RESUMO
OBJECTIVE: To investigate the effects of total saponins from Sanguisorba officinalis (DYS) on hematopoietic cell proliferation, differentiation and the expression level of IL-3R and c-kit. METHOD: Baf3 and 32D cells were cultured with or without IL-3, then the cells were exposed to DYS in different concentrations of 5, 10, 20, 30 and 40 mg x L(-1) for 24, 48, 72 and 96 hours separately. After that, the cell proliferation and differentiation capacity were determinated by the methods of CCK8 and Giemsa staining separately. The effects of DYS on the expression level of IL-3 receptor in Baf3 cells and the expression level of c-kit in 32D cells were determinated using RT-PCR. RESULT: DYS promotes alone proliferation of Baf3 cells and 32D cells after 48 h. In contrast to control cells, 32D cells containing DYS without IL-3 form many large clusters. DYS also increases the proliferation when cultured with IL-3. High concentration of DYS induce alone the differentiation of 32D cells and increase alone the number of the polyploidy megakaryocyte. Moreover, DYS increases alone the expression level of IL-3R in Baf3 cells and the expression level of c-kit in 32D cells separately. CONCLUSION: Our data shows DYS can promote alone proliferation and differentiation of megakaryocyte progenitor cells. The proliferative and differentiative effect of DYS on megakaryocyte progenitor cells is correlated to the up-regulation of IL-3 receptor and c-kit expression.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Progenitoras de Megacariócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/genética , Receptores de Interleucina-3/genética , Sanguisorba/química , Saponinas/farmacologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Interleucina-3/farmacologia , Células Progenitoras de Megacariócitos/metabolismo , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.
Assuntos
Janus Quinase 2/fisiologia , Microdomínios da Membrana/fisiologia , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/enzimologia , Mutação Puntual , Transdução de Sinais/efeitos dos fármacos , Sinvastatina/farmacologia , beta-Ciclodextrinas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Colesterol/análise , Colesterol/fisiologia , Ensaio de Unidades Formadoras de Colônias , Avaliação Pré-Clínica de Medicamentos , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/enzimologia , Humanos , Janus Quinase 2/genética , Células K562/efeitos dos fármacos , Células K562/enzimologia , Leucemia Eritroblástica Aguda/enzimologia , Leucemia Eritroblástica Aguda/patologia , Leucemia Megacarioblástica Aguda/enzimologia , Leucemia Megacarioblástica Aguda/patologia , Células Progenitoras de Megacariócitos/efeitos dos fármacos , Células Progenitoras de Megacariócitos/enzimologia , Lipídeos de Membrana/fisiologia , Microdomínios da Membrana/efeitos dos fármacos , Transtornos Mieloproliferativos/sangue , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fator de Transcrição STAT5/metabolismoRESUMO
BACKGROUND: A novel family of intracellular Ca(2+)-release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca(2+) mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca(2+) release from the acidic compartments. OBJECTIVES: The present study is aimed to investigate NAADP-mediated Ca(2+) release from intracellular stores in the megakaryoblastic cell line MEG01. METHODS: Changes in cytosolic and intraluminal free Ca(2+) concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR. RESULTS: Treatment of MEG01 cells with the H(+)/K(+) ionophore nigericin or the V-type H(+)-ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca(2+) stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca(2+) from acidic lysosomal-like Ca(2+) stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca(2+) efflux from the acidic lysosomal-like Ca(2+) stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca(2+)-induced Ca(2+) release (CICR) from the complementary Ca(2+) compartment. CONCLUSION: Our results show for the first time NAADP-evoked Ca(2+) release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.
Assuntos
Sinalização do Cálcio/fisiologia , Células Progenitoras de Megacariócitos/metabolismo , NADP/análogos & derivados , Sequência de Bases , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Compartimento Celular , Linhagem Celular , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Hidroquinonas/farmacologia , Ionóforos/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Células Progenitoras de Megacariócitos/efeitos dos fármacos , NADP/metabolismo , Nigericina/farmacologia , RNA Interferente Pequeno/genética , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidoresRESUMO
In this unit, protocols to culture human platelet progenitors (Colony Forming Unit-Megakaryocyte; CFU-M) with or without toxicants are described. Platelet progenitors are obtained from human umbilical cord blood. After separation of mononuclear cells, the cell suspension can be cryopreserved or plated immediately. Megakaryocytes are identified by immunocytochemistry. Test chemicals are added to the culture medium before cells are plated. Megakaryocytes are scored after 12 days of culture. IC(10), IC(50) and IC(90) can be calculated by comparison to control cultures. A predictive model is proposed to evaluate the hazard of thrombocytopenia induced by chemicals. When IC(50) and IC(90) are below C(max) in humans, the likelihood of thrombocytopenia is strong.