RESUMO
MicroRNAs (miRNAs or miRs) are approximately 22 nt single-stranded noncoding RNAs that control gene expression in eukaryotes. miRNAs play an essential role in all basic cellular processes including cell development, proliferation, differentiation, and apoptosis. Importantly, miRNAs regulate hematopoietic progenitor cells differentiation toward the different hematopoietic lineages. This occurs through the regulation of key factors involved in hematopoiesis (e.g., transcription factors, growth factor receptors). We, hereby, describe how to investigate the role of miRNAs in monocytopoiesis.
Assuntos
Hematopoese/genética , MicroRNAs/metabolismo , Monócitos/fisiologia , Adulto , Northern Blotting/instrumentação , Northern Blotting/métodos , Diferenciação Celular/genética , Células Cultivadas , Vetores Genéticos , Células-Tronco Hematopoéticas/fisiologia , Humanos , MicroRNAs/genéticaRESUMO
BACKGROUND: The differentiation process, proceeding from stem cells towards the different committed cell types, can be considered as a trajectory towards an attractor of a dynamical process. This view, taking into consideration the transcriptome and miRNome dynamics considered as a whole, instead of looking at few 'master genes' driving the system, offers a novel perspective on this phenomenon. We investigated the 'differentiation trajectories' of the hematopoietic system considering a genome-wide scenario. RESULTS: We developed serum-free liquid suspension unilineage cultures of cord blood (CB) CD34+ hematopoietic progenitor cells through erythroid (E), megakaryocytic (MK), granulocytic (G) and monocytic (Mo) pathways. These cultures recapitulate physiological hematopoiesis, allowing the analysis of almost pure unilineage precursors starting from initial differentiation of HPCs until terminal maturation. By analyzing the expression profile of protein coding genes and microRNAs in unilineage CB E, MK, G and Mo cultures, at sequential stages of differentiation and maturation, we observed a coordinated, fully interconnected and scalable character of cell population behaviour in both transcriptome and miRNome spaces reminiscent of an attractor-like dynamics. MiRNome and transcriptome space differed for a still not terminally committed behaviour of microRNAs. CONCLUSIONS: Consistent with their roles, the transcriptome system can be considered as the state space of a cell population, while the continuously evolving miRNA space corresponds to the tuning system necessary to reach the attractor. The behaviour of miRNA machinery could be of great relevance not only for the promise of reversing the differentiated state but even for tumor biology.
Assuntos
Diferenciação Celular/fisiologia , Genoma/genética , Células-Tronco Hematopoéticas/fisiologia , Modelos Biológicos , Transdução de Sinais/fisiologia , Antígenos CD34/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem da Célula , Biologia Computacional/métodos , Sangue Fetal/citologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genéticaRESUMO
BACKGROUND: The human hemoglobin switch (HbF-->HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor. DESIGN AND METHODS: We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34(+) cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production. RESULTS: In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80-90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects. CONCLUSIONS: Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221-222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.
Assuntos
Hemoglobina Fetal/metabolismo , Hemoglobina A/metabolismo , MicroRNAs/fisiologia , Fator de Células-Tronco/fisiologia , Adulto , Antígenos CD34/sangue , Benzamidas , Ciclo Celular , Células Cultivadas , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Eritropoese/efeitos dos fármacos , Eritropoese/genética , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Citometria de Fluxo , Expressão Gênica , Humanos , Mesilato de Imatinib , Recém-Nascido , MicroRNAs/genética , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-kit/sangue , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Células-Tronco/sangue , Fator de Células-Tronco/genética , Fatores de TempoRESUMO
The pathophysiology of coronary artery disease (CAD) progression is not well understood. Endothelial progenitor cells (EPCs) may have an important role. In the present observational cohort study we assessed the number of circulating EPCs in 136 patients undergoing elective percutaneous coronary intervention and who had at least one major epicardial vessel with a nonsignificant stenosis [<50% diameter stenosis (DS)], and the relationship between plasma EPC levels and the 24-mo progression of the nonsignificant coronary artery lesion. The following cell populations were analyzed: CD34(+), CD133(+), CD34(+)/KDR(+), CD34(+)/VE cadherin(+), and endothelial cell colony-forming units (CFU-ECs). Progression was defined as a >15% DS increase of the objective vessel at follow-up. At 24 mo, 57 patients (42%) experienced significant progression. Independent predictors of disease progression were LDL cholesterol > 100 mg/dl (OR=1.03; 95% CI 1.01-1.04; P=0.001), low plasma levels of CFU-ECs (OR=3.99; 95% CI 1.54-10.37; P=0.005), and male sex (OR=3.42; 95% CI 1.15-10.22; P=0.027). Circulating levels of EPCs are significantly lower in patients with angiographic CAD progression.
Assuntos
Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/patologia , Endotélio Vascular/citologia , Células-Tronco/metabolismo , Células Cultivadas , Estudos de Coortes , Ensaio de Unidades Formadoras de Colônias , Doença da Artéria Coronariana/metabolismo , Progressão da Doença , Endotélio Vascular/metabolismo , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Curva ROC , Fatores de Risco , Taxa de Sobrevida , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
It is generally conceded that selective combinations of transcription factors determine hematopoietic lineage commitment and differentiation. Here we show that in normal human hematopoiesis the transcription factor nuclear factor I-A (NFI-A) exhibits a marked lineage-specific expression pattern: it is upmodulated in the erythroid (E) lineage while fully suppressed in the granulopoietic (G) series. In unilineage E culture of hematopoietic progenitor cells (HPCs), NFI-A overexpression or knockdown accelerates or blocks erythropoiesis, respectively: notably, NFI-A overexpression restores E differentiation in the presence of low or minimal erythropoietin stimulus. Conversely, NFI-A ectopic expression in unilineage G culture induces a sharp inhibition of granulopoiesis. Finally, in bilineage E + G culture, NFI-A overexpression or suppression drives HPCs into the E or G differentiation pathways, respectively. These NFI-A actions are mediated, at least in part, by a dual and opposite transcriptional action: direct binding and activation or repression of the promoters of the beta-globin and G-CSF receptor gene, respectively. Altogether, these results indicate that, in early hematopoiesis, the NFI-A expression level acts as a novel factor channeling HPCs into either the E or G lineage.
Assuntos
Eritrócitos/metabolismo , Regulação da Expressão Gênica , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/citologia , Fatores de Transcrição NFI/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Globinas beta/metabolismo , Antígenos CD34/biossíntese , Diferenciação Celular , Linhagem da Célula , Eritropoetina/metabolismo , Sangue Fetal/metabolismo , Humanos , Modelos Biológicos , Regiões Promotoras GenéticasRESUMO
BACKGROUND: MicroRNAs are small non-coding RNAs that regulate gene expression through mRNA degradation or translational inhibition. MicroRNAs are emerging as key regulators of normal hematopoiesis and hematologic malignancies. Several miRNAs are differentially expressed during hematopoiesis and their specific expression regulates key functional proteins involved in hematopoietic lineage differentiation. This study focused on the functional role of microRNA-223 (miR-223) on erythroid differentiation. DESIGN AND METHODS: Purified cord blood CD34+ hematopoietic progenitor cells were grown in strictly controlled conditions in the presence of saturating dosage of erythropoietin to selectively induce erythroid differentiation. The effects of enforced expression of miR-223 in unilin-eage erythroid cultures were evaluated in liquid phase culture experiments and clonogenic studies. RESULTS: In unilineage erythroid culture of cord blood CD34+ hematopoietic progenitor cells miR-223 is down-regulated, whereas LMO2, an essential protein for erythroid differentiation, is up-regulated. Functional studies showed that enforced expression of miR-223 reduces the mRNA and protein levels of LMO2, by binding to LMO2 3' UTR, and impairs differentiation of erythroid cells. Accordingly, knockdown of LMO2 by short interfering RNA mimics the action of miR-223. Furthermore, hematopoietic progenitor cells transduced with miR-223 showed a significant reduction of their erythroid clonogenic capacity, suggesting that downmodulation of this miRNA is required for erythroid progenitor recruitment and commitment. CONCLUSIONS: These results show that the decline of miR-223 is an important event for erythroid differentiation that leads to the expansion of erythroblast cells at least partially mediated by unblocking LMO2 protein expression.
Assuntos
Proteínas de Ligação a DNA/genética , Eritropoese , Metaloproteínas/genética , MicroRNAs/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Diferenciação Celular , Células Eritroides , Sangue Fetal , Regulação da Expressão Gênica , Humanos , Proteínas com Domínio LIM , Proteínas Proto-OncogênicasRESUMO
miRNAs (microRNAs) are important regulatory molecules that control gene expression in all eukaryotes. miRNAs play an essential role in basic cellular activities such as proliferation, differentiation, morphogenesis and apoptosis. In haemopoiesis, several miRNA-based pathways have been identified. Importantly, miRNA mutations or mis-expression correlate with various human diseases. In cancer, deregulated miRNAs can function as tumour suppressors or oncogenes. The present review focuses on the recent literature concerning the role of miRNAs in three different research areas: haematology, cardiology and oncology, with particular focus on the results obtained by our group.
Assuntos
Cardiomegalia/metabolismo , Hematopoese/fisiologia , MicroRNAs/metabolismo , Neoplasias/metabolismo , Humanos , MicroRNAs/genéticaRESUMO
OBJECTIVES: Chemotherapy is the preferred therapeutic approach for the therapy of advanced ovarian cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs, microRNAs (miRNAs) in cancer development, with several conjectures regarding their possible involvement in the evolution of drug resistance. This work was aimed to identify selected microRNAs involved in the development of chemoresistance in ovarian cancer. METHODS: High-throughput analysis of the miRNA profile in a panel of paclitaxel- (A2780TAX, A2780TC1 and A2780TC3) and cisplatin-resistant (A2780CIS) cells was assessed using a microarray platform and subsequent validation with qPCR and Northern blots. Downstream target validation was performed for miR-130a and the target M-CSF.] RESULTS: Six miRNAs (let-7e, miR-30c, miR-125b, miR-130a and miR-335) were always diversely expressed in all the resistant cell lines. Let-7e was upregulated in A2780TAX cells, while it was downregulated in the other resistant cell lines. The opposite phenomenon was obtained for miR-125b, which was downregulated in A2780TAX and upregulated in the other cell lines. The miR-30c, miR-130a and miR-335 were downregulated in all the resistant cell lines, thereby suggesting a direct involvement in the development of chemoresistance. Finally downstream target validation was proven for the miR-130a, whose downregulation was linked to the translational activation of the M-CSF gene, a known resistance factor for ovarian cancer. CONCLUSIONS: Our results indicate that ovarian cancer drug resistance is associated with a distinct miRNA fingerprint, and miRNA microarrays could represent a prognostic tool to monitor the chemotherapy outcome.
Assuntos
Cisplatino/farmacologia , MicroRNAs/biossíntese , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Paclitaxel/farmacologia , Antineoplásicos/farmacologia , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Biologia Computacional , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Fator Estimulador de Colônias de Macrófagos/biossíntese , Fator Estimulador de Colônias de Macrófagos/genética , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Regulação para CimaRESUMO
MicroRNAs (miRNAs) are noncoding small RNAs that repress protein translation by targeting specific messenger RNAs. miR-15a and miR-16-1 act as putative tumor suppressors by targeting the oncogene BCL2. These miRNAs form a cluster at the chromosomal region 13q14, which is frequently deleted in cancer. Here, we report that the miR-15a and miR-16-1 cluster targets CCND1 (encoding cyclin D1) and WNT3A, which promotes several tumorigenic features such as survival, proliferation and invasion. In cancer cells of advanced prostate tumors, the miR-15a and miR-16 level is significantly decreased, whereas the expression of BCL2, CCND1 and WNT3A is inversely upregulated. Delivery of antagomirs specific for miR-15a and miR-16 to normal mouse prostate results in marked hyperplasia, and knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, which become tumorigenic in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16-1 expression results in growth arrest, apoptosis and marked regression of prostate tumor xenografts. Altogether, we propose that miR-15a and miR-16 act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion. These findings have therapeutic implications and may be exploited for future treatment of prostate cancer.
Assuntos
MicroRNAs/genética , Família Multigênica/genética , Proteínas Oncogênicas/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Humanos , Masculino , Camundongos , Proteínas Oncogênicas/genética , Neoplasias da Próstata/patologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMO
MicroRNAs (miRNAs) control basic biological functions and are emerging as key regulators of haematopoiesis. This study focused on the functional role of MIRN155 on megakaryocytic (MK) differentiation of human cord blood CD34+ haematopoietic progenitor cells (HPCs). MIRN155, abundantly expressed in early HPCs, decreases sharply during MK differentiation. Functional studies showed that enforced expression of MIRN155 impairs proliferation and differentiation of MK cells. Furthermore, HPCs transfected with MIRN155 showed a significant reduction of their MK clonogenic capacity, suggesting that down-modulation of this miRNA favours MK progenitor differentiation. Consistent with this observation, MIRN155 downregulates, by directly binding to their 3'-UTR, the expression of Ets-1 and Meis1, two transcription factors with well-known functions in MK cells. These results show that the decline of MIRN155 is required for MK proliferation and differentiation at progenitors and precursors level and indicate that sustained expression of MIRN155 inhibits megakaryopoiesis.
Assuntos
Proteínas de Homeodomínio/genética , Megacariócitos/citologia , MicroRNAs/fisiologia , Proteínas de Neoplasias/genética , Proteína Proto-Oncogênica c-ets-1/genética , Trombopoese/genética , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Células K562 , MicroRNAs/metabolismo , Proteína Meis1 , Proteína Proto-Oncogênica c-ets-1/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Trombopoese/fisiologia , TransfecçãoRESUMO
MicroRNAs (miRNAs or miRs) regulate diverse normal and abnormal cell functions. We have identified a regulatory pathway in normal megakaryopoiesis, involving the PLZF transcription factor, miR-146a and the SDF-1 receptor CXCR4. In leukaemic cell lines PLZF overexpression downmodulated miR-146a and upregulated CXCR4 protein, whereas PLZF knockdown induced the opposite effects. In vitro assays showed that PLZF interacts with and inhibits the miR-146a promoter, and that miR-146a targets CXCR4 mRNA, impeding its translation. In megakaryopoietic cultures of CD34(+) progenitors, PLZF was upregulated, whereas miR-146a expression decreased and CXCR4 protein increased. MiR-146a overexpression and PLZF or CXCR4 silencing impaired megakaryocytic (Mk) proliferation, differentiation and maturation, as well as Mk colony formation. Mir-146a knockdown induced the opposite effects. Rescue experiments indicated that the effects of PLZF and miR-146a are mediated by miR-146a and CXCR4, respectively. Our data indicate that megakaryopoiesis is controlled by a cascade pathway, in which PLZF suppresses miR-146a transcription and thereby activates CXCR4 translation.
Assuntos
Hematopoese/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Megacariócitos/fisiologia , MicroRNAs/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais/fisiologia , Sequência de Bases , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/genética , Megacariócitos/citologia , MicroRNAs/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteína com Dedos de Zinco da Leucemia Promielocítica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores CXCR4/genética , Células-Tronco/citologia , Células-Tronco/fisiologia , Transcrição GênicaRESUMO
Mesenchymal stromal cells (MSCs) represent a bone marrow (BM) population, classically defined by five functional properties: extensive proliferation, ability to differentiate into osteoblasts, chondrocytes, adipocytes, and stromal cells-supporting hematopoiesis. However, research progress in this area has been hampered by lack of suitable markers and standardized procedures for MSC isolation. We have isolated a CD146(+) multipotent MSC population from 20 human BM donors displaying the phenotype of self-renewing osteoprogenitors; an extensive 12-week proliferation; and the ability to differentiate in osteoblasts, chondrocytes, adipocytes, and stromal cells supporting hematopoiesis. Furthermore, the CD146(+) MSCs secrete a complex combination of growth factors (GFs) controlling hematopoietic stem cells (HSCs) function, while providing a >2-log increase in the long-term culture (LTC) colony output in 8-week LTC over conventional assays. The hematopoietic stromal function exhibited by the MSCs was further characterized by manipulating LTCs with the chemical inhibitors Imatinib or SU-5416, targeting two GF receptors (GFRs), KIT or VEGFR2/1, respectively. Both treatments similarly impaired LTC colony output, indicating key roles for these two GF/GFR interactions to support LTC-initiating cell activity. CD146(+) MSCs may thus represent a tool to explore the MSC-HSC cross-talk in an in vitro surrogate model for HSC "niches," and for regenerative therapy studies. In addition, the MSC microRNA (miRNA) expression profile was analyzed by microarrays in both basic conditions and chondrogenic differentiation. Our analysis revealed that several miRNAs are modulated during chondrogenesis, and many of their putative targets are genes involved in chondrogenic differentiation.
Assuntos
Antígeno CD146/biossíntese , Linhagem Celular , Células-Tronco Mesenquimais/citologia , MicroRNAs/biossíntese , Células Estromais/citologia , Western Blotting , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Separação Celular/métodos , Condrócitos/citologia , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Análise em Microsséries , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismo , Fatores de TempoRESUMO
We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3' UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/uso terapêutico , Neuroblastoma/patologia , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sequência de Bases , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Genes myc , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , MicroRNAs/genética , Neuroblastoma/tratamento farmacológico , RNA Interferente PequenoRESUMO
The incidence of cutaneous melanoma is steadily increasing. Although several molecular abnormalities have been associated with melanoma progression, the mechanisms underlying the differential gene expression are still largely unknown and targeted therapies are not yet available. Noncoding small RNAs, termed microRNAs (miR), have been recently reported to play important roles in major cellular processes, including those involved in cancer development and progression. We have identified the promyelocytic leukemia zinc finger (PLZF) transcription factor as a repressor of miR-221 and miR-222 by direct binding to their putative regulatory region. Specifically, PLZF silencing in melanomas unblocks miR-221 and miR-222, which in turn controls the progression of the neoplasia through down-modulation of p27Kip1/CDKN1B and c-KIT receptor, leading to enhanced proliferation and differentiation blockade of the melanoma cells, respectively. In vitro and in vivo functional studies, including the use of antisense "antagomir" oligonucleotides, confirmed the key role of miR-221/-222 in regulating the progression of human melanoma; this suggests that targeted therapies suppressing miR-221/-222 may prove beneficial in advanced melanoma.
Assuntos
Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição Kruppel-Like/metabolismo , Melanoma Experimental/genética , MicroRNAs/genética , Neoplasias Cutâneas/genética , Dedos de Zinco , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Inibidor de Quinase Dependente de Ciclina p27 , Progressão da Doença , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Nus , Oligonucleotídeos Antissenso/farmacologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
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
In human beta-thalassemia, the imbalance between alpha- and non-alpha-globin chains causes ineffective erythropoiesis, hemolysis, and anemia: this condition is effectively treated by an enhanced level of fetal hemoglobin (HbF). In spite of extensive studies on pharmacologic induction of HbF synthesis, clinical trials based on HbF reactivation in beta-thalassemia produced inconsistent results. Here, we investigated the in vitro response of beta-thalassemic erythroid progenitors to kit ligand (KL) in terms of HbF reactivation, stimulation of effective erythropoiesis, and inhibition of apoptosis. In unilineage erythroid cultures of 20 patients with intermedia or major beta-thalassemia, addition of KL, alone or combined with dexamethasone (Dex), remarkably stimulated cell proliferation (3-4 logs more than control cultures), while decreasing the percentage of apoptotic and dyserythropoietic cells (<5%). More important, in both thalassemic groups, addition of KL or KL plus Dex induced a marked increase of gamma-globin synthesis, thus reaching HbF levels 3-fold higher than in con-trol cultures (eg, from 27% to 75% or 81%, respectively, in beta-thalassemia major). These studies indicate that in beta-thalassemia, KL, alone or combined with Dex, induces an expansion of effective erythropoiesis and the reactivation of gamma-globin genes up to fetal levels and may hence be considered as a potential therapeutic agent for this disease.
Assuntos
Eritropoese/efeitos dos fármacos , Hemoglobina Fetal/fisiologia , Fator de Células-Tronco/farmacologia , Talassemia beta/sangue , Talassemia beta/tratamento farmacológico , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Dexametasona/farmacologia , Quimioterapia Combinada , Eritrócitos/citologia , Eritrócitos/fisiologia , Glucocorticoides/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , HumanosRESUMO
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.