RESUMO
Fanconi anemia (FA) is a disease caused by defective deoxyribonucleic acid (DNA) repair that manifests as bone marrow failure, cancer predisposition, and developmental defects. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wildtype controls with either MET alone, OXM alone, MET+OXM, or placebo diet from age 3 weeks to 18 months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p = .01) and hemoglobin levels (p < .05). In addition, the percentage of quiescent hematopoietic stem cell (HSC) (LSK [Lin-Sca+c-Kit+]) was significantly increased (p = .001) by long-term treatment with MET alone. The combination of metformin and oxymetholone did not result in a significant synergistic effect in any hematopoietic parameter. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration. We conclude that androgen therapy is not a contraindication to concurrent metformin administration in clinical trials. HIGHLIGHTS: Long-term coadministration of metformin in combination with oxymetholone is well tolerated by Fancd2-/- mice. Hematopoietic stem cell quiescence in mutant mice was enhanced by treatment with metformin alone. Metformin treatment caused a partial normalization of gene expression in the livers of mutant mice.
Assuntos
Modelos Animais de Doenças , Quimioterapia Combinada , Anemia de Fanconi , Metformina , Oximetolona , Animais , Metformina/farmacologia , Metformina/administração & dosagem , Camundongos , Anemia de Fanconi/tratamento farmacológico , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Camundongos Knockout , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismoRESUMO
Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fanconi Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis.
Assuntos
Antraciclinas/farmacologia , Antibacterianos/farmacologia , Reparo do DNA/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Peritonite/tratamento farmacológico , Sepse/prevenção & controle , Infecções por Adenoviridae/imunologia , Animais , Antraciclinas/uso terapêutico , Antibacterianos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Proteína 7 Relacionada à Autofagia , Ceco/lesões , Dano ao DNA , Epirubicina/administração & dosagem , Epirubicina/farmacologia , Epirubicina/uso terapêutico , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Inflamação , Mediadores da Inflamação/análise , Injeções Intraperitoneais , Pulmão/metabolismo , Meropeném , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/fisiologia , Especificidade de Órgãos , Peritonite/etiologia , Peritonite/genética , Peritonite/imunologia , Peritonite/fisiopatologia , Infecções Respiratórias/imunologia , Choque Séptico/prevenção & controle , Tienamicinas/uso terapêutico , Irradiação Corporal TotalRESUMO
BACKGROUND: Fanconi anemia is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Currently, no interventions to prevent or delay the formation of solid tumors are available. PROCEDURE: Two of the most important hallmarks of Fanconi anemia are inflammation and oxidative stress. In this study, we administrated the antioxidant atorvastatin and the anti-inflammatory drug celecoxib to cohorts of Fancd2-/- /Trp53+/- mice, a model of Fanconi anemia. Treatment started at weaning and continued until the mice developed a palpable mass or suffered from >20% weight loss. Tumor samples and selected tissues were subjected to histopathological examination. χ2 test was performed to analyze tumor incidence, and Kaplan-Meier survival curves were evaluated with log-rank test. In addition, a small cohort of mice was monitored for the safety of the drugs. RESULTS: The combined oral administration of both drugs significantly delayed tumor onset in Fancd2-/- /Trp53+/- mice. Specifically, the treatment delayed the onset of ovarian tumors in Fancd2-/- /Trp53+/- mice and increased the mean ovarian tumor-free survival time by 17%, whereas this combinatorial drug regimen did not have a significant effect on other tumor types. In addition, no detrimental effects on hematopoiesis from the drug treatment were observed during a 12-month safety monitoring. CONCLUSIONS: The data presented here suggest that a combination of atorvastatin and celecoxib may be a good candidate for chemoprevention in Fanconi anemia.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Modelos Animais de Doenças , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Anemia de Fanconi/tratamento farmacológico , Proteína Supressora de Tumor p53/fisiologia , Animais , Atorvastatina/administração & dosagem , Celecoxib/administração & dosagem , Anemia de Fanconi/patologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Taxa de SobrevidaRESUMO
Fanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2-/- mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2-/- mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2-/-Trp53+/- mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient-derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells.
Assuntos
Carcinogênese/patologia , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/patologia , Hematopoese/efeitos dos fármacos , Metformina/farmacologia , Aldeídos/metabolismo , Animais , Contagem de Células Sanguíneas , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Carcinogênese/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Quebra Cromossômica , Dano ao DNA , Dieta , Anemia de Fanconi/sangue , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Guanidinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Inativação Metabólica/efeitos dos fármacos , Metformina/administração & dosagem , Camundongos , Poli I-C/farmacologiaRESUMO
Stem cell therapy is the administration of stem cells to a patient to treat or prevent a disease. Since stem cells possess the long-term self-renewal capacity and provide daughter cells that differentiate into the specialized cells of each tissue, stem cell therapy will theoretically improve the disease condition for the lifetime of the patient. As the most widely used stem cell therapy, bone marrow transplantation is the treatment of choice for many kinds of blood disorders, including anemias, leukemias, lymphomas, and rare immunodeficiency diseases. For the fatal genetic blood disorder Fanconi anemia, allogeneic bone marrow transplantation has remained the only curative treatment. But the recent advances in stem cell and gene therapy fields may provide promising opportunities for an alternative or even better management of Fanconi anemia. Many of these new ideas and opportunities are also useful for treating other blood diseases that affect hematopoietic stem cells, such as sickle cell anemia, severe combined immunodeficiencies, and beta-thalassemias. In this chapter, these advances along with their challenges and limitations will be thoroughly discussed.
Assuntos
Transplante de Medula Óssea , Anemia de Fanconi/terapia , Transplante de Células-Tronco Hematopoéticas , Terapia Genética , Células-Tronco Hematopoéticas , HumanosRESUMO
Fanconi anemia (FA) patients suffer from progressive bone marrow failure and often develop cancers. Previous studies showed that antioxidants tempol and resveratrol (RV) delayed tumor onset and reduced hematologic defects in FA murine models, respectively. Here we tested whether antioxidants N-acetylcysteine (NAC) or RV could delay cancer in tumor prone Fancd2(-/-) /Trp53(+/-) mice. Unlike tempol, neither compound had any significant chemopreventive effect in this model. We conclude that not all anti-oxidants are chemopreventive in FA. In addition, when given to Fancd2(-/-) mice, NAC helped maintain Fancd2(-/-) KSL cells in quiescence while tempol did not. The mechanisms behind the different actions of these antioxidants await further investigation.
Assuntos
Acetilcisteína/uso terapêutico , Antioxidantes/uso terapêutico , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Anemia de Fanconi/prevenção & controle , Sequestradores de Radicais Livres/uso terapêutico , Estilbenos/uso terapêutico , Proteína Supressora de Tumor p53/fisiologia , Animais , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Citometria de Fluxo , Camundongos , Camundongos Knockout , ResveratrolRESUMO
Fanconi Anemia (FA) is a disease caused by defective DNA repair which manifests as bone marrow failure, cancer predisposition, and developmental defects. Mice containing inactivating mutations in one or more genes in the FA pathway partially mimic the human disease. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow (BM) hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wild-type controls with either MET alone, OXM alone, MET+OXM or placebo diet. Both male and female mice were treated from age 3 weeks to 18 months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p=0.01) and hemoglobin levels (p<0.05). In addition, the percentage of quiescent HSC (LSK) was significantly increased (p=0.001) by long-term treatment with MET alone. However, the absolute number of progenitors, measured by LSK frequency or CFU-S, was not significantly altered by MET therapy. The combination of metformin and oxymetholone did not result in a significant synergistic effect on any parameter. Male animals on MET+OXM or MET alone were significantly leaner than controls at 18 months, regardless of genotype. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration.
RESUMO
Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2(-/-) mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit(+)Sca-1(+)Lineage(-) (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2(-/-) KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2(-/-) mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2(-/-) KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2(-/-) bone marrow cells. We conclude that Fancd2(-/-) mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies.
Assuntos
Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Anemia de Fanconi/tratamento farmacológico , Sistema Hematopoético/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Antioxidantes , Medula Óssea/efeitos dos fármacos , Ciclo Celular , Linhagem da Célula , Ensaio de Unidades Formadoras de Colônias , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Resveratrol , Baço/citologia , Estilbenos/uso terapêutico , Resultado do TratamentoRESUMO
The thermal degradation kinetics and degradation products of IPBC during the heating process are investigated herein. Experiments were conducted at isothermal conditions from 60 °C to 150 °C. The remaining IPBC content was analyzed by high-performance liquid chromatography (HPLC) at specific time intervals for each test, and the kinetic model of IPBC thermal degradation was established. The thermal degradation products of IPBC were studied by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS). The results showed that thermal degradation of IPBC occurred at 70 °C, and the degradation rate increased significantly from 70 °C to 150 °C. The thermal degradation kinetics of IPBC conformed to the first-order reaction and k=3.47×1012e-111125/RT from 60 °C to 150 °C. Seven degradation products such as prop-2-yn-1-yl ethylcarbamate and methyl N-butylcarbamate were identified and the degradation reaction pathway and the mechanism of IPBC were proposed, which involved deiodination, demethylation, deethynylation, deethylation, and hydroxylation processes.
RESUMO
The efficiency of gene repair by homologous recombination in the liver is enhanced by CRISP/Cas9 incision near the mutation. In this study, we explored interventions designed to further enhance in vivo hepatocyte gene repair in a model of hereditary tyrosinemia. A two-AAV system was employed: one virus carried a Staphylococcus pyogenes Cas9 (SpCas9) expression cassette and the other harbored a U6 promoter-driven sgRNA and a fragment of fumarylacetoacetate hydrolase (Fah) genomic DNA as the homologous recombination donor. In neonatal mice, a gene correction frequency of â¼10.8% of hepatocytes was achieved. The efficiency in adult mice was significantly lower at â¼1.6%. To determine whether hepatocyte replication could enhance the targeting frequency, cell division was induced with thyroid hormone T3. This more than doubled the gene correction efficiency to 3.5% (p < 0.005). To determine whether SpCas9 delivery was rate limiting, the gene repair AAV was administered to SpCas9 transgenic mice. However, this did not significantly enhance gene repair. Finally, we tested whether the Fanconi anemia (FA) DNA repair pathway was important in hepatocyte gene repair. Gene correction frequencies were significantly lower in neonatal mice lacking the FA complementation group A (Fanca) gene. Taken together, we conclude that pharmacological induction of hepatocyte replication along with manipulation of DNA repair pathways could be a useful strategy for enhancing in vivo gene correction.
Assuntos
Tirosinemias , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes , Terapia Genética , Hepatócitos , Regeneração Hepática , Camundongos , Tirosinemias/genética , Tirosinemias/terapiaRESUMO
Drosophila Pumilio (Pum) protein is a translational regulator involved in embryonic patterning and germline development. Recent findings demonstrate that Pum also plays an important role in the nervous system, both at the neuromuscular junction (NMJ) and in long-term memory formation. In neurons, Pum appears to play a role in homeostatic control of excitability via down regulation of para, a voltage gated sodium channel, and may more generally modulate local protein synthesis in neurons via translational repression of eIF-4E. Aside from these, the biologically relevant targets of Pum in the nervous system remain largely unknown. We hypothesized that Pum might play a role in regulating the local translation underlying synapse-specific modifications during memory formation. To identify relevant translational targets, we used an informatics approach to predict Pum targets among mRNAs whose products have synaptic localization. We then used both in vitro binding and two in vivo assays to functionally confirm the fidelity of this informatics screening method. We find that Pum strongly and specifically binds to RNA sequences in the 3'UTR of four of the predicted target genes, demonstrating the validity of our method. We then demonstrate that one of these predicted target sequences, in the 3'UTR of discs large (dlg1), the Drosophila PSD95 ortholog, can functionally substitute for a canonical NRE (Nanos response element) in vivo in a heterologous functional assay. Finally, we show that the endogenous dlg1 mRNA can be regulated by Pumilio in a neuronal context, the adult mushroom bodies (MB), which is an anatomical site of memory storage.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Neurônios/química , Neurônios/metabolismo , Análise de Sequência de Proteína/métodos , Sinapses/química , Sinapses/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Dados de Sequência Molecular , Ligação Proteica , Proteínas de Ligação a RNARESUMO
Fanconi anemia is a genetic bone marrow failure syndrome. The current treatment options are suboptimal and do not prevent the eventual onset of aplastic anemia requiring bone marrow transplantation. We previously showed that resveratrol, an antioxidant and an activator of the protein deacetylase Sirt1, enhanced hematopoiesis in Fancd2 mutant mice and improved the impaired stem cell quiescence observed in this disease. Given that Sirt1 is important for the function of hematopoietic stem cells, we hypothesized that Sirt1 activation may improve hematopoiesis. Indeed, Fancd2(-/-) mice and wild-type mice treated with the selective Sirt1 activator SRT3025 had increased numbers of hematopoietic stem and progenitor cells, platelets and white blood cells. SRT3025 was also protective against acetaldehyde-induced hematopoietic damage. Unlike resveratrol, however, SRT3025 did not affect stem cell quiescence, suggesting distinct mechanisms of action. Conditional deletion of Sirt1 in hematopoietic cells did not abrogate the beneficial effects of SRT3025, indicating that the drug did not act by directly stimulating Sirt1 in stem cells, but must be acting indirectly via extra-hematopoietic effects. RNA-Seq transcriptome analysis revealed the down-regulation of Egr1-p21 expression, providing a potential mechanism for improved hematopoiesis. Overall, our data indicate that SRT3025 or related compounds may be beneficial in Fanconi anemia and other bone marrow failure syndromes.
Assuntos
Anilidas/farmacologia , Anemia de Fanconi/patologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Sirtuína 1/metabolismo , Tiazóis/farmacologia , Acetaldeído , Anilidas/administração & dosagem , Animais , Contagem de Células Sanguíneas , Proliferação de Células/efeitos dos fármacos , Dieta , Etanol/farmacologia , Anemia de Fanconi/sangue , Deleção de Genes , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Camundongos Transgênicos , Análise de Sequência de RNA , Tiazóis/administração & dosagem , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Androgens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2(-/-) mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2(-/-) mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug's action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure.
Assuntos
Ciclo Celular/efeitos dos fármacos , Anemia de Fanconi/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Osteopontina/genética , Oximetolona/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Contagem de Células Sanguíneas , Medula Óssea/patologia , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Regulação da Expressão Gênica , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Humanos , Camundongos , Camundongos Knockout , Oximetolona/uso terapêutico , Pancitopenia/sangue , Pancitopenia/genética , Pancitopenia/patologia , Análise de Sequência de RNA , Fatores de TempoRESUMO
The 19F nuclear magnetic resonance (NMR) spectra of 4-fluorotryptophan (4-F-Trp)-labeled Escherichia coli arginyl-tRNA synthetase (ArgRS) show that there are distinct conformational changes in the catalytic core and tRNA anticodon stem and loop-binding domain of the enzyme, when arginine and tRNA(Arg) are added to the unliganded enzyme. We have assigned five fluorine resonances of 4-F-Trp residues (162, 172, 228, 349 and 446) in the spectrum of the fluorinated enzyme by site-directed mutagenesis. The local conformational changes of E. coli ArgRS induced by its substrates observed herein by 19F NMR are similar to those of crystalline yeast homologous enzyme.
Assuntos
Arginina-tRNA Ligase/química , Arginina-tRNA Ligase/metabolismo , Escherichia coli/enzimologia , Triptofano/análogos & derivados , Anticódon/genética , Arginina/metabolismo , Sítios de Ligação , Domínio Catalítico , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , RNA de Transferência de Arginina/metabolismo , Especificidade por SubstratoRESUMO
Fanconi anemia patients suffer from progressive bone marrow failure. An overactive p53 response to DNA damage contributes to the progressive elimination of Fanconi anemia hematopoietic stem and progenitor cells (HSPC), and hence presents a potential target for therapeutic intervention. To investigate whether the cell cycle regulatory protein p21 is the primary mediator of the p53-dependent stem cell loss, p21/Fancd2 double-knockout mice were generated. Surprisingly double mutant mice displayed even more severe loss of HSPCs than Fancd2(-/-) single mutants. p21 deletion did not rescue the abnormal cell cycle profile and had no impact on the long-term repopulating potential of Fancd2(-/-) bone marrow cells. Collectively, our data indicate that p21 has an indispensable role in maintaining a normal HSPC pool and suggest that other p53-targeted factors, not p21, mediate the progressive elimination of HSPC in Fanconi anemia.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco/citologia , Animais , Tamanho Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco/metabolismoRESUMO
Fanconi anemia (FA) is an inherited DNA repair deficiency syndrome. FA patients undergo progressive bone marrow failure (BMF) during childhood, which frequently requires allogeneic hematopoietic stem cell transplantation. The pathogenesis of this BMF has been elusive to date. Here we found that FA patients exhibit a profound defect in hematopoietic stem and progenitor cells (HSPCs) that is present before the onset of clinical BMF. In response to replicative stress and unresolved DNA damage, p53 is hyperactivated in FA cells and triggers a late p21(Cdkn1a)-dependent G0/G1 cell-cycle arrest. Knockdown of p53 rescued the HSPC defects observed in several in vitro and in vivo models, including human FA or FA-like cells. Taken together, our results identify an exacerbated p53/p21 "physiological" response to cellular stress and DNA damage accumulation as a central mechanism for progressive HSPC elimination in FA patients, and have implications for clinical care.
Assuntos
Medula Óssea/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Células-Tronco Hematopoéticas/patologia , Proteína Supressora de Tumor p53/metabolismo , Adolescente , Adulto , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/patologia , Envelhecimento/patologia , Animais , Medula Óssea/metabolismo , Criança , Pré-Escolar , Modelos Animais de Doenças , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/patologia , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Pontos de Checagem da Fase G2 do Ciclo Celular , Técnicas de Silenciamento de Genes , Inativação Gênica , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Camundongos , Pessoa de Meia-Idade , Fase SRESUMO
BACKGROUND: Retinitis pigmentosa (RP) is characterized by progressive night blindness, visual field loss, altered vascular permeability and loss of central vision. Currently there is no effective treatment available except gene replacement therapy has shown promise in a few patients with specific gene defects. There is an urgent need to develop therapies that offer generic neuro-and vascular-protective effects with non-invasive intervention. Here we explored the potential of systemic administration of pluripotent bone marrow-derived mesenchymal stem cells (MSCs) to rescue vision and associated vascular pathology in the Royal College Surgeons (RCS) rat, a well-established animal model for RP. METHODOLOGY/PRINCIPAL FINDINGS: Animals received syngeneic MSCs (1x10(6) cells) by tail vein at an age before major photoreceptor loss. PRINCIPAL RESULTS: both rod and cone photoreceptors were preserved (5-6 cells thick) at the time when control animal has a single layer of photoreceptors remained; Visual function was significantly preserved compared with controls as determined by visual acuity and luminance threshold recording from the superior colliculus; The number of pathological vascular complexes (abnormal vessels associated with migrating pigment epithelium cells) and area of vascular leakage that would ordinarily develop were dramatically reduced; Semi-quantitative RT-PCR analysis indicated there was upregulation of growth factors and immunohistochemistry revealed that there was an increase in neurotrophic factors within eyes of animals that received MSCs. CONCLUSIONS/SIGNIFICANCE: These results underscore the potential application of MSCs in treating retinal degeneration. The advantages of this non-invasive cell-based therapy are: cells are easily isolated and can be expanded in large quantity for autologous graft; hypoimmunogenic nature as allogeneic donors; less controversial in nature than other stem cells; can be readministered with minor discomfort. Therefore, MSCs may prove to be the ideal cell source for auto-cell therapy for retinal degeneration and other ocular vascular diseases.
Assuntos
Modelos Animais de Doenças , Transplante de Células-Tronco Mesenquimais/métodos , Degeneração Retiniana/cirurgia , Doenças Vasculares/cirurgia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Fator Neurotrófico Ciliar/genética , Fator Neurotrófico Ciliar/metabolismo , Expressão Gênica , Humanos , Imuno-Histoquímica , Ratos , Retina/metabolismo , Retina/patologia , Degeneração Retiniana/complicações , Retinose Pigmentar/patologia , Retinose Pigmentar/cirurgia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resultado do Tratamento , Doenças Vasculares/complicações , Acuidade VisualRESUMO
Fanconi anemia (FA) is a genetic disorder characterized by congenital abnormalities, bone marrow failure, and marked cancer susceptibility. FA patients have an elevated risk of developing hematologic malignancies and solid tumors. Using Fancd2(-/-) knockout mice as a model of FA, we examined the potential of tempol, a nitroxide antioxidant and a superoxide dismutase mimetic, as a tumor-delaying agent for solid tumors. Dietary tempol increased the mean tumor-free survival time of Fancd2(-/-) Trp53(+/-) mice by 27% (P < 0.01), from 308 to 390 days, without changing the overall tumor spectrum. More strikingly, tempol delayed the onset of epithelial tumors and increased the mean epithelial tumor-free survival time by 38% (P < 0.0001), from 312 to 432 days, in Fancd2(-/-) Trp53(+/-) mice. These results show that tempol can significantly delay tumor formation in Fancd2(-/-) Trp53(+/-) mice. Furthermore, tempol treatment did not adversely affect the repopulating ability of FA hematopoietic stem cells. The reduction in oxidative DNA damage in tempol-treated FA fibroblasts and mice suggests that its tumor-delaying function may be attributed to its antioxidant activity.
Assuntos
Antioxidantes/farmacologia , Óxidos N-Cíclicos/farmacologia , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/prevenção & controle , Animais , Antioxidantes/metabolismo , Dano ao DNA , Modelos Animais de Doenças , Intervalo Livre de Doença , Feminino , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Estresse Oxidativo , Oxigênio/metabolismo , Marcadores de Spin , Superóxido Dismutase/metabolismoRESUMO
Telomerase is a ribonucleoprotein enzyme complex that reverse-transcribes an integral RNA template to add short DNA repeats to the 3'-ends of telomeres. G-quadruplex structure in a DNA substrate can block its extension by telomerase. We have found that hnRNP A1--which was previously implicated in telomere length regulation--binds to both single-stranded and structured human telomeric repeats, and in the latter case, it disrupts their higher-order structure. Using an in vitro telomerase assay, we observed that depletion of hnRNP A/B proteins from 293 human embryonic kidney cell extracts dramatically reduced telomerase activity, which was fully recovered upon addition of purified recombinant hnRNP A1. This finding suggests that hnRNP A1 functions as an auxiliary, if not essential, factor of telomerase holoenzyme. We further show, using chromatin immunoprecipitation, that hnRNP A1 associates with human telomeres in vivo. We propose that hnRNP A1 stimulates telomere elongation through unwinding of a G-quadruplex or G-G hairpin structure formed at each translocation step.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Sequência de Bases , Células Cultivadas , Imunoprecipitação da Cromatina , DNA/química , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Ligação ProteicaRESUMO
Previous investigations show that tRNA(Arg)-induced conformational changes of arginyl-tRNA synthetase (ArgRS) Omega-loop region (Escherichia coli (E. coli), Ala451-Ala457) may contribute to the productive conformation of the enzyme catalytic core, and E. coli tRNA(2)(Arg)(ICG)-bound and -free conformations of the Omega-loop exchange at an intermediate rate on NMR timescale. Herein, we report that E. coli ArgRS catalyzes tRNA(2)(Arg)(ICG) and tRNA(4)(Arg)(UCU) with similar efficiencies. However, 19F NMR spectroscopy of 4-fluorotryptophan-labeled E. coli ArgRS reveals that the tRNA(4)(Arg)(UCU)-bound and -free conformations of the Omega-loop region interconvert very slowly and the lifetime of bound conformation is much longer than 0.33 ms. Therefore, tRNA(4)(Arg)(UCU) differs from tRNA(2)(Arg)(ICG) in the conformation-exchanging rate of the Omega-loop. Comparative structure model of E. coli ArgRS is presented to rationalize these 19F NMR data. Our 19F NMR and catalytic assay results suggest that the tRNA(Arg)-induced conformational changes of Omega-loop little contribute to the productive conformation of ArgRS catalytic core.