RESUMO
Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a low-oxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Estresse Oxidativo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Western Blotting , Hipóxia Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Ácido Dicloroacético/farmacologia , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Leucemia/genética , Leucemia/patologia , Camundongos Knockout , Camundongos Transgênicos , Proteína Meis1 , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Fosforilação Oxidativa/efeitos dos fármacos , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , TranscriptomaRESUMO
Pentavalent antimonials have been the first-line treatment for leishmaniasis for decades. However, the development of resistance to sodium stibogluconate (SSG) has limited its use, especially for treating visceral leishmaniasis (VL). The present work aims to optimize a cationic liposomal formulation of SSG for the treatment of both SSG-sensitive (AG83) and SSG-resistant (GE1F8R and CK1R) Leishmania donovani infections. Parasite killing was determined by the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic counting of Giemsa-stained macrophages. Macrophage uptake studies were carried out by confocal microscopic imaging. Parasite-liposome interactions were visualized through transmission electron microscopy. Toxicity tests were performed using assay kits. Organ parasite burdens were determined by microscopic counting and limiting dilution assays. Cytokines were measured by enzyme-linked immunosorbent assays (ELISAs) and flow cytometry. Although all cationic liposomes studied demonstrated leishmanicidal activity, phosphatidylcholine (PC)-dimethyldioctadecylammonium bromide (DDAB) vesicles were most effective, followed by PC-stearylamine (SA) liposomes. Since entrapment of SSG in PC-DDAB liposomes demonstrated enhanced ultrastructural alterations in promastigotes, PC-DDAB-SSG vesicles were further investigated in vitro and in vivo. PC-DDAB-SSG could effectively alleviate SSG-sensitive and SSG-resistant L. donovani infections in the liver, spleen, and bone marrow of BALB/c mice at a dose of SSG (3 mg/kg body weight) not reported previously. The parasiticidal activity of these vesicles was attributed to better interactions with the parasite membranes, resulting in direct killing, and generation of a strong host-protective environment, necessitating a very low dose of SSG for effective cures.
Assuntos
Gluconato de Antimônio e Sódio/uso terapêutico , Antiprotozoários/uso terapêutico , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Lipossomos/uso terapêutico , Animais , Gluconato de Antimônio e Sódio/química , Gluconato de Antimônio e Sódio/farmacologia , Antiprotozoários/química , Antiprotozoários/farmacologia , Medula Óssea/parasitologia , Cricetinae , Resistência a Medicamentos , Humanos , Leishmaniose Visceral/parasitologia , Lipossomos/farmacologia , Fígado/parasitologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Parasitária , Baço/parasitologiaRESUMO
The transcription factor Meis1 is expressed preferentially in hematopoietic stem cells (HSCs) and overexpressed in certain leukemias. However, the functions of Meis1 in hematopoiesis remain largely unknown. In the present study, we found that Meis1 is required for the maintenance of hematopoiesis under stress and over the long term, whereas steady-state hematopoiesis was sustained in the absence of Meis1 in inducible knock-out mice. BM cells of Meis1-deficient mice showed reduced colony formation and contained significantly fewer numbers of long-term HSCs, which exhibited loss of quiescence. Further, we found that Meis1 deletion led to the accumulation of reactive oxygen species in HSCs and decreased expression of genes implicated in hypoxia response. Finally, reactive oxygen species scavenging by N-acetyl cysteine or stabilization of hypoxia signaling by knockdown of the von-Hippel-Lindau (VHL) protein led to reversal of the effects of Meis1 deletion. The results of the present study demonstrate that Meis1 protects and preserves HSCs by restricting oxidative metabolism.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Estresse Oxidativo , Animais , Ciclo Celular , Hipóxia Celular , Deleção de Genes , Regulação da Expressão Gênica , Hematopoese , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Meis1 , Proteínas de Neoplasias/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVES: Lipid-associated formulations of antileishmanial agents have proved to be more effective therapies with reduced toxicities. Previous studies from our group and others revealed that liposomes bearing phosphatidylcholine and stearylamine (SA) themselves kill Leishmania and other protozoan parasites in vitro and in vivo, without causing any adverse effect on host. In the present study, we offer detailed insights into the mechanism of action of these liposomes. METHODS: Mechanism study was carried out using fluorometric, confocal and electron microscopic methods. RESULTS: Herein, we provide evidence for induction of membrane disruption by specific interaction with surface phosphatidylserine (PS) of Leishmania promastigotes and amastigotes, phospholipids normally not found on mammalian cell surface, with SA-containing liposomes. Cell surface PS on different forms of Leishmania facilitated liposome-induced parasite killing. The target selectivity of the liposomes was further proved through inhibition of antileishmanial activity with annexinV, and strong affinity with anionic PS rather than phosphatidic acid-containing liposomes for leishmanicidal activity. CONCLUSIONS: SA-bearing liposomes specifically kill Leishmania, but are non-toxic to murine peritoneal macrophages and human erythrocytes.
Assuntos
Aminas/farmacologia , Antiprotozoários/farmacologia , Membrana Celular/efeitos dos fármacos , Leishmania donovani/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Trifosfato de Adenosina/metabolismo , Aminas/administração & dosagem , Aminas/efeitos adversos , Animais , Anexina A5/metabolismo , Antiprotozoários/administração & dosagem , Antiprotozoários/efeitos adversos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Composição de Medicamentos , Eletroquímica , Leishmania donovani/isolamento & purificação , Leishmania donovani/metabolismo , Leishmania donovani/ultraestrutura , Lipossomos , Macrófagos Peritoneais/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Consumo de Oxigênio/efeitos dos fármacos , Fosfatidilserinas/química , Ligação ProteicaRESUMO
Many patients with histiocytic disorders such as Langerhans cell histiocytosis (LCH) or Erdheim-Chester disease (ECD) have treatment-refractory disease or suffer recurrences. Recent findings of gene mutations in histiocytoses have generated options for targeted therapies. We sought to determine the utility of prospective sequencing of select genes to further characterize mutations and identify targeted therapies for patients with histiocytoses. Biopsies of 72 patients with a variety of histiocytoses underwent comprehensive genomic profiling with targeted DNA and RNA sequencing. Fifteen patients (21%) carried the known BRAF V600E mutation, and 11 patients (15%) carried various mutations in MAP2K1, which we confirm induce constitutive activation of extracellular signal-regulated kinase (ERK) and were sensitive to inhibitors of mitogen-activated protein kinase kinase (MEK, the product of MAP2K1). We also identified recurring ALK rearrangements, and 4 LCH patients with an uncommon in-frame deletion in BRAF (N486_P490del or N486_T491>K), resulting in constitutive activation of ERK with resistance to V600E-specific inhibitors. We subsequently describe clinical cases where patients with aggressive multisystem LCH experience dramatic and sustained responses to monotherapy with either dabrafenib or trametinib. These findings support our conclusion that comprehensive genomic profiling should be regularly applied to these disorders at diagnosis, and can positively impact clinical care.
Assuntos
Quinase do Linfoma Anaplásico/genética , Histiocitose/tratamento farmacológico , MAP Quinase Quinase 1/genética , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas B-raf/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Células 3T3 , Adulto , Animais , Resistencia a Medicamentos Antineoplásicos , Feminino , Histiocitose/genética , Histiocitose/patologia , Humanos , Imidazóis/administração & dosagem , Imidazóis/uso terapêutico , Lactente , Masculino , Camundongos , Mutação , Oximas/administração & dosagem , Oximas/uso terapêutico , Estudos Prospectivos , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/administração & dosagem , Piridonas/uso terapêutico , Pirimidinonas/administração & dosagem , Pirimidinonas/uso terapêutico , Resultado do Tratamento , Adulto JovemRESUMO
PURPOSE: We examined the effect of aging on Fas ligand (FasL) function in a mouse model of choroidal neovascularization (CNV). METHODS: Young and aged mice were laser treated to induce CNV. Bone marrow chimeras were performed between young and aged mice. FasL protein expression was examined in the eye and soluble FasL (sFasL) was measured in the blood. Young and aged mice were treated with a matrix metalloprotease (MMP) inhibitor and systemic sFasL was neutralized by antibody treatment. Macrophages from young and aged mice were tested for sFasL-mediated cytokine production and migration. RESULTS: The elevated CNV response observed with aging was dependent on bone marrow-derived cells. FasL expression in the eye was increased with age, but decreased following laser treatment. Aged mice had higher levels of sFasL in the blood compared to young mice. Systemic treatment with an MMP inhibitor decreased bloodborne sFasL, and reduced CNV in young and aged mice. Systemic neutralization of sFasL reduced CNV only in aged mice. sFasL increased cytokine production in aged macrophages and proangiogenic M2 macrophages. Aged M2 macrophages had elevated Fas (CD95) expression and displayed increased migration in response to sFasL compared to M1 macrophages derived from young animals. CONCLUSIONS: Age modulates FasL function where increased MMP cleavage leads to a loss of function in the eye. The released form of FasL (sFasL) preferentially induces the migration of proangiogenic M2 macrophages into the laser lesions and increases proangiogenic cytokines promoting CNV. FasL may be a viable target for therapeutic intervention in aged-related neovascular disease.
Assuntos
Envelhecimento/imunologia , Neovascularização de Coroide/imunologia , Proteína Ligante Fas/metabolismo , Imunidade Celular , Macrófagos/imunologia , Degeneração Macular/imunologia , Envelhecimento/patologia , Animais , Western Blotting , Movimento Celular , Células Cultivadas , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Proteína Ligante Fas/imunologia , Macrófagos/metabolismo , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
PURPOSE: We noted an unexpected inheritance pattern of lesions in several strains of gene-manipulated mice with ocular phenotypes. The lesions, which appeared at various stages of backcross to C57BL/6, bore resemblance to the rd8 retinal degeneration phenotype. We set out to examine the prevalence of this mutation in induced mutant mouse lines, vendor C57BL/6 mice and in widely used embryonic stem cells. METHODS: Ocular lesions were evaluated by fundus examination and histopathology. Detection of the rd8 mutation at the genetic level was performed by PCR with appropriate primers. Data were confirmed by DNA sequencing in selected cases. RESULTS: Analysis of several induced mutant mouse lines with ocular disease phenotypes revealed that the disease was associated 100% with the presence of the rd8 mutation in the Crb1 gene rather than with the gene of interest. DNA analysis of C57BL/6 mice from common commercial vendors demonstrated the presence of the rd8 mutation in homozygous form in all C57BL/6N substrains, but not in the C57BL/6J substrain. A series of commercially available embryonic stem cells of C57BL/6N origin and C57BL/6N mouse lines used to generate ES cells also contained the rd8 mutation. Affected mice displayed ocular lesions typical of rd8, which were detectable by funduscopy and histopathology as early as 6 weeks of age. CONCLUSIONS: These findings identify the presence of the rd8 mutation in the C57BL/6N mouse substrain used widely to produce transgenic and knockout mice. The results have grave implications for the vision research community who develop mouse lines to study eye disease, as presence of rd8 can produce significant disease phenotypes unrelated to the gene or genes of interest. It is suggested that researchers screen for rd8 if their mouse lines were generated on the C57BL/6N background, bear resemblance to the rd8 phenotype, or are of indeterminate origin.
Assuntos
Células-Tronco Embrionárias , Camundongos Endogâmicos C57BL/genética , Mutação , Proteínas do Tecido Nervoso/genética , Degeneração Retiniana/genética , Animais , Linhagem Celular , Análise Mutacional de DNA , Homozigoto , Camundongos , Fenótipo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Tomografia de Coerência ÓpticaRESUMO
BACKGROUND: Resistance of Leishmania donovani to pentavalent antimonials, the first-line treatment of visceral leishmaniasis (VL), has become a critical issue worldwide. Second-line and new drugs are also not devoid of limitations. Suitable drug-delivery systems can improve the mode of administration and action of the existing antimonials, thus increasing their clinical life. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the efficacy of sodium stibogluconate (SSG) in phosphatidylcholine (PC)-stearylamine-bearing liposomes (PC-SA-SSG), PC-cholesterol liposomes (PC-Chol-SSG) and free amphotericin B (AmB) against SSG-resistant L. donovani strains in 8-wk infected BALB/c mice. Animals were sacrificed and parasites in liver, spleen and bone marrow were estimated 4-wk post-treatment by microscopic examination of stamp smears and limiting dilution assay. A set of PC-SA-SSG and AmB treated mice were further studied for protection against reinfection. Serum antibodies and cytokine profiles of ex-vivo cultured splenocytes were determined by ELISA. Uptake of free and liposomal SSG in intracellular amastigotes was determined by atomic absorption spectroscopy. Rhodamine 123 and 5-carboxyfluorescein, known substrates of Pgp and MRP transporter proteins, respectively, were used in free and liposomal forms for efflux studies to estimate intracellular drug retention. Unlike free and PC-Chol-SSG, PC-SA-SSG was effective in curing mice infected with two differentially originated SSG-unresponsive parasite strains at significantly higher levels than AmB. Successful therapy correlated with complete suppression of disease-promoting IL-10 and TGF-ß, upregulation of Th1 cytokines and expression of macrophage microbicidal NO. Cure due to elevated accumulation of SSG in intracellular parasites, irrespective of SSG-resistance, occurs as a result of increased drug retention and improved therapy when administered as PC-SA-SSG versus free SSG. CONCLUSIONS/SIGNIFICANCE: The design of this single-dose combination therapy with PC-SA-SSG for VL, having reduced toxicity and long-term efficacy, irrespective of SSG-sensitivity may prove promising, not only to overcome SSG-resistance in Leishmania, but also for drugs with similar resistance-related problems in other diseases.
Assuntos
Aminas/química , Gluconato de Antimônio e Sódio/farmacologia , Gluconato de Antimônio e Sódio/uso terapêutico , Resistência a Medicamentos/efeitos dos fármacos , Leishmania donovani/efeitos dos fármacos , Lipossomos/química , Animais , Antígenos de Protozoários/imunologia , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Citocinas/biossíntese , Fluoresceínas/metabolismo , Imunidade Humoral/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Leishmania donovani/imunologia , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/biossíntese , Fosfatidilcolinas/química , Rodamina 123/metabolismo , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
PURPOSE. Current studies suggest that the immune system plays a critical role in blinding eye disorders. The eye is an immune-privileged site, and FasL expression is a major part of that mechanism because Fas/FasL interactions regulate inflammation and neovascularization, preventing damage to delicate ocular structures. These studies were undertaken to test the idea that modulating immune privilege might be an effective therapeutic approach to pathogenic angiogenesis in the eye. METHODS. C57BL/6 mice or FasL-defective B6-gld mice were laser treated to induce choroidal neovascularization (CNV). Mice were injected with cytotoxic FasL in the vitreous cavity or were treated with oral doxycycline in the drinking water. They were evaluated for CNV 7 days later. In some experiments eye tissue was harvested and evaluated for FasL expression, macrophage influx by immunohistochemistry, and release of sFasL. RESULTS. Injection of cytotoxic FasL successfully prevented neovascularization in a mouse model of CNV. Oral doxycycline increased functional FasL in the eye and substantially inhibited neovascularization. Doxycycline treatment increased FasL expression on the RPE cells and reduced circulating and tissue-associated sFasL. Treatment was ineffective in B6-gld mice, demonstrating that CNV inhibition was mediated by FasL. CONCLUSIONS. Targeting immune privilege using cytotoxic molecules or by increasing expression of the proapoptotic protein FasL may be a viable approach to treating neovascular eye disease.