RESUMO
Dominant optic atrophy (DOA) is a rare progressive and irreversible blinding disease which is one of the most frequent forms of hereditary optic neuropathy. DOA is mainly caused by dominant mutation in the OPA1 gene encoding a large mitochondrial GTPase with crucial roles in membrane dynamics and cell survival. Hereditary optic neuropathies are commonly characterized by the degeneration of retinal ganglion cells, leading to the optic nerve atrophy and the progressive loss of visual acuity. Up to now, despite increasing advances in the understanding of the pathological mechanisms, DOA remains intractable. Here, we tested the efficiency of gene therapy on a genetically-modified mouse model reproducing DOA vision loss. We performed intravitreal injections of an Adeno-Associated Virus carrying the human OPA1 cDNA under the control of the cytomegalovirus promotor. Our results provide the first evidence that gene therapy is efficient on a mouse model of DOA as the wild-type OPA1 expression is able to alleviate the OPA1-induced retinal ganglion cell degeneration, the hallmark of the disease. These results displayed encouraging effects of gene therapy for Dominant Optic Atrophy, fostering future investigations aiming at clinical trials in patients.
Assuntos
GTP Fosfo-Hidrolases/genética , Terapia Genética/métodos , Mitocôndrias/genética , Atrofia Óptica Autossômica Dominante/terapia , Células Ganglionares da Retina/metabolismo , Baixa Visão/terapia , Animais , Morte Celular , Citomegalovirus/genética , Citomegalovirus/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Feminino , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Injeções Intravítreas , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mutação , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/patologia , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Regiões Promotoras Genéticas , Células Ganglionares da Retina/patologia , Transgenes , Baixa Visão/genética , Baixa Visão/metabolismo , Baixa Visão/patologiaRESUMO
Pluripotency is at the crossroads of stem cell research and biology of reproduction. The mature metaphase II oocyte contains the key factors for pluripotency induction and maintenance as assessed by its capacity to reprogram somatic nuclei. The cumulus cells (CCs) niche that surrounds the oocyte is crucial for its maturation and presumably for the oocyte to acquire its competence to confer pluripotency. In this study, we examined whether cells cultured from the human mature metaphase II oocyte CC niche (hCC) could be used as feeders for the propagation of human induced pluripotent stem cells. The induced pluripotent (iPS) cells cultured on hCC (hCC-iPS) were assessed for their pluripotency potential by their expression of pluripotency-associated genes such as Oct4, Nanog, and TRA1-60 and their competence to differentiate into the three germ layers in vitro (embryoid bodies) as well as in vivo (teratoma formation). We show that not only the hCC-iPS cells maintained their pluripotency potential, but they also exhibited much better self-renewal performance in terms of proliferation rate compared to the same cells cultured on human foreskin fibroblast (hFF) feeders (hFF-iPS). A comparative gene expression profile study of hCC and hFF revealed significant differences (P < 0.05) in expression of cellular matrix components and an upregulation in hCC of genes known to be important players in cell proliferation such as interleukin 6 gene (IL6).
Assuntos
Proliferação de Células , Células do Cúmulo/citologia , Células Alimentadoras/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Células do Cúmulo/metabolismo , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Molécula de Adesão da Célula Epitelial , Células Alimentadoras/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Oócitos/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Teratoma/genética , Teratoma/metabolismo , Teratoma/patologia , Transplante Heterólogo , Vimentina/genética , Vimentina/metabolismoRESUMO
Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM (-/y) patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE). This iPSC-derived RPE is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype. The high, and unmatched, in vitro transduction efficiency is likely aided by phagocytosis and mimics the scenario that an AAV vector encounters in vivo in the subretinal space. We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC-derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model.
RESUMO
In culture, human pluripotent stem cells (PSCs) are phenotypically (for instance, the SSEA3 expression level) and functionally (capacity to survive after single-cell dissociation) heterogeneous. We report here that the side scatter (SSC) signal measured by flow cytometry, a variable correlated with membrane irregularity and cell granularity, is very high in PSCs, even higher than in blood polymorphonuclear cells, and markedly heterogeneous. Moreover, SSC intensity rapidly and strongly decreases upon PSC differentiation into any of the three germ layers. PSCs with high SSC (HSSC cells) or low SSC (LSSC cells) values both express pluripotency markers, but HSSC cells are characterized by more frequent simultaneous expression of the membrane pluripotency factors SSEA3, SSEA4, TRA-1-81, TRA-1-60, and CD24 and by a higher mitochondrial content. Functionally, HSSC cells are more likely to generate colonies upon single-cell passage than LSSC cells. SSC monitoring might provide a simple, but robust and rapid method to estimate pluripotency variations in culture and unveils a new phenotypic and functional heterogeneity in PSCs.
Assuntos
Células-Tronco Embrionárias/citologia , Heterogeneidade Genética , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Antígeno CD24/genética , Antígeno CD24/metabolismo , Diferenciação Celular , Linhagem Celular , Células Clonais , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Expressão Gênica , Camadas Germinativas/metabolismo , Humanos , Camundongos , Camundongos SCID , Células-Tronco Pluripotentes/metabolismo , Proteoglicanas/genética , Proteoglicanas/metabolismo , Antígenos Embrionários Estágio-Específicos/genética , Antígenos Embrionários Estágio-Específicos/metabolismo , Teratoma/metabolismo , Teratoma/patologiaRESUMO
The generation of specific T lymphocyte subsets is under the strict control of specific transcription factors, as this has been shown by knockout experiments in mice. Here, we show that siRNAs that specifically target the transcription factor Gata3 (which is required for the development of T helper 1 cells) or T-Bet (which is required for the development of T helper 2 cells) can be effective in vivo. Thus, the intraperitoneal injection of siRNAs specific for Gata3 or t-Bet leads to the specific depletion of their target gene products in vivo, in the spleen and in the lymph nodes of mice. The immunomodulatory action of these siRNAs was validated in a model of anti-tumor vaccination in which colorectal cancer cells that succumb to anthracyclin-induced immunogenic cell death were injected subcutaneously into one flank, in the absence of any adjuvant and live tumor cells were injected simultaneously in the opposite flank of immunocompetent mice. In this setting, the siRNA targeting t-Bet was able to accelerate tumor growth while the siRNA targeting Gata3 significantly reduced the proliferation of cancer cells in vivo. These effects were dependent on the immune response elicited by dying tumor cells because both siRNAs failed to modulate the growth of tumors in non-vaccinated mice. The immune response-dependent anticancer effect of the Gata3-specific siRNA was not due to the induction of class I interferons and could be fully abolished by co-injection of t-Bet-specific siRNA. These results demonstrate the possibility to use siRNAs for immunomodulaton in vivo and illustrate the antagonistic implication of distinct T helper populations in anti-cancer immune responses.
Assuntos
Fator de Transcrição GATA3/antagonistas & inibidores , Interferência de RNA , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Camundongos , RNA Interferente Pequeno/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
The susceptibility of cells to apoptosis induction is deeply influenced by their position in the cell cycle. Unfortunately, however, current methods for the enrichment of cells in defined phases of the cell cycle are mostly based on the synchronization of cells by agents or conditions that are intrinsically toxic and induce apoptosis on their own. We developed a novel procedure for the purification of cells in distinct phases of the cell cycle. This method is based on the stable transfection of cells with a chimeric protein made up by histone H2B and green fluorescent protein (GFP). Cytofluorometric purification of cells defined by their size and their H2B-GFP-dependent fluorescence (which reflects chromatin and hence DNA content) allowed for the efficient separation of diploid and tetraploid cells in the fluorescence-activated cell sorter (FACS). Moreover, when applied to diploid cells, this method allowed for the enrichment of live, functional cells in the G1, S and G2 phases of the cell cycle. FACS-purified cells were viable and readily resumed the cell cycle upon reculture. While staurosporine was equally toxic for cells in any phase of the cell cycle, camptothecin was particularly toxic for cells in the S phase. Moreover, BAY11-7082, a specific inhibitor of the IKK complex required for NF-kappaB activation, exhibited a particular cell cycle-specific profile of toxicity (G2>S>G1). These results delineate a novel procedure for studying the intersection between cell cycle regulation and cell death mechanisms.
Assuntos
Apoptose , Neoplasias do Colo/patologia , Citometria de Fluxo/métodos , Interfase , Apoptose/efeitos dos fármacos , Bromodesoxiuridina/metabolismo , Camptotecina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Humanos , Interfase/efeitos dos fármacos , Nitrilas/farmacologia , Ploidias , Estaurosporina/farmacologia , Sulfonas/farmacologiaRESUMO
Tetraploidy can result in cancer-associated aneuploidy. As shown here, freshly generated tetraploid cells arising due to mitotic slippage or failed cytokinesis are prone to undergo Bax-dependent mitochondrial membrane permeabilization and subsequent apoptosis. Knockout of Bax or overexpression of Bcl-2 facilitated the survival of tetraploid cells at least as efficiently as the p53 or p21 knockout. When tetraploid cells were derived from diploid p53 and Bax-proficient precursors, such cells exhibited an enhanced transcription of p53 target genes. Tetraploid cells exhibited an enhanced rate of spontaneous apoptosis that could be suppressed by inhibition of p53 or by knockdown of proapoptotic p53 target genes such as BBC3/Puma, GADD45A and ferredoxin reductase. Unexpectedly, tetraploid cells were more resistant to DNA damaging agents (cisplatin, oxaliplatin and camptothecin) than their diploid counterparts, and this difference disappeared upon inhibition of p53 or knockdown of p53-inducible ribonucleotide reductase. Tetraploid cells were also more resistant against UVC and gamma-irradiation. These data indicate the existence of p53-dependent alterations in apoptosis regulation in tetraploid cells.
Assuntos
Apoptose/fisiologia , Neoplasias/genética , Poliploidia , Animais , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Cisplatino/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Dano ao DNA , Feminino , Deleção de Genes , Humanos , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Nocodazol/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The interferon (IFN)-gamma-induced TRAIL effector mechanism is a vital component of cancer immunosurveillance by natural killer (NK) cells in mice. Here we show that the main source of IFN-gamma is not the conventional NK cell but a subset of B220(+)Ly6C(-) dendritic cells, which are atypical insofar as they express NK cell-surface molecules. Upon contact with a variety of tumor cells that are poorly recognized by NK cells, B220(+)NK1.1(+) dendritic cells secrete high levels of IFN-gamma and mediate TRAIL-dependent lysis of tumor cells. Adoptive transfer of these IFN-producing killer dendritic cells (IKDCs) into tumor-bearing Rag2(-/-)Il2rg(-/-) mice prevented tumor outgrowth, whereas transfer of conventional NK cells did not. In conclusion, we identified IKDCs as pivotal sensors and effectors of the innate antitumor immune response.
Assuntos
Células Dendríticas/classificação , Células Dendríticas/imunologia , Neoplasias Experimentais/imunologia , Transferência Adotiva , Animais , Apresentação de Antígeno , Antígenos Ly , Antígenos de Superfície/metabolismo , Proteínas Reguladoras de Apoptose/imunologia , Antígeno CD11c/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Células Dendríticas/ultraestrutura , Feminino , Interferon gama/biossíntese , Subunidade gama Comum de Receptores de Interleucina , Células Matadoras Naturais/imunologia , Lectinas Tipo C/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Microscopia Eletrônica , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Receptores de Interleucina/deficiência , Receptores de Interleucina/genética , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Systemic anticancer chemotherapy is immunosuppressive and mostly induces nonimmunogenic tumor cell death. Here, we show that even in the absence of any adjuvant, tumor cells dying in response to anthracyclins can elicit an effective antitumor immune response that suppresses the growth of inoculated tumors or leads to the regression of established neoplasia. Although both antracyclins and mitomycin C induced apoptosis with caspase activation, only anthracyclin-induced immunogenic cell death was immunogenic. Caspase inhibition by Z-VAD-fmk or transfection with the baculovirus inhibitor p35 did not inhibit doxorubicin (DX)-induced cell death, yet suppressed the immunogenicity of dying tumor cells in several rodent models of neoplasia. Depletion of dendritic cells (DCs) or CD8+T cells abolished the immune response against DX-treated apoptotic tumor cells in vivo. Caspase inhibition suppressed the capacity of DX-killed cells to be phagocytosed by DCs, yet had no effect on their capacity to elicit DC maturation. Freshly excised tumors became immunogenic upon DX treatment in vitro, and intratumoral inoculation of DX could trigger the regression of established tumors in immunocompetent mice. These results delineate a procedure for the generation of cancer vaccines and the stimulation of anti-neoplastic immune responses in vivo.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Doxorrubicina/farmacologia , Mitomicina/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Antibióticos Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Inibidores de Caspase , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Doxorrubicina/uso terapêutico , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Camundongos , Mitomicina/uso terapêutico , Neoplasias/prevenção & controle , Ratos , Vacinação/métodos , Proteínas Virais/genética , Proteínas Virais/farmacologiaRESUMO
Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.
Assuntos
Proteínas de Membrana/deficiência , Trifosfato de Adenosina/biossíntese , Animais , Apoptose , Fator de Indução de Apoptose , Encéfalo/metabolismo , Células Cultivadas , Complexo I de Transporte de Elétrons/biossíntese , Complexo III da Cadeia de Transporte de Elétrons/biossíntese , Flavoproteínas/genética , Flavoproteínas/metabolismo , Glucose/metabolismo , Humanos , Ácido Láctico/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Especificidade de Órgãos , Fosforilação Oxidativa , Filogenia , RNA Interferente Pequeno/metabolismo , Retina/metabolismo , Leveduras/genética , Leveduras/crescimento & desenvolvimento , Leveduras/metabolismoRESUMO
We and others have previously reported in an in vivo rat colon cancer cell model that cell death precedes and is necessary for the development of a specific antitumoral immune response. To sensitize colon cancer cells to death, we depleted cytochrome c by stable transfection with an antisense construct. Cytochrome c depletion sensitizes human and rat colon cancer cells to a nonapoptotic, nonautophagic death induced by various stimuli. This increased sensitization to a necrosis-like cell death may be related to a decrease in cellular ATP levels and an increase in reactive oxygen species production caused by cytochrome c depletion. In vivo, depletion of cytochrome c decreases the tumorigenicity of colon cancer cells in syngeneic rats without influencing their growth in immune-deficient animals. Furthermore, decreased expression of cytochrome c in tumor cells facilitates in vivo "necrotic" cell death and the induction of a specific immune response. These results delineate a novel strategy to sensitize colon cancer cells to chemotherapy and to increase their immunogenicity in immuno-competent hosts.
Assuntos
Neoplasias do Colo/imunologia , Citocromos c/deficiência , Citocromos c/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cisplatino/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Citocromos c/biossíntese , Citocromos c/genética , DNA Antissenso/genética , Regulação para Baixo , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Células HCT116 , Células HT29 , Humanos , Macrófagos/imunologia , Camundongos , Ratos , Estaurosporina/farmacologia , TransfecçãoRESUMO
Cystinosis is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The causative gene, CTNS, encodes cystinosin, a seven-transmembrane-domain protein, which we recently showed to be a lysosomal cystine transporter. The most severe and frequent form of cystinosis, the infantile form, appears around 6 to 12 months, with a proximal tubulopathy (de Toni-Debré-Fanconi syndrome) and ocular damage. End-stage renal failure is reached by 10 years of age. Accumulation of cystine in all tissues eventually leads to multisystemic disease. Treatment with cysteamine, which reduces the concentration of intracellular cystine, delays disease progression but has undesirable side effects. We report the first Ctns knockout mouse model generated using a promoter trap approach. We replaced the last four Ctns exons by an internal ribosome entry site-betagal-neo cassette and showed that the truncated protein was mislocalized and nonfunctional. Ctns(-/-) mice accumulated cystine in all organs tested, and cystine crystals, pathognomonic of cystinosis, were observed. Ctns(-/-) mice developed ocular changes similar to those observed in affected individuals, bone defects and behavioral anomalies. Interestingly, Ctns(-/-) mice did not develop signs of a proximal tubulopathy, or renal failure. A preliminary therapeutic trial using an oral administration of cysteamine was carried out and demonstrated the efficiency of this treatment for cystine clearance in Ctns(-/-) mice. This animal model will prove an invaluable and unique tool for testing emerging therapeutics for cystinosis.