RESUMO
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
Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinase-expressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.
Assuntos
Técnicas de Cultura de Células/métodos , Proteínas de Ligação a DNA/genética , Hepatócitos/citologia , Hepatócitos/transplante , Hidrolases/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Engenharia Tecidual/métodos , Animais , Humanos , Camundongos , Camundongos KnockoutRESUMO
PURPOSE: Fanconi anemia is a genetically heterogeneous chromosomal breakage disorder exhibiting a high degree of clinical variability. Clinical diagnoses are confirmed by testing patient cells for increased sensitivity to crosslinking agents. Fanconi anemia complementation group assignment, essential for efficient molecular diagnosis of the disease, had not been validated for clinical application before this study. The purpose of this study was (1) confirmation of the accuracy of Fanconi anemia complementation group assignment to Group A (FANCA) and (2) development of a rapid mutation detection strategy that ensures the efficient capture of all FANCA mutations. METHODS: Using fibroblasts from 29 patients, diagnosis of Fanconi anemia and assignment to complementation Group A was made through breakage analysis studies. FANCA coding and flanking sequences were analyzed using denaturing high pressure liquid chromatography, sequencing, and multiplex ligation-dependent probe amplification. Patients in which two mutations were not identified were analyzed by cDNA sequencing. Patients with no mutations were sequenced for mutations in FANCC, G, E, and F. RESULTS: Of the 56 putative mutant alleles studied, 89% had an identifiable FANCA pathogenic mutation. Eight unique novel mutations were identified. CONCLUSION: Complementation assignment to Group A was validated in a clinical laboratory setting using our FANCA rapid molecular testing strategy.
Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Mutação , Adolescente , Linhagem Celular , Criança , Análise Citogenética , Análise Mutacional de DNA/métodos , Anemia de Fanconi/classificação , Anemia de Fanconi/diagnóstico , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
The Fanconi anemia proteins participate in a canonical pathway that repairs cross-linking agent-induced DNA damage. Cells with inactivated Fanconi anemia genes are universally hypersensitive to such agents. Fanconi anemia-deficient hematopoietic stem cells are also hypersensitive to inflammatory cytokines, and, as importantly, Fanconi anemia macrophages overproduce such cytokines in response to TLR4 and TLR7/8 agonists. We questioned whether TLR-induced DNA damage is the primary cause of aberrantly regulated cytokine production in Fanconi anemia macrophages by quantifying TLR agonist-induced TNF-α production, DNA strand breaks, crosslinker-induced chromosomal breakage, and Fanconi anemia core complex function in Fanconi anemia complementation group C-deficient human and murine macrophages. Although both M1 and M2 polarized Fanconi anemia cells were predictably hypersensitive to mitomycin C, only M1 macrophages overproduced TNF-α in response to TLR-activating signals. DNA damaging agents alone did not induce TNF-α production in the absence of TLR agonists in wild-type or Fanconi anemia macrophages, and mitomycin C did not enhance TLR responses in either normal or Fanconi anemia cells. TLR4 and TLR7/8 activation induced cytokine overproduction in Fanconi anemia macrophages. Also, although TLR4 activation was associated with induced double strand breaks, TLR7/8 activation was not. That DNA strand breaks and chromosome breaks are neither necessary nor sufficient to account for the overproduction of inflammatory cytokines by Fanconi anemia cells suggests that noncanonical anti-inflammatory functions of Fanconi anemia complementation group C contribute to the aberrant macrophage phenotype and suggests that suppression of macrophage/TLR hyperreactivity might prevent cytokine-induced stem cell attrition in Fanconi anemia.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Anemia de Fanconi/imunologia , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Polaridade Celular , Células Cultivadas , Dano ao DNA , Proteína do Grupo de Complementação C da Anemia de Fanconi/fisiologia , Histonas/análise , Humanos , Imidazóis/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Mitomicina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Toll-Like/fisiologiaRESUMO
The disease Fanconi anemia is a genome instability syndrome characterized by cellular sensitivity to DNA interstrand cross-linking agents, manifest by decreased cellular survival and chromosomal aberrations after such treatment. There are at least 13 proteins acting in the pathway, with the FANCD2 protein apparently functioning as a late term effecter in the maintenance of genome stability. We find that the chromatin remodeling protein, Tip60, interacts directly with the FANCD2 protein in a yeast two-hybrid system. This interaction has been confirmed by co-immunoprecipitation and co-localization using both endogenous and epitope-tagged FANCD2 and Tip60 from human cells. The observation of decreased cellular survival after exposure to mitomycin C in normal fibroblasts depleted for Tip60 indicates a direct function in interstrand cross-link repair. The coincident function of Tip60 and FANCD2 in one pathway is supported by the finding that depletion of Tip60 in Fanconi anemia cells does not increase sensitivity to DNA cross-links. However, depletion of Tip60 did not reduce monoubiquitination of FANCD2 or its localization to nuclear foci following DNA damage. The observations indicate that Fanconi anemia proteins act in concert with chromatin remodeling functions to maintain genome stability after DNA cross-link damage.
Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Reparo do DNA/fisiologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Histona Acetiltransferases/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Linhagem Celular , Núcleo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Histona Acetiltransferases/genética , Humanos , Lisina Acetiltransferase 5 , Mitomicina/farmacologia , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/fisiologiaRESUMO
Repopulation by transplanted cells can result in effective therapy for several regenerative organs including blood, liver, and skin. In contrast, cell therapies for renal diseases are not currently available. Here we developed an animal model in which cells genetically resistant to a toxic intermediate of tyrosine metabolism, homogentisic acid (HGA), were able to repopulate the damaged proximal tubule epithelium of mice with fumarylacetoacetate hydrolase (Fah) deficiency. HGA resistance was achieved by two independent mechanisms. First, Fah+ transplanted bone marrow cells produced significant replacement of damaged proximal tubular epithelium (up to 50%). The majority of bone marrow-derived epithelial cells were generated by cell fusion, not transdifferentiation. In addition to regeneration by fusion-derived epithelial cells, proximal tubular repopulation was also observed by host epithelial cells, which had lost the homogentisic acid dioxygenase gene. These data demonstrate that extensive regeneration of the renal proximal tubule compartment can be achieved through genetic selection of functional cells.
Assuntos
Transplante de Medula Óssea , Modelos Animais de Doenças , Ácido Homogentísico/metabolismo , Hidrolases/metabolismo , Nefropatias/patologia , Túbulos Renais Proximais/patologia , Doença Aguda , Animais , Diferenciação Celular , Fusão Celular , Doença Crônica , Homogentisato 1,2-Dioxigenase/genética , Hidrolases/genética , Nefropatias/enzimologia , Nefropatias/fisiopatologia , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Necrose , Regeneração , Tirosina/metabolismo , Urotélio/enzimologia , Urotélio/patologiaRESUMO
Phenylalanine homeostasis in mammals is primarily controlled by liver phenylalanine hydroxylase (PAH) activity. Inherited PAH deficiency (phenylketonuria or PKU) leads to hyperphenylalaninemia in both mice and humans. A low level of residual liver PAH activity ensures near-normal dietary protein tolerance with normal serum phenylalanine level, but the precise threshold for normal phenylalanine clearance is unknown. We employed hepatocyte transplantation under selective growth conditions to investigate the minimal number of PAH-expressing hepatocytes necessary to prevent hyperphenylalaninemia in mice. Serum phenylalanine levels remained normal in mice exhibiting nearly complete liver repopulation with PAH-deficient hepatocytes (<5% residual wild-type liver PAH activity). Conversely, transplantation of PAH-positive hepatocytes into PAH-deficient Pah(enu2) mice, a model of human PKU, yielded a significant decrease in serum phenylalanine (<700 muM) when liver repopulation exceeded approximately 5%. These data suggest that restoration of phenylalanine homeostasis requires PAH activity in only a minority of hepatocytes.
Assuntos
Hepatócitos/transplante , Fenilalanina Hidroxilase/metabolismo , Fenilalanina/sangue , Fenilcetonúrias/metabolismo , Animais , Modelos Animais de Doenças , Hepatócitos/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Mutantes , Fenilalanina Hidroxilase/deficiência , Fenilcetonúrias/prevenção & controleRESUMO
The appearance of bipotential oval cells in chronic liver injury suggests the existence of hepatocyte progenitor/stem cells. To study the origin and properties of this cell population, oval cell proliferation was induced in adult mouse liver by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and a method for their isolation was developed. Transplantation into fumarylacetoacetate hydrolase (Fah) deficient mice was used to determine their capacity for liver repopulation. In competitive repopulation experiments, hepatic oval cells were at least as efficient as mature hepatocytes in repopulating the liver. In mice with chimeric livers, the oval cells were not derived from hepatocytes but from liver nonparenchymal cells. This finding supports a model in which intrahepatic progenitors differentiate into hepatocytes irreversibly. To determine whether oval cells originated from stem cells residing in the bone marrow, bone marrow transplanted wild-type mice were treated with DDC for 8 months and oval cells were then serially transferred into Fah mutants. The liver repopulating cells in these secondary transplant recipients lacked the genetic markers of the original bone marrow donor. We conclude that hepatic oval cells do not originate in bone marrow but in the liver itself, and that they have valuable properties for therapeutic liver repopulation.
Assuntos
Hepatócitos/citologia , Células-Tronco Multipotentes/citologia , Animais , Sequência de Bases , Diferenciação Celular , DNA/genética , Dicarbetoxi-Di-Hidrocolidina/toxicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/transplante , Hidrolases/deficiência , Hidrolases/genética , Fígado/efeitos dos fármacos , Fígado/lesões , Fígado/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/transplante , Proteínas/genética , RNA não Traduzido , Tirosinemias/patologia , Tirosinemias/terapiaRESUMO
Recent work has convincingly demonstrated that adult bone marrow contains cells capable of differentiating into liver epithelial cells in vivo. However, the frequency and time course with which fully functional hepatocytes emerge after bone marrow transplantation remained controversial. Here, we used the fumarylacetoacetate hydrolase knockout mouse to determine the kinetics of hepatocyte replacement after complete hematopoietic reconstitution. Single donor-derived hepatocytes were first detected 7 weeks after lethal irradiation and bone marrow transplantation. Liver disease was not required for this transdifferentiation. In the presence of selective pressure the single cells evolved into hepatocyte nodules by 11 weeks after transplantation and resulted in >30% overall liver repopulation by 22 weeks. The frequency with which hepatocytes were produced was between 10(-4) and 10(-6), resulting in only 50 to 500 repopulation events per liver. Hepatic engraftment was not observed without previous hematopoietic reconstitution even in the presence of liver injury. In addition, significant liver repopulation was completely dependent on hepatocyte growth selection. We conclude that hepatocyte replacement by bone marrow cells is a slow and rare event. Significant improvements in the efficiency of this process will be needed before clinical success can be expected.
Assuntos
Transplante de Medula Óssea , Diferenciação Celular , Células-Tronco Hematopoéticas/patologia , Hepatócitos/patologia , Animais , Divisão Celular , Hidrolases , Fígado/patologia , Camundongos , Camundongos KnockoutRESUMO
The murine model of hereditary tyrosinemia type 1 (HT1) was used to analyze the relationship between chronic liver disease and programmed cell death in vivo. In healthy fumarylacetoacetate hydrolase deficient mice (Fah(-/-)), protected from liver injury by the drug 2-(2- nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), the tyrosine metabolite homogentisic acid (HGA) caused rapid hepatocyte death. In contrast, all mice survived the same otherwise lethal dose of HGA if they had preexisting liver damage induced by NTBC withdrawal. Similarly, Fah(-/-) animals with liver injury were also resistant to apoptosis induced by the Fas ligand Jo-2 and to necrosis-like cell death induced by acetaminophen (APAP). Molecular studies revealed a marked up-regulation of the antiapoptotic heat shock proteins (Hsp) 27, 32, and 70 and of c-Jun in hepatocytes of stressed mice. In addition, the p38 and Jun N-terminal kinase (JNK) stress-activated kinase pathways were markedly impaired in the cell-death resistant liver. In conclusion, these results provide evidence that chronic liver disease can paradoxically result in cell death resistance in vivo. Stress-induced failure of cell death programs may lead to an accumulation of damaged cells and therefore enhance the risk for cancer as observed in HT1 and other chronic liver diseases.
Assuntos
Hidrolases/genética , Falência Hepática Aguda/metabolismo , Tirosinemias/metabolismo , Tirosinemias/patologia , Acetaminofen , Analgésicos não Narcóticos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Sobrevivência Celular/fisiologia , Depressores do Sistema Nervoso Central/farmacologia , Doença Crônica , Cicloexanonas/farmacologia , Inibidores Enzimáticos/farmacologia , Etanol/farmacologia , Proteína Ligante Fas , Glutationa/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Ácido Homogentísico/metabolismo , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/patologia , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Mutantes , NF-kappa B/metabolismo , Necrose , Nitrobenzoatos/farmacologia , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Regulação para Cima , Receptor fas/metabolismoRESUMO
Gene therapy applications of naked DNA constructs for genetic disorders have been limited because of lack of permanent transgene expression. This limitation, however, can be overcome by the Sleeping Beauty (SB) transposable element, which can achieve permanent transgene expression through genomic integration from plasmid DNA. To date, only one example of an in vivo gene therapy application of this system has been reported. In this report, we have further defined the activity of the SB transposon in vivo by analyzing the expression and integration of a fumarylacetoacetate hydrolase (FAH) transposon in FAH-deficient mice. In this model, stably corrected FAH(+) hepatocytes are clonally selected and stable integration events can therefore be quantified and characterized at the molecular level. Herein, we demonstrate that SB-transposon-transfected hepatocytes can support significant repopulation of the liver, resulting in long-lasting correction of the FAH-deficiency phenotype. A single, combined injection of an FAH-expressing transposon plasmid and a transposase expression construct resulted in stable FAH expression in approximately 1% of transfected hepatocytes. The average transposon copy number was determined to be approximately 1/diploid genome and expression was not silenced during serial transplantation. Molecular analysis indicated that high-efficiency DNA-mediated transposition into the mouse genome was strictly dependent on the expression of wild-type transposase.
Assuntos
Elementos de DNA Transponíveis/genética , Terapia Genética/métodos , Hidrolases/genética , Hidrolases/metabolismo , Recombinação Genética/genética , Tirosinemias/genética , Tirosinemias/terapia , Animais , DNA/genética , DNA/metabolismo , Fígado/enzimologia , Fígado/patologia , Camundongos , Camundongos Knockout , Fenótipo , Taxa de Sobrevida , Fatores de TempoRESUMO
Evidence suggests that haematopoietic stem cells might have unexpected developmental plasticity, highlighting therapeutic potential. For example, bone-marrow-derived hepatocytes can repopulate the liver of mice with fumarylacetoacetate hydrolase deficiency and correct their liver disease. To determine the underlying mechanism in this murine model, we performed serial transplantation of bone-marrow-derived hepatocytes. Here we show by Southern blot analysis that the repopulating hepatocytes in the liver were heterozygous for alleles unique to the donor marrow, in contrast to the original homozygous donor cells. Furthermore, cytogenetic analysis of hepatocytes transplanted from female donor mice into male recipients demonstrated 80,XXXY (diploid to diploid fusion) and 120,XXXXYY (diploid to tetraploid fusion) karyotypes, indicative of fusion between donor and host cells. We conclude that hepatocytes derived form bone marrow arise from cell fusion and not by differentiation of haematopoietic stem cells.