RESUMO
FMS-related tyrosine kinase 3 (FLT3) mutations are found in 30% of cases of acute myeloid leukaemia (AML). In addition, recent studies have lead to the identification of about 10-15% of AML patients displaying high expression of FLT3, not associated with mutations of the receptor (FLT3 Wild-type High, FLT3WTH). These AMLs, as well as those displaying internal tandem duplication (ITD) are associated with an unfavourable prognosis. However, the biological features of these AMLs are poorly characterized. The present study explored the immunophenotypic features of FLT3WTH AMLs in 94 de novo cases of AML. The levels of FLT3 expression, as assessed by flow cytometry and FLT3 mutational status, was used to identify four AML subgroups: FLT3WTH (14/94); FLT3 Wild-type low (FLT3WTL, 48/94); FLT3 internal tandem duplication (FLT3ITD 26/94); FLT3 aspartic acid 835 (FLT3D835, 6/94). FLT3WTH and FLT3ITD were characterized by: high white blast cell counts; predominance of M4 and M5 French-American-British classification subtypes and associated expression of myelo-monocytic markers; high expression of CD123 and TRAIL-Rs; high expression of receptors for angiogenic growth factors. Addition of FLT3 Ligand to human CD34(+) or monocytic cells stimulated CD123 and TRAIL-R expression. These findings are of potential value for the development of new therapeutic strategies.
Assuntos
Leucemia Mieloide Aguda/imunologia , Tirosina Quinase 3 Semelhante a fms/metabolismo , Antígenos CD34/análise , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-3/genética , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas de Membrana/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Mutação , Receptor TIE-2/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células Tumorais Cultivadas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Previous studies suggested an important role for vascular endothelial growth factor (VEGF) and its receptors in postnatal haemopoiesis. However, it is unclear how VEGF receptor (VEGFR) signalling could interact with that issued from the activation of haematopoietic growth factor receptors. To elucidate this point we explored VEGF-R2 and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) membrane localization and cell signalling in TF1-KDR cells (TF1 leukaemic cells that overexpress VEGF-R2/KDR). Activation of either GM-CSFR or VEGF-R2 was shown to determine the migration of both receptor elements (VEGF-R2 and the common beta-chain of the GM-CSFR) to lipid rafts. The study of receptor phosphorylation showed that GM-CSF induced the phosphorylation of its own receptor and the transphosphorylation of VEGF-R2; on the other hand, VEGF triggered the phosphorylation of its receptor and transphosphorylated the beta-chain of the GM-CSFR. Co-stimulation of TF1-KDR cells with both GM-CSF and VEGF-A resulted in massive migration of both the common GM-CSFR beta-chain and VEGF-R2 to lipid rafts and sustained p38 mitogen-activated protein kinase activation. Disruption of lipid rafts inhibited the capacity of both GM-CSF and VEGF-A to activate p38. Experiments with specific p38 inhibitors showed that p38 activation was required to sustain the VEGF- and GM-CSF-dependent proliferation of TF1-KDR and the survival of primary acute myeloid leukaemia blasts.
Assuntos
Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Interleucina-3/metabolismo , Leucemia Mieloide Aguda/metabolismo , Microdomínios da Membrana/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Apoptose , Transporte Biológico , Western Blotting/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Ativação Enzimática , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Imidazóis/farmacologia , Imunofenotipagem , Imunoprecipitação , Leucemia Mieloide Aguda/patologia , Fosforilação , Piridinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
The common beta chain subunit (beta(c)), also known as CDw131, shared by the interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor (GM-CSF) and IL-5 receptors, is required for high-affinity ligand binding and signal transduction. The present study explored the expression of CDw131 in 105 de novo cases of acute myeloid leukaemia (AML). The levels of CDw131 expression were used to identify two AML subgroups characterized by low (75/105) and high (30/105) expression of this receptor chain. It was observed that (i) the level of CDw131 expression strictly correlated with the level of CD116 (GM-CSFalpha receptor chain) and CD123 (IL-3Ralpha chain); (ii) AMLs with high CDw131 expression were characterized by low CD34 expression and usually high CD11b, CD14 expression; (iii) AMLs with high CDw131 expression frequently co-expressed receptors for angiogenic growth factors (vascular endothelial growth factor R2, Tie-2); (iv) AMLs with high CDw131 expression were more cycling than those with low CDw131 expression; (v) AMLs with high CDw131 frequently displayed Feline Murine Sarcoma (FMS-related) tyrosine kinase 3 (FLT3) internal tandem duplication and constitutively activated Signal Transducer and Activator of Transcription-5 (STAT5). In conclusion, the analysis of the level of CDw131 expression enabled the identification of a subset of AMLs characterized by a high cycling status, the expression of myelo-monocytic markers, mutated FLT3 and the co-expression of receptors for angiogenic growth factors. These findings are of value for the development of new therapeutic strategies for the treatment of these AMLs.
Assuntos
Subunidade beta Comum dos Receptores de Citocinas/análise , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/imunologia , Mutação , Tirosina Quinase 3 Semelhante a fms/genética , Biomarcadores/análise , Western Blotting/métodos , Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Citometria de Fluxo , Humanos , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-3/análise , Subunidade alfa de Receptor de Interleucina-5/metabolismo , Contagem de Leucócitos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Translocação Genética , Células Tumorais Cultivadas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análiseRESUMO
The synthetic triterpenoid CDDO-Im-induced apoptosis of patient-derived AML blasts: 11/25 AMLs were highly sensitive, while the remaining were moderately sensitive to CDDO-Im. The addition of TRAIL significantly potentiated the cytotoxic effect of CDDO-Im, through mechanisms involving the induction of TRAIL-R1/TRAIL-R2 and downmodulation of TRAIL-R3/TRAIL-R4. Biochemical studies showed that CDDO-Im: induced a rapid and marked GSH depletion and antioxidants (GSH or NAC) completely inhibited its pro-apoptotic effect; sequentially activated caspase-8, -9 and -3; caspase inhibitors partially protected AML blasts from CDDO-Im-induced apoptosis; resistance of AML blasts to CDDO-Im-induced apoptosis correlated with low caspase-8/FADD and high Bcl-X(L) expression in leukemic blasts.
Assuntos
Caspase 8/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Imidazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Ácido Oleanólico/análogos & derivados , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Humanos , Ácido Oleanólico/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/análise , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Células Tumorais CultivadasRESUMO
Chronic obstructive pulmonary disease (COPD) patients have reduced circulating hemopoietic progenitors. We hypothesized that severity of COPD parallels the decrease in progenitors and that the reduction in body mass index (BMI) could be associated with more severe bone marrow dysfunction. We studied 39 patients with moderate to very severe COPD (18 with low-BMI and 21 with normal-BMI) and 12 controls. Disease severity was associated to a greater reduction in circulating progenitors. Proangiogenetic and inflammatory markers correlated with disease severity parameters. Compared to normal-BMI patients, low-BMI patients showed: greater reduction in circulating progenitors; higher VEGF-A, VEGF-C, HGF, Ang-2, TNF-alpha, IL-6 and MCP-1 levels. Furthermore, among patients with similar pulmonary impairment, those who displayed low-BMI had a more markedly reduced number of CD34(+) cells and late endothelial progenitors. We show that the reduction in hematopoietic and endothelial progenitor cells correlates with COPD severity. Our findings also indicate that, in severe low-BMI COPD patients, bone marrow function seems to be further impaired and may lead to reduced reparative capacity.
Assuntos
Índice de Massa Corporal , Transplante de Medula Óssea/métodos , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/cirurgia , Idoso , Análise de Variância , Antígenos CD/metabolismo , Contagem de Células Sanguíneas/métodos , Estudos de Casos e Controles , Ensaio de Unidades Formadoras de Colônias/métodos , Creatina Quinase/sangue , Citocinas/sangue , Células Endoteliais/fisiologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lactato Desidrogenases/sangue , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Estatística como AssuntoRESUMO
We investigated the expression of Tie-2 in primary blasts from 111 patients with acute myeloid leukemia (AML) to evaluate a possible linkage between the expression of this receptor and the immunophenotypic and biologic properties of leukemic blasts. Tie-2 was expressed at moderate and high levels in 39 and 23 of 111 AMLs, respectively. The analysis of the immunophenotype clearly showed that Tie-2 expression in AML was associated with monocytic features. Interestingly, Tie-2 expression on AML blasts was associated with concomitant expression of other receptors for endothelial growth factors, such as vascular endothelial growth factor receptor 1 (VEGF-R1), -R2, and -R3. Tie-2(+) AMLs were characterized by high blast cell counts at diagnosis, a high frequency of Flt3 mutations, and increased Flt3 expression. The survival of Tie-2(+) AMLs is sustained through an autocrine pattern involving Angiopoietin-1 and Tie-2, as suggested by experiments showing induction of apoptosis in Tie-2(+) AMLs by agents preventing the binding of angiopoietins to Tie-2. Finally, the in vitro growth of Tie-2(+) AMLs in endothelial culture medium supplemented with VEGF and angiopoietins resulted in their partial endothelial differentiation. These observations suggest that Tie-2(+) AMLs pertain to a mixed monocytic/endothelial lineage, derived from the malignant transformation of the normal counterpart represented by monocytic cells expressing endothelial markers. The autocrine angiopoietin/Tie-2 axis may represent a promising therapeutic target to improve the outcome of patients with monocytic AML. Disclosure of potential conflicts of interest is found at the end of this article.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Células Precursoras de Granulócitos/metabolismo , Leucemia Mieloide/metabolismo , Monócitos/metabolismo , Receptor TIE-2/metabolismo , Doença Aguda , Angiopoietina-1/metabolismo , Angiopoietina-1/farmacologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular , Células Endoteliais/citologia , Células Precursoras de Granulócitos/citologia , Células Precursoras de Granulócitos/efeitos dos fármacos , Células Precursoras de Granulócitos/patologia , Humanos , Imunofenotipagem , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/patologia , Contagem de Leucócitos , Mutação , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/farmacologia , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
The present study explored the sensitivity of leukaemic blasts derived from 30 acute myeloid leukaemia (AML) patients to Bortezomib. Bortezomib induced apoptosis of primary AML blasts: 18/30 AMLs were clearly sensitive to the proapoptotic effects of Bortezomib, while the remaining cases were moderately sensitive to this molecule. The addition of tumour necrosis factor-related-apoptosis-inducing ligand, when used alone, did not induce apoptosis of AML blasts and further potentiated the cytotoxic effects of Bortezomib. The majority of AMLs sensitive to Bortezomib showed immunophenotypic features of the M4 and M5 French-American-British classification subtypes and displayed myelomonocytic features. All AMLs with mutated FLT3 were in the Bortezomib-sensitive group. Biochemical studies showed that: (i) Bortezomib activated caspase-8 and caspase-3 and decreased cellular FLICE [Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme]-inhibitory protein (c-FLIP) levels in AML blasts; (ii) high c-FLIP levels in AML blasts were associated with low Bortezomib sensitivity. Finally, analysis of the effects of Bortezomib on leukaemic cells displaying high aldehyde dehydrogenase activity suggested that this drug induced in vitro killing of leukaemic stem cells. The findings of the present study, further support the development of Bortezomib as an anti-leukaemic drug and provide simple tools to predict the sensitivity of AML cells to this drug.
Assuntos
Ácidos Borônicos/uso terapêutico , Leucemia Monocítica Aguda/tratamento farmacológico , Leucemia Mielomonocítica Aguda/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , Pirazinas/uso terapêutico , Aldeído Desidrogenase/metabolismo , Apoptose , Bortezomib , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/análise , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Proteína de Domínio de Morte Associada a Fas/análise , Proteína de Domínio de Morte Associada a Fas/metabolismo , Citometria de Fluxo , Humanos , Imunofenotipagem , Leucemia Monocítica Aguda/patologia , Leucemia Mielomonocítica Aguda/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/análise , Células-Tronco/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/análise , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/análise , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
We have investigated the expression of podocalyxin in primary cultures of leukemic blast cells from 73 patients with acute myeloid leukemia. Podocalyxin was expressed at moderate levels in 15 patients and at high levels in 13 patients. The analysis of membrane markers showed that Podocalyxin expression in leukemic blasts was associated with a monocytic immunophenotype. Cases of podocalyxin-positive acute myelogenous leukemia had high blast cell counts at diagnosis and elevated CD123, CD135, VLA-4 and CXCR4 expression, features associated with poor prognosis. Podocalyxin expression in leukemic blasts was coupled with the concomitant expression of VEGF-R1, -R2, -R3 and Tie-2, the capacity to release VEGF-A and angiopoietin1 and the ability to differentiate into endothelial cells under appropriate culture conditions. These findings show that podocalyxin is a marker of acute myeloid leukemia with a monocytic phenotype and suggest that podocalyxin-positive cases of acute myeloid leukemia originate from the malignant transformation of progenitors common to the myeloid and endothelial lineages. These observations suggest a possible relationship between the monocytic lineage and podocytes.