RESUMO
The early events that drive myeloid oncogenesis are not well understood. Most studies focus on the cell-intrinsic genetic changes and how they impact cell fate decisions. We consider how chronic exposure to the proinflammatory cytokine, interleukin-1ß (IL-1ß), impacts Cebpa-knockout hematopoietic stem and progenitor cells (HSPCs) in competitive settings. Surprisingly, we found that Cebpa loss did not confer a hematopoietic cell-intrinsic competitive advantage; rather chronic IL-1ß exposure engendered potent selection for Cebpa loss. Chronic IL-1ß augments myeloid lineage output by activating differentiation and repressing stem cell gene expression programs in a Cebpa-dependent manner. As a result, Cebpa-knockout HSPCs are resistant to the prodifferentiative effects of chronic IL-1ß, and competitively expand. We further show that ectopic CEBPA expression reduces the fitness of established human acute myeloid leukemias, coinciding with increased differentiation. These findings have important implications for the earliest events that drive hematologic disorders, suggesting that chronic inflammation could be an important driver of leukemogenesis and a potential target for intervention.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Interleucina-1beta/metabolismo , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem da Célula/fisiologia , Expressão Gênica/fisiologia , Células HEK293 , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Inflamação/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismoRESUMO
Here, we report a unique acute myeloid leukemia (AML) bone marrow-derived mesenchymal stem cell (MSC) with both mesenchymal and endothelial potential, which we have named Mesenchymal Cancer Stem Cells (MCSCs). These MCSCs are CD90-CD13-CD44+ and differ from MSCs in isolation, expansion, differentiation, immunophenotype, and cytokine release profile. Furthermore, blocking CD44 inhibited the proliferation and cluster formation of early MCSCs with lower ICAM-1 protein levels. Similar CD90-CD44+ cancer stem cells have been reported in both gastric and breast cancers, which grew in floating spheres in vitro and exhibited mesenchymal features and high metastatic/tumorigenic capabilities in vivo. Our novel discovery provides the first evidence that certain AMLs may be comprised of both hematopoietic and stromal malignant cells. Targeting MCSCs and their cytokine release has potential as a novel therapeutic approach in AML.
Assuntos
Antígenos de Neoplasias/análise , Medula Óssea/patologia , Receptores de Hialuronatos/análise , Leucemia Mielomonocítica Aguda/patologia , Células-Tronco Mesenquimais/patologia , Células-Tronco Neoplásicas/patologia , Proteínas Angiogênicas/metabolismo , Adesão Celular , Separação Celular , Citocinas/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/química , Células-Tronco Neoplásicas/classificação , Células-Tronco Neoplásicas/metabolismo , Células Tumorais CultivadasAssuntos
Leucemia Mielomonocítica Aguda , Mutação , Proteína de Leucina Linfoide-Mieloide , Proteínas de Fusão Oncogênica , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Lactente , Leucemia Mielomonocítica Aguda/genética , Leucemia Mielomonocítica Aguda/metabolismo , Masculino , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Acute myeloid leukaemia (AML) is a genetically heterogeneous, severe and rapidly progressing disease triggered by blocking granulocyte or monocyte differentiation and maturation. Overexpression of myeloid cell leukaemia-1 (Mcl-1) and Survivin is associated with drug resistance, tumour progression and inhibition of apoptotic mechanisms in leukaemia and several cancers. In the present study, we examined the combined effect of etoposide and dual siRNA-mediated silencing of Mcl-1 and Survivin on U-937 AML cells. The AML cells were co-transfected with Mcl-1 and Survivin-specific siRNAs and genes silencing were confirmed by quantitative real-time PCR and Western blotting. Subsequently, MTT assay was used for the evaluation of cytotoxic effects by dual siRNA and etoposide on their own and in combination. For the studying of apoptosis, DNA-histone ELISA and annexin-V/FITC assays were performed. Co-transfection of Mcl-1 and Survivin siRNA significantly blocked their expression at the mRNA and protein levels, leading to the induction of apoptosis and strong inhibition of growth (p < .05). Besides, combined treatment of etoposide with Mcl-1 and Survivin siRNAs co-transfection leads to synergistically enhance etoposide-induced cytotoxic and apoptotic effects (p < .05). The results showed that Mcl-1 and Survivin play a major role in the U937 cells survival and their resistance relative to etoposide. Thus, Mcl-1 and Survivin can be considered as promising molecular targets for the treatment of AML. The combination treatment with etoposide, and siRNA-mediated silencing of corresponding genes may be a novel strategy in chemoresistance AML treatment.
Assuntos
Apoptose , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Inativação Gênica , Leucemia Mielomonocítica Aguda/terapia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Survivina/antagonistas & inibidores , Humanos , Leucemia Mielomonocítica Aguda/genética , Leucemia Mielomonocítica Aguda/metabolismo , Leucemia Mielomonocítica Aguda/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , RNA Interferente Pequeno/genética , Survivina/biossíntese , Survivina/genética , Células U937Assuntos
Proteínas de Ligação a Calmodulina/genética , Transformação Celular Neoplásica/genética , Leucemia Mielomonocítica Aguda/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Animais , Transformação Celular Neoplásica/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mielomonocítica Aguda/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Suppressor with morphogenetic effect on genitalia family member (SMG1) belongs to a family of phosphoinositide 3-kinase-related kinases and is the main kinase involved in nonsense-mediated mRNA decay. Recently, SMG1 was suggested as a novel potential tumor suppressor gene, particularly in hypoxic tumors. To investigate the function of SMG1 in acute myeloid leukemia (AML), we performed methylation-specific polymerase chain reaction and found that SMG1 was hypermethylated in the promoter region. SMG1 hypermethylation was found in 66% (33/50) of AML samples compared with none (0/14) of the normal controls. SMG1 mRNA was down-regulated in AML patients with hypermethylation status whereas it was readily expressed in patients without methylation. Moreover, treatment of AML cells with demethylating agent 5-aza-2'-deoxycytidine (decitabine) inhibited AML cell growth and induced apoptosis by reversing SMG1 methylation status and restoring SMG1 expression. On the other hand, knockdown of SMG1 by RNA interference inhibited apoptosis. We also found that mTOR expression level was negatively correlated to SMG1 expression in AML patients which indicated that SMG1 and mTOR maybe act antagonistically to regulate AML cell growth. In conclusion, our results indicate that SMG1 acts as a potential tumor suppressor with epigenetic regulation in AML.
Assuntos
Metilação de DNA , DNA de Neoplasias/genética , Regulação Leucêmica da Expressão Gênica/fisiologia , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Medula Óssea/metabolismo , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Decitabina , Regulação para Baixo , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mielomonocítica Aguda/genética , Leucemia Mielomonocítica Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Serina-Treonina Quinases TOR/fisiologia , Proteínas Supressoras de Tumor/genética , Adulto JovemRESUMO
Leukemic stem cells (LSCs) isolated from acute myeloid leukemia (AML) patients are more sensitive to nuclear factor κB (NF-κB) inhibition-induced cell death when compared with hematopoietic stem and progenitor cells (HSPCs) in in vitro culture. However, inadequate anti-leukemic activity of NF-κB inhibition in vivo suggests the presence of additional survival/proliferative signals that can compensate for NF-κB inhibition. AML subtypes M3, M4, and M5 cells produce endogenous tumor necrosis factor α (TNF). Although stimulating HSPC with TNF promotes necroptosis and apoptosis, similar treatment with AML cells (leukemic cells, LCs) results in an increase in survival and proliferation. We determined that TNF stimulation drives the JNK-AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC. We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF-JNK-AP1 signaling pathway. Our data suggest that co-inhibition of both TNF-JNK-AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia Mieloide Aguda/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Antracenos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Células K562 , Leucemia Monocítica Aguda/genética , Leucemia Monocítica Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Mielomonocítica Aguda/genética , Leucemia Mielomonocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Nitrilas/farmacologia , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sulfonas/farmacologia , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Células U937RESUMO
Anaplastic lymphoma receptor tyrosine kinase (ALK) is located on chromosome 2p23; the chromosomal rearrangements of this gene are common genetic alterations, resulting in the creation of multiple fusion genes involved in tumorigenesis. However, the presence of an ALK fusion in myeloid malignancies is extremely rare. We report a case of acute myelomonocytic leukemia in a 31-year-old woman with an unusual rearrangement between RAN-binding protein 2 (RANBP2) and ALK and a karyotype of 45,XX,inv(2)(p23q21),-7[20]. We detected an ALK rearrangement using fluorescence in situ hybridization, identified the ALK fusion partner by using RNA transcriptome sequencing, and demonstrated the RANBP2-ALK fusion transcript by reverse transcriptase--PCR and Sanger sequencing. Immunohistochemistry for ALK showed strong staining of the nuclear membrane in leukemic cells. The patient had an unfavorable clinical course. Our results, together with a literature review, suggest the RANBP2-ALK fusion combined with monosomy 7 may be related to a unique clonal hematologic disorder of childhood and adolescence, characterized by myelomonocytic leukemia and a poor prognosis.
Assuntos
Inversão Cromossômica , Leucemia Mielomonocítica Aguda/genética , Chaperonas Moleculares/metabolismo , Monossomia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Adulto , Quinase do Linfoma Anaplásico , Células da Medula Óssea/citologia , Cromossomos Humanos Par 7 , Evolução Fatal , Feminino , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Mielomonocítica Aguda/metabolismo , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Prognóstico , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Comunicação Celular , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Linfócitos/metabolismo , Células Mieloides/metabolismo , Segunda Neoplasia Primária/metabolismo , Idoso , Medula Óssea/patologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Mielomonocítica Aguda/diagnóstico , Leucemia Mielomonocítica Aguda/etiologia , Masculino , Segunda Neoplasia Primária/diagnóstico , Segunda Neoplasia Primária/etiologia , Microambiente TumoralRESUMO
Myb-transformed haematopoietic cell (MTHC) lines have been developed and clones selected based on their ability to respond to tumor necrosis factor (TNF)-α. MTHC-A cells underwent apoptosis in response to TNF-α (MTHC-A). The apoptotic effect of TNF-α in MTHC-A was mimicked by a specific inhibitor of protein kinase A (PKI 5-24) and by the tyrosine kinase inhibitor genistein, suggesting that phosphorylation of tyrosine and PKA activity were important in protecting MTHC from apoptosis. Agents that elevate intracellular levels of cAMP, e.g. cholera toxin and dibuteryl cAMP, protected MTHC-A from the apoptotic effects of TNF-α, and also reduced the apoptotic effects of PKI and genistein. MTHC-A thus provides a useful model for investigating the role of TNF-mediated apoptosis in regulation of the myeloid lineage of cells.
Assuntos
Apoptose/genética , Transformação Celular Neoplásica/genética , Proteínas Proto-Oncogênicas c-myb/genética , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Leucemia Mielomonocítica Aguda/genética , Leucemia Mielomonocítica Aguda/metabolismo , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and protein synthesis inhibition downstream of mammalian target of rapamycin complex 1 (mTORC1) has shown promise as a possible therapy for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor formation in a mouse AML xenotransplantation model. l-asparaginase (l-ase) is an anticancer agent also harboring glutaminase activity. We show that l-ases from both Escherichia coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition correlates with their glutaminase activity levels and produces a strong apoptotic response in primary AML cells. We further show that l-ases upregulate glutamine synthase (GS) expression in leukemic cells and that a GS knockdown enhances l-ase-induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon l-ase treatment. These results suggest that l-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML.
Assuntos
Glutamina/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Sistema ASC de Transporte de Aminoácidos/antagonistas & inibidores , Sistema ASC de Transporte de Aminoácidos/genética , Animais , Apoptose/efeitos dos fármacos , Asparaginase/isolamento & purificação , Asparaginase/farmacologia , Autofagia/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Dickeya chrysanthemi/enzimologia , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Escherichia coli/farmacologia , Feminino , Glutaminase/isolamento & purificação , Glutaminase/farmacologia , Glutamina/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucemia Mielomonocítica Aguda/tratamento farmacológico , Leucemia Mielomonocítica Aguda/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor , Complexos Multiproteicos/antagonistas & inibidores , Biossíntese de Proteínas/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
The AML1-ETO fusion transcription factor generated by the t(8;21) translocation is considered to deregulate the expression of genes that are crucial for normal differentiation and proliferation of hematopoietic progenitors, resulting in acute myelogenous leukemia by recruiting co-repressor complexes to DNA. To investigate the role of AML1-ETO in leukemogenesis, we transfected the cloned AML1-ETO cDNA and expressed the AML1-ETO protein in U937 myelomonocytic leukemia cells. By focusing on the anti-apoptotic gene Bcl-2, the key regulator gene of granulocytic differentiation CCAAT/enhancer-binding protein α (CEBPA) and the tumor suppressor gene p14(ARF), we found that both AML1-ETO-expressing cell lines and t(8;21) leukemia samples displayed low levels of these three genes. Chromatin immunoprecipitation assays demonstrated that Bcl-2, CEBPA and p14(ARF) were direct transcriptional targets of AML1-ETO. The universal binding of AML1-ETO to genomic DNA resulted in recruitment of methyl-CpG binding protein 2 (MeCP2), reduction of histone H3 or H4 acetylation and increased trimethylation of histone H3 lysine 9 as well as lysine 27 indicating that AML1-ETO induced heterochromatic silencing of Bcl-2, CEBPA and p14(ARF). These results suggested that the aberrant transcription factor AML1-ETO epigenetically silenced the function of the Bcl-2, CEBPA and p14(ARF) genes by inducing repressed chromatin configurations at their promoters through histone modifications.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Supressora de Tumor p14ARF/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mielomonocítica Aguda/genética , Proteína 1 Parceira de Translocação de RUNX1 , Células U937RESUMO
The concept of reactive oxygen species (ROS) being produced via the activation of specific oncogenes provides a basis for generating genomic instability and pro-survival signalling in tumour cells. The purpose of this study was to identify downstream targets of NADPH oxidase (Nox)-derived ROS signalling in acute myeloid leukaemia cells, by performing a proteomic analysis utilizing two-dimensional phosphotyrosine immunoblotting. The majority of the targets identified were cytoskeletal-associated proteins including Ezrin, a known regulator of the cytoskeleton, which was examined further. The study demonstrated that inhibition of Nox enzymes, using diphenyleneiodonium chloride in the acute myeloid leukaemia cell line MOLM-13, resulted in a decrease in Ezrin tyrosine phosphorylation and also triggered a shift in Ezrin sub-cellular localization as detected by immunofluorescence. The change in Ezrin localization coincided with altered cell morphology, observed using scanning electron microscopy and a decreased ability to migrate through a polycarbonate transwell membrane. Similar effects were observed upon inhibition of the oncogenic receptor tyrosine kinase FLT3 using the staurosporine derivate PKC412, implicating a role for FLT3 as an upstream regulator of Ezrin. Our results indicate that FLT3 drives production of ROS by Nox, which stimulates changes in Ezrin tyrosine phosphorylation and localization via redox regulation of Src. Furthermore, inhibition of FLT3 signalling leads to alterations in MOLM-13 cell morphology and has a significant influence on cell motility.
Assuntos
Movimento Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Leucemia Mielomonocítica Aguda/enzimologia , Leucemia Mielomonocítica Aguda/patologia , Tirosina Quinase 3 Semelhante a fms/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Células HL-60 , Humanos , Leucemia Mielomonocítica Aguda/genética , Leucemia Mielomonocítica Aguda/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Oxirredução , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Transfecção , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Activation of NRas signaling is frequently found in human myeloid leukemia and can be induced by activating mutations as well as by mutations in receptors or signaling molecules upstream of NRas. To study NRas-induced leukemogenesis, we retrovirally overexpressed wild-type NRas in a murine bone marrow transplantation (BMT) model in C57BL/6J mice. Overexpression of wild-type NRas caused myelomonocytic leukemias â¼3 months after BMT in the majority of mice. A subset of mice (30%) developed malignant histiocytosis similar to mice that received mutationally activated NRas(G12D)-expressing bone marrow. Aberrant Ras signaling was demonstrated in cells expressing mutationally active or wild-type NRas, as increased activation of Erk and Akt was observed in both models. However, more NRas(G12D) were found to be in the activated, GTP-bound state in comparison with wild-type NRas. Consistent with observations reported for primary human myelomonocytic leukemia cells, Stat5 activation was also detected in murine leukemic cells. Furthermore, clonal evolution was detected in NRas wild-type-induced leukemias, including expansion of clones containing activating vector insertions in known oncogenes, such as Evi1 and Prdm16. In vitro cooperation of NRas and Evi1 improved long-term expansion of primary murine bone marrow cells. Evi1-positive cells upregulated Bcl-2 and may, therefore, provide anti-apoptotic signals that collaborate with the NRas-induced proliferative effects. As activation of Evi1 has been shown to coincide with NRAS mutations in human acute myeloid leukemia, our murine model recapitulates crucial events in human leukemogenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Fatores de Transcrição/metabolismo , Proteínas ras/metabolismo , Animais , Apoptose , Transplante de Medula Óssea , Linhagem Celular Tumoral , Proliferação de Células , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Genes ras , Humanos , Leucemia Mielomonocítica Aguda/genética , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proto-Oncogenes , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Proteínas ras/genéticaRESUMO
Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against acute promyelocytic leukemia. However, organic arsenic derivatives (OAD) have a more favorable toxicity profile than ATO. We herein characterized dipropil-S-glycerol arsenic (GMZ27), a novel OAD. GMZ27 had potent antiproliferative activity against human acute myeloid leukemia (AML) cell lines that was higher than that of ATO. In contrast to ATO, GMZ27 only marginally induced maturation of leukemia cells and had no effect on the cell cycle. The anti-leukemia activity of GMZ27 against AML cells was independent of the presence of the PML-RARα fusion protein. GMZ27 dissipates mitochondrial transmembrane potential, and induces cleavage of caspase 9 and activation of caspase 3 without altering the expression levels of (BCL-2), BAX and BCL-xl. GMZ27 induces the formation of intracellular superoxide, a reactive oxygen species (ROS) which plays a major role in the antileukemia activity of this OAD. In addition to ROS generation, GMZ27 concomitantly reduces intracellular glutathione which markedly weakens the cellular antioxidant capacity, thus enhancing the detrimental intracellular effects of ROS production. These results indicate that GMZ27 induces apoptosis in AML cells in a PML-RARα-independent fashion, through the induction of ROS production. This activity provides the rationale for the testing of GMZ27 in patients with AML.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Leucemia Mielomonocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/tratamento farmacológico , Proteínas de Fusão Oncogênica/biossíntese , Animais , Apoptose/fisiologia , Trióxido de Arsênio , Caspase 9/metabolismo , Ciclo Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Células HL-60 , Humanos , Leucemia Mielomonocítica Aguda/metabolismo , Leucemia Mielomonocítica Aguda/patologia , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Camundongos , Óxidos/farmacologia , Oxigênio/metabolismoRESUMO
CCAAT/enhancer binding protein alpha (C/EBPα) induction induces monocytic differentiation even in acute myeloid leukaemia (AML). In this study, the induction/activation of C/EBPα in myelomonocytic AML was investigated using a combination of all-trans retinoic acid (ATRA) and RAD001 (Everolimus), a mammalian target of rapamycin complex 1 (mTORC1) inhibitor. Combining these agents increased PU.1, C/EBPε and C/EBPα expression, increased the p42/p30 C/EBPα ratio, and decreased C/EBPα phosphorylation at serine 21, and was accompanied by growth inhibition, induction of CD11b expression and apoptosis in AML cell lines. Thus, agents that induce sufficient levels of C/EBPα expression might be useful in treating AML.
Assuntos
Apoptose/genética , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Diferenciação Celular/genética , Leucemia Mielomonocítica Aguda/genética , Monócitos/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Criança , Pré-Escolar , Everolimo , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/fisiologia , Humanos , Imunossupressores/farmacologia , Leucemia Mielomonocítica Aguda/metabolismo , Leucemia Mielomonocítica Aguda/patologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosforilação/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/fisiologia , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Tretinoína/farmacologia , Células U937RESUMO
AIMS: In the absence of adequate aspirate films and touch imprints, distinction of chronic myelomonocytic leukaemia (CMML) from acute myeloid leukaemia with monocytic differentiation (Mo-AML) may be difficult solely on the basis of bone marrow biopsy morphological features. The aim of this study was to evaluate the diagnostic utility of a novel immunohistochemical panel for the diagnosis of acute and chronic myelomonocytic leukaemias in bone marrow biopsies. METHODS AND RESULTS: Immunohistochemical labelling for CD14, CD123, CD33, myeloperoxidase (MPO) and CD68R was assessed in 49 myeloid neoplasms with monocytic differentiation (24 CMMLs and 25 Mo-AMLs) and compared with that of 15 non-monocytic acute myeloid leukaemias (NM-AMLs) and 17 non-neoplastic controls. More than 20% CD14 immunohistochemistry (IHC)+ cells were seen only in Mo-AMLs and CMMLs, although Mo-AMLs showed wide variability and overlapped with other categories. More than 20% CD68R IHC+ cells had the highest sensitivity and specificity for Mo-AML. Discrepant MPO-/CD33+ expression was specific for Mo-AML but insensitive. A subset of blasts in Mo-AMLs and NM-AMLs were weakly CD123+. CONCLUSIONS: A significantly increased number of CD14+ cells raises the possibility of a myelomonocytic neoplasm but does not distinguish between CMML and Mo-AML. Significantly increased numbers of CD68R IHC+ cells and a discrepant MPO-/CD33+ staining pattern are specific for Mo-AML but are best utilized in a comprehensive panel.
Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Imuno-Histoquímica/métodos , Leucemia Mielomonocítica Aguda/diagnóstico , Leucemia Mielomonocítica Crônica/diagnóstico , Peroxidase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Diferenciação Mielomonocítica/metabolismo , Criança , Pré-Escolar , Humanos , Lactente , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Leucemia Mielomonocítica Crônica/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Pessoa de Meia-Idade , Estudos Retrospectivos , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Adulto JovemRESUMO
Resting human NK cells require a two-stage activation process that we have previously described as "priming" and "triggering." NK-sensitive tumor cells provide both priming and triggering signals. NK-resistant tumors evade lysis, mostly by failure to prime; however, we recently reported a tumor cell line (CTV-1) that primes resting NK cells but fails to trigger lysis. In this article, we report two additional leukemia cell lines that prime NK cells but are resistant to lysis. Tumor-mediated NK priming is via CD2 binding to a ligand within CD15 on the tumor cell. NK-resistant RAJI cells became susceptible to NK lysis following transfection and expression of CD15. Blockade of CD15 on K562 cells or on CD15(+) RAJI cells significantly inhibited lysis, as did blockade of CD2 on resting NK cells. NK priming via CD2 induced CD16 shedding, releasing CD3ζ to the CD2, leading to its phosphorylation and the subsequent phosphorylation of linker for activation of T cells and STAT-5 and synthesis of IFN-γ. Blockade of C-type lectin receptors significantly suppressed the tumor-mediated priming of NK cells, whereas blockade of Ig-superfamily-like receptors had no effect at the NK-priming stage. Tumor priming of resting NK cells was irrespective of HLA expression, and blockade of HLA-killer Ig-like receptor interactions did not influence the incidence or degree of priming. However, CD15-CD2 interactions were critical for NK priming and were required, even in the absence of HLA-mediated NK inhibition. Tumor-mediated priming led to a sustained primed state, and the activated NK cells retained the ability to lyse NK-resistant tumors, even after cryopreservation.
Assuntos
Antígenos CD2/metabolismo , Citotoxicidade Imunológica , Fucosiltransferases/fisiologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Antígenos CD15/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Receptores KIR/fisiologia , Fase de Repouso do Ciclo Celular/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Linhagem da Célula/imunologia , Técnicas de Cocultura , Resistência à Doença , Fucosiltransferases/metabolismo , Humanos , Células Matadoras Naturais/citologia , Leucemia Monocítica Aguda/imunologia , Leucemia Monocítica Aguda/metabolismo , Leucemia Monocítica Aguda/patologia , Leucemia Mielomonocítica Aguda/imunologia , Leucemia Mielomonocítica Aguda/metabolismo , Leucemia Mielomonocítica Aguda/patologia , Antígenos CD15/metabolismo , Ligantes , Ativação Linfocitária/imunologia , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/metabolismo , Linfoma não Hodgkin/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Ligação Proteica/imunologia , Transdução de Sinais/imunologiaAssuntos
Síndrome de Down/complicações , Síndrome de Down/genética , Leucemia Mielomonocítica Aguda/complicações , Leucemia Mielomonocítica Aguda/genética , Adolescente , Idade de Início , Antígenos CD7/metabolismo , Aberrações Cromossômicas , Cromossomos Humanos Par 21 , Síndrome de Down/epidemiologia , Síndrome de Down/metabolismo , Humanos , Leucemia Mielomonocítica Aguda/metabolismoRESUMO
Leukemic cells responding to apoptosis-inducing drugs can be varied in terms of the mechanisms of action. Fenretinide, a synthetic retinoid, is worth of study as a promising candidate for apoptosis-based therapy of leukemia. Yet, it remains unclear whether this drug exerts the similar mechanisms on different leukemic cells. Here, we report a comparative analysis of fenretinide-induced apoptosis in three acute myeloid leukemic (AML) cell lines including HL60, NB4 and U937. Through a series of antagonist assays, we revealed similarities and differences of mechanisms involved in these three cell lines. Antioxidant vitamin C completely abrogated fenretinide-induced apoptosis in all cell lines, demonstrating that ROS is an essential and common mediator. However, the apoptotic effects of fenretinide could be blocked by ceramide synthase inhibitor fumonisin B1 only in HL60 rather than the other two. Moreover, fumonisin B1 was unable to inhibit the generation of ROS in fenretinide-treated HL60 cells, indicating that ROS may function as upstream stimulus of ceramide-mediated apoptosis. These comparative results strongly suggest that the apoptotic response induced by fenretinide in HL60 involves both ROS and ceramide, whereas drug-induced apoptosis in NB4 and U937 requires ROS but is independent of ceramide. Differentiated modes of action exerting on AML may guide the use of this apoptosis-inducing drug, and hence advance our knowledge about the nature of cancer-specific responses to this drug.