility of these models in preclinical compound testing. The HIM model is an experimentally tractable human in vivo system that holds great potential for advancing our basic understanding of cancer biology and for the discovery and testing of targeted therapies.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Transformación Celular Neoplásica/metabolismo , Ciclina D1/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasas/metabolismo , Interferencia de ARN , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Virus 40 de los Simios/genética , Virus 40 de los Simios/metabolismo , Telomerasa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Therapies that target the EGF receptor (EGFR), such as gefitinib (IRESSA), are effective in a subset of patients with advanced non-small cell lung cancer (NSCLC). The differences in intracellular signaling networks between gefitinib-sensitive and -resistant NSCLCs remain poorly understood. In this study, we observe that gefitinib reduces phospho-Akt levels only in NSCLC cell lines in which it inhibits growth. To elucidate the mechanism underlying this observation, we compared immunoprecipitates of phosphoinositide 3-kinase (PI3K) between gefitinib-sensitive and -resistant NSCLC cell lines. We observe that PI3K associates with ErbB-3 exclusively in gefitinib-sensitive NSCLC cell lines. Gefitinib dissociates this complex, thereby linking EGFR inhibition to decreased Akt activity. In contrast, gefitinib-resistant cells do not use ErbB-3 to activate the PI3K/Akt pathway. In fact, abundant ErbB-3 expression is detected only in gefitinib-sensitive NSCLC cell lines. Two gefitinib-sensitive NSCLC cell lines with endogenous distinct activating EGFR mutations (L858R and Del747-749), frequently observed in NSCLC patients who respond to gefitinib, also use ErbB-3 to couple to PI3K. Down-regulation of ErbB-3 by means of short hairpin RNA leads to decreased phospho-Akt levels in the gefitinib-sensitive NSCLC cell lines, Calu-3 (WT EGFR) and H3255 (L858R EGFR), but has no effect on Akt activation in the gefitinib-resistant cell lines, A549 and H522. We conclude that ErbB-3 is used to couple EGFR to the PI3K/Akt pathway in gefitinib-sensitive NSCLC cell lines harboring WT and mutant EGFRs.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/fisiología , Quinazolinas/farmacología , Receptor ErbB-3/fisiología , Animales , Células CHO , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Cricetinae , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/fisiología , Gefitinib , Humanos , Neoplasias Pulmonares/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-aktRESUMEN
Multidrug resistance (MDR) confers resistance to anticancer drugs and reduces therapeutic efficiency. It is often characterized by the expression of the MDR1 gene product P-glycoprotein (or gp170) at the membrane of tumor cells. To further propose a potential complementary tool in cancer treatment, the sensitivity of gp170 tumor cells to NK-dependent lysis was investigated. Two kinds of cells were generated from wild-type K562 erythroleukemic cells: the first were derived from Taxol-selected cells and cloned, whereas the second were retrovirally transduced by the cDNA of the MDR1 gene. The last process was also applied to the human embryonal carcinoma cells called Tera-2 cells. First, both cloned and MDR-1 K562 cells appeared highly susceptible to naive NK cell killing. Interestingly, in addition, Tera-2 cells that were not sensitive to NK lysis could be killed when they expressed gp170 at their membranes. In previous data, we demonstrated that NK cell release of bimolecular complexes composed of perforin and platelet-activating factor (PAF) interacting with the PAF-R, which has to be expressed on the target cell membranes, were components of NK tumor cell killing. In the present study, we show that gp170 has the capacity to drive constitutive PAF-R expression on tumor cells, which could be responsible for hypersensitivity to NK lysis and accelerated cell death.
Asunto(s)
Citotoxicidad Inmunológica , Resistencia a Antineoplásicos/inmunología , Glicoproteínas/fisiología , Células Asesinas Naturales/inmunología , Factor de Activación Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Receptores Acoplados a Proteínas G/biosíntesis , Subfamilia B de Transportador de Casetes de Unión a ATP , Carcinoma Embrionario/inmunología , Carcinoma Embrionario/metabolismo , Carcinoma Embrionario/patología , Línea Celular Tumoral , Membrana Celular/inmunología , Membrana Celular/metabolismo , Membrana Celular/patología , Células Clonales , Citotoxicidad Inmunológica/efectos de los fármacos , Genes MDR/inmunología , Glicoproteínas/biosíntesis , Humanos , Concentración de Iones de Hidrógeno , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Leucemia Eritroblástica Aguda/inmunología , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patología , Paclitaxel/farmacología , Retroviridae/genética , Teratoma/inmunología , Teratoma/metabolismo , Teratoma/patología , Transducción Genética , TransfecciónRESUMEN
Clinical translation of gene-based therapies for arthritis could be accelerated by vectors capable of efficient intra-articular gene delivery and long-term transgene expression. Previously, we have shown that lentiviral vectors transduce rat synovium efficiently in vivo. Here, we evaluated the functional capacity of transgene expression provided by lentiviral-mediated gene delivery to the joint. To do this, we measured the ability of a lentiviral vector containing the cDNA for human interleukin-1 receptor antagonist (LV-hIL-1Ra) to suppress intra-articular responses to IL-1beta. Groups of rats were injected in one knee with 5 x 10(7) infectious units of LV-IL-1Ra. After 24 h, a range of doses of fibroblasts (3 x 10(3), 10(4), 3 x 10(4), or 10(5) cells) genetically modified to overexpress IL-1beta was injected into both knees. Intra-articular delivery of LV-hIL-1Ra strongly prevented swelling in all treated knees, even in those receiving the greatest dose of IL-1beta(+) cells. Cellular infiltration, cartilage erosion, and invasiveness of inflamed synovium were effectively prevented in LV-hIL-1Ra-treated knees and were significantly inhibited in contralateral joints. Beneficial effects were also observed systemically in the lentivirus-treated animals. Interestingly, intra-articular expression of the IL-1Ra transgene was found to increase in relation to the number of IL-1beta(+) cells injected. Further experiments using GFP suggest this is due to the proliferation of cells, stably modified by the integrative lentivirus, in response to inflammatory stimulation.
Asunto(s)
Artritis Experimental/terapia , Lentivirus/genética , Sialoglicoproteínas/genética , Membrana Sinovial/metabolismo , Animales , Artritis Experimental/metabolismo , Artritis Experimental/patología , Vectores Genéticos/administración & dosificación , Miembro Posterior/metabolismo , Miembro Posterior/patología , Humanos , Inyecciones Intraarticulares , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/genética , Interleucina-1/metabolismo , Articulaciones/metabolismo , Articulaciones/patología , Masculino , Microscopía Fluorescente , Ratas , Ratas Wistar , Sialoglicoproteínas/metabolismoRESUMEN
The delivery of anti-arthritic genes to the synovial lining of joints is being explored as a strategy for the treatment of rheumatoid arthritis. In this study, we have investigated the use of VSV-G pseudotyped, HIV-1-based lentiviral vectors for gene delivery to articular tissues. Recombinant lentivirus containing a beta-galactosidase/neomycin resistance fusion gene under control of the elongation factor (EF) 1alpha promoter efficiently transduced human and rat synoviocytes and chondrocytes in cell culture. When directly injected into the knees of rats, this vector transduced synovial lining cells, but not other articular tissues such as cartilage. We also constructed a lentiviral vector containing the human interleukin-1 receptor antagonist (IL1RA) cDNA and examined transgene expression in vitro and in vivo following injection into the knee joints of rats. In immunocompetent animals, intra-articular IL1RA expression was high and persisted, at a sharply declining rate, for approximately 20 days. In immunocompromised rats, however, lentivirus-mediated intra-articular expression of human IL1RA was found to persist for at least 6 weeks. Extra-articular expression of the transgene was minimal. These results indicate that lentiviral vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing long-term expression for gene-based treatments of arthritis.