RESUMO
A fundamental property of leukemic stem cells is clonal dominance of the bone marrow microenvironment. Truncation mutations of CSF3R, which encodes the G-CSF receptor (G-CSFR), are implicated in leukemic progression in patients with severe congenital neutropenia. Here we show that expression of a truncated mutant Csf3r in mice confers a strong clonal advantage at the HSC level that is dependent upon exogenous G-CSF. G-CSF-induced proliferation, phosphorylation of Stat5, and transcription of Stat5 target genes were increased in HSCs isolated from mice expressing the mutant Csf3r. Conversely, the proliferative advantage conferred by the mutant Csf3r was abrogated in myeloid progenitors lacking both Stat5A and Stat5B, and HSC function was reduced in mice expressing a truncated mutant Csf3r engineered to have impaired Stat5 activation. These data indicate that in mice, inappropriate Stat5 activation plays a key role in establishing clonal dominance by stem cells expressing mutant Csf3r.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Mutação , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Fator de Transcrição STAT5/fisiologia , Animais , Proliferação de Células/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neutropenia/congênito , Fator de Transcrição STAT3/fisiologiaRESUMO
Inhibitors of phenoloxidase are used routinely to characterise the structural and functional properties of phenoloxidases. Hemocyanin-derived phenoloxidase activity is also sensitive to standard phenoloxidase inhibitors. In this study, we characterise the effects of a number of phenoloxidase inhibitors on hemocyanin-derived phenoloxidase activity from the chelicerate, Limulus polyphemus. Both inhibition type and K(i) values were similar to those observed for hemocyanin-derived phenoloxidase from another chelicerate, Eurypelma californicum. In addition, substrate inhibition was observed at concentrations above 2mM dopamine. The conformation in which two of the inhibitors, namely tropolone and kojic acid, would bind near the Cu(II) centre of hemocyanin is proposed.
Assuntos
Quelantes/farmacologia , Inibidores Enzimáticos/farmacologia , Hemocianinas/antagonistas & inibidores , Hemocianinas/metabolismo , Caranguejos Ferradura/enzimologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Animais , Ligação Competitiva , Quelantes/metabolismo , Cobre/metabolismo , Dopamina/metabolismo , Inibidores Enzimáticos/metabolismo , Hemocianinas/química , Hexilresorcinol/metabolismo , Hexilresorcinol/farmacologia , Cinética , Modelos Moleculares , Feniltioureia/metabolismo , Feniltioureia/farmacologia , Conformação Proteica , Pironas/metabolismo , Pironas/farmacologia , Tropolona/metabolismo , Tropolona/farmacologiaRESUMO
Expression of the constitutively activated TEL/PDGFbetaR fusion protein is associated with the t(5;12)(q33;p13) chromosomal translocation found in a subset of patients with chronic myelomonocytic leukemia. TEL/PDGFbetaR activates multiple signal transduction pathways in cell-culture systems, and expression of the TEL-PDGFRB fusion gene induces myeloproliferative disease (MPD) in mice. We used gene-targeted mice to characterize the contribution of signal transducer and activator of transcription (Stat) and Src family genes to TEL-PDGFRB-mediated transformation in methylcellulose colony and murine bone marrow transduction/transplantation assays. Fetal liver hematopoietic stem and progenitor cells harboring targeted deletion of both Stat5a and Stat5b (Stat5ab(null/null)) genes were refractory to transformation by TEL-PDGFRB in methylcellulose colony assays. Notably, these cell populations were maintained in Stat5ab(null/null) fetal livers and succumbed to transformation by c-Myc. Surprisingly, targeted disruption of either Stat5a or Stat5b alone also impaired TEL-PDGFRB-mediated transformation. Survival of TPiGFP-->Stat5a(-/-) and TPiGFP-->Stat5a(+/-) mice was significantly prolonged, demonstrating significant sensitivity of TEL-PDGFRB-induced MPD to the dosage of Stat5a. TEL-PDGFRB-mediated MPD was incompletely penetrant in TPiGFP-->Stat5b(-/-) mice. In contrast, Src family kinases Lyn, Hck, and Fgr and the Stat family member Stat1 were dispensable for TEL-PDGFRB disease. Together, these data demonstrate that Stat5a and Stat5b are dose-limiting mediators of TEL-PDGFRB-induced myeloproliferation.
Assuntos
Dosagem de Genes , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas de Fusão Oncogênica/biossíntese , Proteínas Proto-Oncogênicas c-ets/biossíntese , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Proteínas Repressoras/biossíntese , Fator de Transcrição STAT5/biossíntese , Transdução de Sinais , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Regulação Leucêmica da Expressão Gênica/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos Knockout , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-ets/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Repressoras/genética , Fator de Transcrição STAT5/deficiência , Transdução de Sinais/genética , Translocação Genética/genética , Ensaio Tumoral de Célula-Tronco , Quinases da Família src/metabolismo , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The TEL-PDGFRB fusion oncogene is associated with chronic myelomonocytic leukemia (CMML) and results in the expression of a constitutively active tyrosine kinase. SU11657 is a multitargeted selective inhibitor of class III/V receptor tyrosine kinases, including the platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptors KIT and FLT3. SU11657 inhibited TEL/PDGFbetaR kinase activity at nanomolar concentrations and inhibited TELPDGFRB-mediated factor-independent growth in myeloblastic 32D cells. Daily oral administration of SU11657 at 40 mg/kg suppressed myeloproliferation and significantly prolonged survival in TELPDGFRB mice treated prior to disease development, as well as in those with large tumor burdens. Our findings suggest that SU11657 or similar agents may have therapeutic potential in humans with hematologic malignancies expressing PDGFR fusion oncogenes.