RESUMO
The NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome is a node of intracellular stress pathways and a druggable target which integrates mitochondrial stress and inflammatory cascades. While a body of evidence suggests the involvement of the NLRP3 inflammasome in numerous diseases, a lack of reliable measurement techniques highlights the need for a robust assay using small quantities of biological samples. We present a literature overview on peripheral activation of the NLRP3 inflammasome in mood disorders, then outline a process to develop and validate a robust assay to measure baseline and activated intracellular levels of "apoptosis-associated speck-like protein containing a CARD" (ASC) as a key component of an inflammatory profile in peripheral blood mononuclear cells (PBMC). A consistent association between high NLRP3 mRNA levels and relevant cytokines was seen in the literature. Using our method to measure ASC, stimulation of PBMC with lipopolysaccharide and nigericin or adenosine triphosphate resulted in microscopic identification of intracellular ASC specks, as well as interleukin 1 (IL-1) beta and caspase-1 p10 in the periphery. This was abolished by dose-dependent pre-treatment with 100 nM MCC950. We also report the use of this technique in a small pilot sample from patients with bipolar disorder and depressive disorders. The results show that levels of intracellular ASC and IL-1 beta are sensitive to change upon activation and maintained over time, which may be used to improve the detection of NLRP3 activation and guide personalized therapeutic strategy in the treatment of patients.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/sangue , Inflamação/sangue , Interleucina-1beta/sangue , Transtornos do Humor/sangue , Proteína 3 que Contém Domínio de Pirina da Família NLR/sangue , Adolescente , Animais , Apoptose/genética , Caspase 1/sangue , Feminino , Humanos , Inflamassomos/sangue , Inflamassomos/genética , Inflamação/genética , Inflamação/patologia , Leucócitos Mononucleares/metabolismo , Masculino , Mitocôndrias/genética , Transtornos do Humor/genética , Transtornos do Humor/patologiaRESUMO
Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML.
Assuntos
DNA Mitocondrial/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Fosforilação Oxidativa , Fosfotransferases/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Replicação do DNA , Humanos , Camundongos SCID , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Zalcitabina/metabolismoRESUMO
Human pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.
Assuntos
AMP Cíclico/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Ativinas/metabolismo , Agregação Celular , Técnicas de Cultura de Células , Diferenciação Celular , Endoderma/citologia , Endoderma/metabolismo , Humanos , Proteína Nodal/metabolismo , Transdução de Sinais , TranscriptomaRESUMO
Mitochondria contain multiple copies of their own 16.6 kb circular genome. To explore the impact of mitochondrial DNA (mtDNA) damage on mitochondrial (mt) function and viability of AML cells, we screened a panel of DNA damaging chemotherapeutic agents to identify drugs that could damage mtDNA. We identified bleomycin as an agent that damaged mtDNA in AML cells at concentrations that induced cell death. Bleomycin also induced mtDNA damage in primary AML samples. Consistent with the observed mtDNA damage, bleomycin reduced mt mass and basal oxygen consumption in AML cells. We also demonstrated that the observed mtDNA damage was functionally important for bleomycin-induced cell death. Finally, bleomycin delayed tumor growth in xenograft mouse models of AML and anti-leukemic concentrations of the drug induced mtDNA damage in AML cells preferentially over normal lung tissue. Taken together, mtDNA-targeted therapy may be an effective strategy to target AML cells and bleomycin could be useful in the treatment of this disease.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Leucemia Mieloide Aguda/metabolismo , Animais , Antibióticos Antineoplásicos/uso terapêutico , Bleomicina/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Transplante de NeoplasiasRESUMO
Current drug screening methods are insufficiently predictive of clinical toxicity and efficacy. Recent advances in stem cell technology have the potential to improve drug screening. For tests of cardiotoxicity and efficacy of cardioactive drugs, cardiomyocytes derived from human embryonic stem cells and/or human induced pluripotent stem cells, collectively termed human pluripotent stem cells (hPSCs), have been utilized as model alternatives to current drug screening platforms. In this review, we report on recent advances in the differentiation of hPSCs to cardiomyocytes and summarize the evidence for pharmacological responses in hPSC-derived cardiomyocytes.
Assuntos
Fármacos Cardiovasculares/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Embrionárias/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologiaRESUMO
Xeroderma pigmentosum variant (XP-V) cells lack the damage-specific DNA polymerase eta and have normal excision repair but show defective DNA replication after UV irradiation. Previous studies using cells transformed with SV40 or HPV16 (E6/E7) suggested that the S-phase response to UV damage is altered in XP-V cells with non-functional p53. To investigate the role of p53 directly we targeted p53 in normal and XP-V fibroblasts using short hairpin RNA. The shRNA reduced expression of p53, and the downstream cell cycle effector p21, in control and UV irradiated cells. Cells accumulated in late S phase after UV, but after down-regulation of p53 they accumulated earlier in S. Cells in which p53 was inhibited showed ongoing genomic instability at the replication fork. Cells exhibited high levels of UV induced S-phase gammaH2Ax phosphorylation representative of exposed single strand regions of DNA and foci of Mre11/Rad50/Nbs1 representative of double strand breaks. Cells also showed increased variability of genomic copy numbers after long-term inhibition of p53. Inhibition of p53 expression dominated the DNA damage response. Comparison with earlier results indicates that in virally transformed cells cellular targets other than p53 play important roles in the UV DNA damage response.
Assuntos
Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Raios Ultravioleta , Sequência de Bases , Células Cultivadas , Primers do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Citometria de Fluxo , Instabilidade Genômica , Imuno-Histoquímica , Fosforilação , Interferência de RNA , Fase SRESUMO
Replication and maintenance of mitochondrial DNA (mtDNA) is essential for cellular function, yet few DNA polymerases are known to function in mitochondria. Here, we conclusively demonstrate that DNA polymerase θ (Polθ) localizes to mitochondria and explore whether this protein is overexpressed in patient-derived cells and tumors. Polθ appears to play an important role in facilitating mtDNA replication under conditions of oxidative stress, and this error-prone polymerase was found to introduce mutations into mtDNA. In patient-derived cells bearing a pathogenic mtDNA mutation, Polθ expression levels were increased, indicating that the oxidative conditions in these cells promote higher expression levels for Polθ. Heightened Polθ expression levels were also associated with elevated mtDNA mutation rates in a selected panel of human tumor tissues, suggesting that this protein can influence mutational frequencies in tumors. The results reported indicate that the mitochondrial function of Polθ may have relevance to human disease.
Assuntos
DNA Mitocondrial/genética , DNA Polimerase Dirigida por DNA/fisiologia , Taxa de Mutação , Animais , Linhagem Celular Tumoral , Replicação do DNA , Humanos , Neoplasias/genética , Estresse Oxidativo , DNA Polimerase tetaRESUMO
Three series of isomeric pyrrolo- and furo-fused 7-deazapurine ribonucleosides were synthesized and screened for cytostatic and antiviral activity. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions. The pyrrolo[2',3':4,5]pyrrolo[2,3- d]pyrimidine and furo[2',3':4,5]pyrrolo[2,3- d]pyrimidine ribonucleosides were found to be potent cytostatics, whereas the isomeric pyrrolo[3',2',4,5]pyrrolo[2,3- d]pyrimidine nucleosides were inactive. The most active were the methyl, methoxy, and methylsulfanyl derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to both RNA and DNA, where they cause DNA damage. They represent a new type of promising candidates for preclinical development toward antitumor agents.
Assuntos
Furanos/química , Purinas/química , Pirróis/química , Ribonucleosídeos/síntese química , Ribonucleosídeos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Linhagem Celular Tumoral , Técnicas de Química Sintética , Humanos , Ribonucleosídeos/química , Relação Estrutura-AtividadeRESUMO
In order to compare the abilities of arctic and temperate fish to accumulate PCBs we conduct a metabolic analysis to determine how process rates in a mathematical fish contaminant model change with temperature. We evaluate the model by applying the original and adapted models to estimate PCB concentrations in lake trout (Salvelinus namaycush) in Trout Lake, Ontario, Canada, and in arctic char (Salvelinus alphinus) in Lake Øyangen, in the Norwegian high arctic. Modeled concentrations are, for the most part, within 50% of mean measured values and are comparable to the error associated with the fish data. In order to evaluate differences in fish bioaccumulation processes, the model is applied to hypothetical arctic and temperate systems, assuming the same contaminant input values in water and diet. The model predicts that temperate salmonids are able to biomagnify PCBs 6-60% more than arctic salmonids. For all congeners, the lower BMF(MAX) of arctic fish contribute to their lower concentrations. For congeners with log K(ow) < 6.0, the lower concentrations in arctic fish are also attributed to faster loss due to gill ventilation. Faster growth rates for temperate fish reduce the difference in bioaccumulation for congeners with log K(ow) > 7.0. These processes are controlled by the influence of lipid in the fish and their diet as well as the dependence of growth on temperature. We suggest that fish models originally calibrated for temperate systems may be directly applied to arctic lakes after accounting for the lipid content of the fish and their diet as well as water temperature.
Assuntos
Modelos Teóricos , Bifenilos Policlorados/análise , Truta/metabolismo , Poluentes Químicos da Água/análise , Animais , Chironomidae/química , Clima , Dieta , Monitoramento Ambiental , Fezes/química , Brânquias/metabolismo , Noruega , Ontário , Bifenilos Policlorados/metabolismo , Poluentes Químicos da Água/metabolismo , Zooplâncton/químicaRESUMO
The lesion-specific DNA polymerase POLH gene is mutated in xeroderma pigmentosum variant (XP-V) patients who exhibit an increased skin cancer incidence from UV exposure. Normal cells in which POLH expression was reduced using short interfering RNAs (siRNAs) were compared with the XP-V cellular phenotype that results from naturally occurring inactivating mutations. Stable clones expressing siRNA had partially reduced POLH protein levels, and intermediate levels of UV sensitivity and S phase checkpoint activation, but similar levels of Mre11 foci as in XP-V cells. Therefore, suppression of POLH expression levels by siRNA recapitulates most of the phenotypes seen in cells from XP-V patients with inactivating mutations in POLH.
Assuntos
Inibidores da Síntese de Ácido Nucleico , RNA Interferente Pequeno/genética , Xeroderma Pigmentoso/enzimologia , Xeroderma Pigmentoso/genética , Cafeína/farmacologia , Linhagem Celular Transformada , DNA Polimerase Dirigida por DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Fibroblastos/efeitos da radiação , Humanos , Plasmídeos/genética , Fase S/genética , Fase S/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Transfecção , Raios Ultravioleta/efeitos adversos , Xeroderma Pigmentoso/prevenção & controleRESUMO
Cockayne syndrome (CS) is a severe neurodevelopmental disorder characterized by growth abnormalities, premature aging, and photosensitivity. Mutation of Cockayne syndrome B (CSB) affects neuronal gene expression and differentiation, so we attempted to bypass its function by expressing downstream target genes. Intriguingly, ectopic expression of Synaptotagmin 9 (SYT9), a key component of the machinery controlling neurotrophin release, bypasses the need for CSB in neuritogenesis. Importantly, brain-derived neurotrophic factor (BDNF), a neurotrophin implicated in neuronal differentiation and synaptic modulation, and pharmacological mimics such as 7,8-dihydroxyflavone and amitriptyline can compensate for CSB deficiency in cell models of neuronal differentiation as well. SYT9 and BDNF are downregulated in CS patient brain tissue, further indicating that sub-optimal neurotrophin signaling underlies neurological defects in CS. In addition to shedding light on cellular mechanisms underlying CS and pointing to future avenues for pharmacological intervention, these data suggest an important role for SYT9 in neuronal differentiation.
Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Amitriptilina/farmacologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Síndrome de Cockayne/metabolismo , Síndrome de Cockayne/patologia , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/genética , Regulação para Baixo/efeitos dos fármacos , Flavonas/farmacologia , Humanos , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/metabolismo , Microscopia de Fluorescência , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor trkB , Sinaptotagminas/antagonistas & inibidores , Sinaptotagminas/genética , Sinaptotagminas/metabolismoRESUMO
Developmental pathologies may result from endogenous or xenobiotic-enhanced formation of reactive oxygen species (ROS), which oxidatively damage cellular macromolecules and/or alter signal transduction. This minireview focuses upon several model drugs (phenytoin, thalidomide, methamphetamine), environmental chemicals (benzo[a]pyrene) and gamma irradiation to examine this hypothesis in vivo and in embryo culture using mouse, rat and rabbit models. Embryonic prostaglandin H synthases (PHSs) and lipoxygenases bioactivate xenobiotics to free radical intermediates that initiate ROS formation, resulting in oxidation of proteins, lipids and DNA. Oxidative DNA damage and embryopathies are reduced in PHS knockout mice, and in mice treated with PHS inhibitors, antioxidative enzymes, antioxidants and free radical trapping agents. Thalidomide causes embryonic DNA oxidation in susceptible (rabbit) but not resistant (mouse) species. Embryopathies are increased in mutant mice deficient in the antioxidative enzyme glucose-6-phosphate dehydrogenase (G6PD), or by glutathione (GSH) depletion, or inhibition of GSH peroxidase or GSH reductase. Inducible nitric oxide synthase knockout mice are partially protected. Inhibition of Ras or NF-kB pathways reduces embryopathies, implicating ROS-mediated signal transduction. Atm and p53 knockout mice deficient in DNA damage response/repair are more susceptible to xenobiotic or radiation embryopathies, suggesting a teratological role for DNA damage, consistent with enhanced susceptibility to methamphetamine in ogg1 knockout mice with deficient repair of oxidative DNA damage. Even endogenous embryonic oxidative stress carries a risk, since untreated G6PD- or ATM-deficient mice have increased embryopathies. Thus, embryonic processes regulating the balance of ROS formation, oxidative DNA damage and repair, and ROS-mediated signal transduction may be important determinants of teratological risk.
Assuntos
Exposição Materna , Animais , Feminino , Feto/efeitos dos fármacos , Substâncias Perigosas/toxicidade , Humanos , Gravidez , Zinco/deficiênciaRESUMO
ATM (ataxia-telangiectasia mutated) is a genotoxic stress transducer. In this first report of Atm-dependent birth defects, Atm-null embryos were uniquely susceptible to low-dose (0.5 Gy) radiation, exhibiting severe runting, tail anomalies, and lethality, independent of cell cycle arrest or insulin-like growth factor 1. This treatment enhanced levels of p53 protein and central nervous system (CNS) apoptosis in wild-type mice, but not Atm-null mutants, at 6 h postirradiation. At 48 h, however, this pattern was reversed, with Atm-null mice exhibiting high levels of a hybrid form of programmed cell death within the CNS. Even heterozygous Atm-deficient embryos were radiosensitive to a higher radiation dose of 2 Gy. These results show that Atm is a novel teratologic suppressor gene protecting embryos from pathological cell death and teratogenesis initiated by even mild DNA damage.
Assuntos
Anormalidades Induzidas por Radiação/genética , Apoptose/genética , Desenvolvimento Embrionário e Fetal/efeitos da radiação , Tolerância a Radiação/genética , Anormalidades Induzidas por Radiação/embriologia , Anormalidades Induzidas por Radiação/patologia , Animais , Apoptose/efeitos da radiação , Morte Celular , Divisão Celular/efeitos da radiação , DNA/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Embrião de Mamíferos/patologia , Embrião de Mamíferos/efeitos da radiação , Células Epiteliais/efeitos da radiação , Feminino , Reabsorção do Feto/etiologia , Genes p53/efeitos da radiação , Genótipo , Idade Gestacional , Masculino , Camundongos , Camundongos Knockout , Necrose , Neurônios/efeitos da radiação , Gravidez , Cauda/anormalidades , Telencéfalo/embriologia , Telencéfalo/patologia , Telencéfalo/efeitos da radiaçãoRESUMO
8-Oxo-deoxyguanosine (8-oxo-dG) is a pervasive oxidative DNA lesion formed by endogenous oxidative stress and enhanced by drugs and environmental chemicals. This lesion results in transcriptional errors and mutations and is linked to neurodegeneration, teratogenesis, cancer, and other pathologies. We demonstrate that the neonatal central nervous system of transgenic mice carrying the tetracycline-regulable DNA repair gene formamidopyrimidine DNA glycosylase (fpg) has a 50% reduction in 8-oxo-dG levels. This enhanced DNA repair is suppressed by treatment with doxycycline. For the first time, this murine model permits the level of a specific DNA oxidation product to be regulated in a temporally and spatially specific manner, allowing its role as a primary or secondary factor in neurodegenerative disease to be determined in vivo.
Assuntos
Dano ao DNA , Reparo do DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , N-Glicosil Hidrolases/genética , Estresse Oxidativo , Tetraciclina/farmacologia , 8-Hidroxi-2'-Desoxiguanosina , Actinas/genética , Animais , Células CHO , Linhagem Celular , Córtex Cerebral/metabolismo , Cricetinae , DNA-Formamidopirimidina Glicosilase , Doxiciclina/farmacologia , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , N-Glicosil Hidrolases/metabolismo , Regiões Promotoras Genéticas , Elementos de RespostaRESUMO
Acute myeloid leukemia (AML) cells have high oxidative phosphorylation and mitochondrial mass and low respiratory chain spare reserve capacity. We reasoned that targeting the mitochondrial RNA polymerase (POLRMT), which indirectly controls oxidative phosphorylation, represents a therapeutic strategy for AML. POLRMT-knockdown OCI-AML2 cells exhibited decreased mitochondrial gene expression, decreased levels of assembled complex I, decreased levels of mitochondrially-encoded Cox-II and decreased oxidative phosphorylation. POLRMT-knockdown cells exhibited an increase in complex II of the electron transport chain, a complex comprised entirely of subunits encoded by nuclear genes, and POLRMT-knockdown cells were resistant to a complex II inhibitor theonyltrifluoroacetone. POLRMT-knockdown cells showed a prominent increase in cell death. Treatment of OCI-AML2 cells with 10-50 µM 2-C-methyladenosine (2-CM), a chain terminator of mitochondrial transcription, reduced mitochondrial gene expression and oxidative phosphorylation, and increased cell death in a concentration-dependent manner. Treatment of normal human hematopoietic cells with 2-CM at concentrations of up to 100 µMdid not alter clonogenic growth, suggesting a therapeutic window. In an OCI-AML2 xenograft model, treatment with 2-CM (70 mg/kg, i.p., daily) decreased the volume and mass of tumours to half that of vehicle controls. 2-CM did not cause toxicity to major organs. Overall, our results in a preclinical model contribute to the functional validation of the utility of targeting the mitochondrial RNA polymerase as a therapeutic strategy for AML.
Assuntos
Adenosina/análogos & derivados , Antineoplásicos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Adenosina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Relação Dose-Resposta a Droga , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos SCID , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Terapia de Alvo Molecular , Fosforilação Oxidativa , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Carga Tumoral/efeitos dos fármacos , Células U937 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neurodevelopmental defects are observed in the hereditary disorder Cockayne syndrome (CS). The gene most frequently mutated in CS, Cockayne Syndrome B (CSB), is required for the repair of bulky DNA adducts in transcribed genes during transcription-coupled nucleotide excision repair. CSB also plays a role in chromatin remodeling and mitochondrial function. The role of CSB in neural development is poorly understood. Here we report that the abundance of neural progenitors is normal in Csb(-/-) mice and the frequency of apoptotic cells in the neurogenic niche of the adult subependymal zone is similar in Csb(-/-) and wild type mice. Both embryonic and adult Csb(-/-) neural precursors exhibited defective self-renewal in the neurosphere assay. In Csb(-/-) neural precursors, self-renewal progressively decreased in serially passaged neurospheres. The data also indicate that Csb and the nucleotide excision repair protein Xpa preserve embryonic neural stem cell self-renewal after UV DNA damage. Although Csb(-/-) neural precursors do not exhibit altered neuronal lineage commitment after low-dose UV (1J/m(2)) in vitro, neurons differentiated in vitro from Csb(-/-) neural precursors that had been irradiated with 1J/m(2) UV exhibited defective neurite outgrowth. These findings identify a function for Csb in neural precursors.
Assuntos
Enzimas Reparadoras do DNA/genética , Células-Tronco Neurais/citologia , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Proliferação de Células , Dano ao DNA , Epêndima/citologia , Camundongos , Camundongos Knockout , Células-Tronco Neurais/efeitos da radiação , Neurogênese/genética , Neurogênese/efeitos da radiação , Proteínas de Ligação a Poli-ADP-Ribose , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/genéticaRESUMO
An analog of the anticancer drug cisplatin (mtPt) was delivered to mitochondria of human cells using a peptide specifically targeting this organelle. mtPt induces apoptosis without damaging nuclear DNA, indicating that mtDNA damage is sufficient to mediate the activity of a platinum-based chemotherapeutic. This study demonstrates the specific delivery of a platinum drug to mitochondria and investigates the effects of directing this agent outside the nucleus.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , DNA Mitocondrial/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Antineoplásicos/química , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/química , Relação Dose-Resposta a Droga , Feminino , Humanos , Conformação Molecular , Neoplasias Ovarianas/patologia , Relação Estrutura-AtividadeRESUMO
Recent studies have demonstrated a role for telomerase in driving tumor progression, but its mechanism of action remains unclear. Here we show that stable, ribozyme-mediated suppression of mouse telomerase RNA reduced telomerase RNA expression, telomerase activity, and telomere length, which significantly reduced tumor invasion and metastatic potential. Our studies reveal that previously unidentified effects of telomerase may mediate its tumor-promoting effects. First, reducing telomerase activity induced a more dendritic morphology, accompanied by increased melanin content and increased expression of tyrosinase, a key enzyme in melanin biosynthesis. Second, gene expression profiling revealed that telomerase targeting down-regulated expression of several glycolytic pathway genes, with a corresponding decrease in glucose consumption and lactate production. Thus, telomerase activity controls the glycolytic pathway, potentially altering the energy state of tumor cells and thereby modulating tyrosinase activity and melanin production. These studies have important implications for understanding the mechanisms by which telomerase promotes tumor invasion and metastasis.