RESUMO
The blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of crucial transcription factors. One of the key master regulators in the hematopoietic systems is PU.1. Reduced levels of PU.1 are characteristic for human acute myeloid leukemia (AML) and are known to induce AML in mouse models. Here, we show that transcriptional downregulation of PU.1 is an active process involving an alternative promoter in intron 3 that is induced by RUNX transcription factors driving noncoding antisense transcription. Core-binding factor (CBF) fusions RUNX1-ETO and CBFß-MYH11 in t(8;21) and inv(16) AML, respectively, activate the PU.1 antisense promoter that results in a shift from sense toward antisense transcription and myeloid differentiation blockade. In patients with CBF-AML, we found that an elevated antisense/sense transcript and promoter accessibility ratio represents a hallmark compared with normal karyotype AML or healthy CD34+ cells. Competitive interaction of an enhancer with the proximal or the antisense promoter forms a binary on/off switch for either myeloid or T-cell development. Leukemic CBF fusions thus use a physiological mechanism used by T cells to decrease sense transcription. Our study is the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. Hence, this disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Elementos Antissenso (Genética)/genética , Linhagem Celular Tumoral , Fusão Gênica , Humanos , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas , Proteína 1 Parceira de Translocação de RUNX1/genética , Células Tumorais CultivadasRESUMO
Tumor necrosis factor (TNF) inhibits granulocyte-colony-stimulating factor (G-CSF)-induced human acute myeloid leukemia (AML) growth in vitro. Incubation of blasts from three patients with AML in serum-free medium with TNF (10(3) U/ml), and subsequent binding studies using 125I-G-CSF reveal that TNF downregulates the numbers of G-CSF receptors by approximately 70%. G-CSF receptor numbers on purified blood granulocytes are also downmodulated by TNF. Downregulation of G-CSF receptor expression becomes evident within 10 min after incubation of the cells with TNF at 37 degrees C and is not associated with an apparent change of the dissociation constant (Kd). The TNF effect does not occur at 0 degrees C and cannot be induced by IL-2, IL-6, or GM-CSF. TNF probably exerts its effect through activation of protein kinase C (PKC) as the TNF effect on G-CSF receptor levels can be mimicked by 12-O-tetradecanoylphorbol-13- acetate. The PKC inhibitor Staurosporine (Sigma Chemical Co., St. Louis, MO) as well as protease inhibitors can completely prevent G-CSF receptor downmodulation. Thus, it appears TNF may act as a regulator of G-CSF receptor expression in myeloid cells and shut off G-CSF dependent hematopoiesis. The regulatory ability of TNF may explain the antagonism between TNF and G-CSF stimulation.
Assuntos
Fator Estimulador de Colônias de Granulócitos/metabolismo , Granulócitos/fisiologia , Leucemia Mieloide/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Fator de Necrose Tumoral alfa/farmacologia , Doença Aguda , Alcaloides/farmacologia , Regulação para Baixo/efeitos dos fármacos , Granulócitos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Inibidores de Proteases/farmacologia , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
Expression of the Evi-1 gene is activated in murine myeloid leukemias by retroviral insertions and in human acute myelogenous leukemia by translocations and inversions involving chromosome band 3q26 where the gene resides. Aberrant expression of the Evi-1 gene has been shown to interfere with myeloid differentiation, which is proposed to be the basis for its role in leukemias. The Evi-1 gene encodes a 145-kDa DNA-binding protein containing two domains of seven and three Cys2-His2 zinc fingers. Previous studies identified a portion of the consensus DNA-binding sequence for the first domain of zinc fingers. The experiments presented here extend these studies and demonstrate that the first domain recognizes a consensus of 15 nucleotides consisting of GA(C/T)AAGA(T/C)AAGATAA. The first three fingers of the first domain do not detectably bind DNA but contribute to the binding by conferring a relative specificity for GACAA verses GATAA in the first position. The first three fingers also contribute to optimal binding of the 15-nucleotide consensus sequence.
Assuntos
Proteínas de Ligação a DNA/genética , Proto-Oncogenes , Fatores de Transcrição , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Consenso , DNA , Proteínas de Ligação a DNA/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
The mechanism underlying p210(BCR/ABL) oncoprotein-mediated transformation in chronic myelogenous leukemia (CML) is not fully understood. We hypothesized that p210(BCR/ABL) suppresses expression of genes which may explain at least some of the pathogenetic features of CML. A subtractive cDNA library was created between BCR/ABL-enhanced-green-fluorescent-protein (GFP)-transduced umbilical cord blood (UCB) CD34+ cells and GFP-transduced UCB CD34+ cells to identify genes whose expression is downregulated by p210(BCR/ABL). At least 100 genes were identified. We have confirmed for eight of these genes that expression was suppressed by quantitative real-time-RT-PCR (Q-RT-PCR) of additional p210(BCR/ABL)-transduced CD34+ UCB cells as well as primary early chronic phase (CP) bone marrow (BM) CML CD34+ cells. Imatinib mesylate reversed downregulation of some genes, to approximately normal levels. Several of the genes are implicated in cell adhesion and motility, including L-selectin, intercellular adhesion molecule-1 (ICAM-1), and the chemokine receptor, CCR7, consistent with the known defect in adhesion and migration of CML cells. Compared with GFP UCB or normal (NL) BM CD34+ cells, p210 UCB and CML CD34+ cells migrated poorly towards the CCR7 ligands, CCL19 and CCL21, suggesting a possible role for CCR7 in the abnormal migratory behavior of CML CD34+ cells.
Assuntos
Movimento Celular/fisiologia , Quimiocinas CC/fisiologia , Proteínas de Fusão bcr-abl/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Receptores de Quimiocinas/fisiologia , Adesão Celular/genética , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/genética , Quimiocina CCL19 , Quimiocina CCL21 , Quimiocinas CC/genética , Regulação para Baixo , Proteínas de Fusão bcr-abl/genética , Humanos , Molécula 1 de Adesão Intercelular/genética , Células K562 , Selectina L/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Ligantes , RNA Mensageiro/genética , Receptores CCR7 , Receptores de Quimiocinas/genéticaRESUMO
The EVI1 gene is activated by chromosomal translocations and inversions in approximately 5% of human acute myeloid leukemia (AML) and by retroviral insertion in approximately 20% of murine myeloid leukemias. EVI1 encodes a nuclear DNA-binding protein having 10 zinc finger motifs in two noncontiguous domains consisting of an amino-terminal domain of seven fingers and a carboxyl domain containing three fingers. To evaluate the sequence specificity of Evi-1 binding and potentially identify genomic targets, whole-genome PCR was utilized to isolate multiple Sau3A fragments which specifically bind to the amino-terminal zinc finger domain. The majority of these clones represented single copy sequences and virtually all contained variable numbers of repeats of the GATA motif, the target sequence for the erythroid-specific transcription factor GATA-1. GST/Evi-1 fusion proteins containing the amino-terminal domain of zinc fingers bound the GATA motif in these clones as well as to those present in the human gamma-globin promoter, similar to the binding of purified GATA-1 protein. By obtaining corresponding large genomic clones for eight of these fragments, transcription units were found associated with two. One corresponded to the glyceraldehyde-3-phosphate dehydrogenase gene and its expression was not affected by Evi-1. The second is a novel gene whose expression is repressed in murine myeloid cell lines that express Evi-1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proto-Oncogenes , Fatores de Transcrição/metabolismo , Dedos de Zinco , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Globinas/genética , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido NucleicoRESUMO
Previous studies have shown that the phenotypes of progenitors of human AML (AML-CFU) are variable, reflecting arrests at different stages of maturation. We were interested to seek discrepancies between the surface properties of AML precursors and normal bone marrow colony formers in order to detect minimal numbers of AML cells among normal bone marrow cells in remission bone marrow. Therefore, we selected two surface markers, the MoAb CD34, reactive with blast cells, and Vim-2, a surface marker reactive with mature myeloid cells, and determined the antigen density of these markers (relative fluorescence intensity using fluorescence-activated cell sorting) for normal marrow and AML progenitors. While these markers defined an identical phenotype (CD34++/Vim-2-/+) for a broad spectrum of normal progenitors, i.e., CFU-GEMM, BFU-e, day 15 CFU-GM, and day 7 CFU-GM, referred to as the "normal" progenitor phenotype, AML progenitors frequently exhibited different phenotypes. In 12 of 20 cases the phenotypes of the majority of AML progenitors were discrepant from the normal surface profile, i.e., according to one marker in 8 cases (CD34-/+/Vim-2-/+ or CD34++/Vim-2++) and two markers in 4 cases (CD34-/+/Vim-2++). Since these data indicate that AML and normal progenitors were frequently distinguishable, we then determined the potential utility of these phenotypic dissimilarities for detection of minimal disease. Artificial mixtures of normal bone marrow and minimal numbers (0.1-1%) of AML cells were prepared. Based upon the phenotypic discrepancies, AML metaphases were successfully demonstrated in these mixtures following cell sorting and culture. Thus, it appears that minimal numbers of AML mitoses can be identified with an approximate 10(-2) to 10(-3) sensitivity by taking advantage of differential coexpression of surface antigens.
Assuntos
Exame de Medula Óssea/métodos , Medula Óssea/patologia , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/patologia , Monitorização Imunológica , Células-Tronco Neoplásicas/patologia , Anticorpos Monoclonais , Antígenos de Neoplasias/análise , Diferenciação Celular , Separação Celular , Citometria de Fluxo , Células-Tronco Hematopoéticas/classificação , Humanos , FenótipoRESUMO
Monitoring of daunorubicin (DNR) concentrations in leukemic cells in blood and bone marrow in vivo of patients with acute myeloid leukemia may yield insight into the interindividual variations of the clinical response to treatment. We evaluated the applicability of flow cytometry for measuring DNR uptake in direct comparison with high performance liquid chromatography (HPLC). In vitro studies revealed good correlations between the mean cellular fluorescence measured by flow cytometry and the cellular DNR concentrations determined with HPLC. In vivo cell measurements were then obtained in 17 evaluable patients during their first remission induction treatment with DNR and cytosine arabinoside. The results indicate that: (a) DNR fluorescence of leukemic blast cells is intermediate between the smaller lymphocytes and the approximately equally large granulocytes; (b) DNR fluorescence of peripheral blast cells and bone marrow blast cells correlate well (p less than 0.001); and (c) patients reaching complete remission show a tendency of higher DNR fluorescence of leukemic blast cells than do partial responders.
Assuntos
Daunorrubicina/farmacocinética , Citometria de Fluxo , Leucemia Mieloide Aguda/metabolismo , Adolescente , Adulto , Idoso , Medula Óssea/metabolismo , Cromatografia Líquida de Alta Pressão , Daunorrubicina/uso terapêutico , Feminino , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucócitos/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
We have investigated the stimulative effects of mast cell growth factor (MGF) in primary acute myeloid leukemia (AML) in vitro. MGF stimulated DNA synthesis of purified leukemic blasts in eight out of 10 cases and colony formation in four cases in serum-free (SF) culture. MGF synergized with interleukin-3 (IL-3; four out of 10 cases), granulocyte-macrophage colony-stimulating factor (GM-CSF; three out of 10 cases), granulocyte colony-stimulating factor (G-CSF; six out of 10 cases), macrophage colony-stimulating factor (M-CSF; one out of 10 cases) and erythropoietin (EPO; one out of 10 cases) when added to culture in combination. Synergistic effects of MGF in combination with other CSFs were also seen in the colony assay. Antibodies against GM-CSF, M-CSF, G-CSF, and IL-6 did not inhibit the MGF response, suggesting that the stimulative effect of MGF was not mediated through autocrine release of those cytokines. Cell recovery data in liquid cultures that contained MGF, IL-3, or MGF + IL-3, indicated that both MGF and IL-3 augmented the maintenance of clonogenic cells as compared to nonsupplemented cultures, but the effect of the combination of IL-3 + MGF did not show synergy. In contrast, activation of DNA synthesis by MGF was abrogated in the presence of tumor necrosis factor (TNF; four out of 10 cases) and interleukin-4 (IL-4; two out of 10 cases). Fluorescence-activated cell sorting (FACS) analysis with anti c-kit antibodies revealed MGF receptor expression in eight out of nine cases, often in a subpopulation of the cells. Scatchard analysis of MGF receptors in two cases indicated the presence of 1460 and 41,500 (mean) binding sites, respectively, of high affinity (Kd 40-160 pmol/l). The MGF dose-response curve in the presence of IL-3 or GM-CSF resulted in a higher plateau of DNA synthesis, however no shift in the dose response was apparent. The respective reciprocal dose response relations to GM-CSF, IL-3, or G-CSF were similarly elevated when MGF was added. MGF did not alter IL-3 and GM-CSF receptor expression, nor did IL-3, GM-CSF, G-CSF, TNF, or IL-4 influence MGF binding to AML cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Citocinas/farmacologia , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Leucemia Mieloide/tratamento farmacológico , Doença Aguda , Anticorpos/farmacologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Interações Medicamentosas , Sinergismo Farmacológico , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fatores de Crescimento de Células Hematopoéticas/imunologia , Fatores de Crescimento de Células Hematopoéticas/fisiologia , Humanos , Interleucina-3/farmacologia , Leucemia Monocítica Aguda/tratamento farmacológico , Leucemia Monocítica Aguda/patologia , Leucemia Mieloide/patologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucemia Mielomonocítica Aguda/tratamento farmacológico , Leucemia Mielomonocítica Aguda/patologia , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-kit , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/fisiologia , Receptores de Interleucina-3/efeitos dos fármacos , Receptores de Interleucina-3/fisiologia , Sensibilidade e Especificidade , Fator de Células-Tronco , Células Tumorais Cultivadas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Interleukin-1 (IL-1) has hemopoietin-1 (H-1) activity, i.e., it synergizes with macrophage-colony stimulating factor (M-CSF), granulocyte-macrophage-CSF (GM-CSF) and interleukin-3 (IL-3) in stimulating in vitro colony formation of hematopoietic progenitor cells. In this study the synergistic activity of IL-1 was investigated on IL-3 and GM-CSF induced growth of acute myeloid leukemia colony forming cells (AML-CFU) in vitro. Among 12 cases of human AML, IL-1 significantly elevated IL-3 stimulated colony numbers in eight instances and enhanced GM-CSF induced colony growth in five cases. As IL-1 is an inducer of cytokine production and since tumor necrosis factor (TNF) elevates IL-3 or GM-CSF induced proliferation of AML-CFU, we examined whether IL-1 enhanced AML-CFU growth via the induction of TNF production. Neutralizing anti-TNF-alpha antibodies significantly decreased IL-1/IL-3 or IL-1/GM-CSF stimulated colony numbers in six of seven cases studied, whereas anti-TNF-beta had no effect, indicating that endogenously produced TNF-alpha costimulated the growth of AML-CFU. Furthermore, AML blast cells stimulated by IL-1 released increased amounts of TNF-alpha (between 25 and 533 pg/ml; median 255 pg/ml) into the culture medium (TNF-alpha specific radioimmunoassay) as compared with noninduced AML cells (less than 1 to 149 pg TNF-alpha/ml; median 31 pg/ml). Thus, the effect of IL-1 on AML-CFU proliferation is not the result of direct activation of AML progenitors, but IL-1 stimulates the release of TNF-alpha by AML cells and endogenous TNF subsequently synergizes with IL-3 or GM-CSF.
Assuntos
Fatores Estimuladores de Colônias/farmacologia , Substâncias de Crescimento/farmacologia , Interleucina-1/farmacologia , Interleucina-3/farmacologia , Leucemia Mieloide Aguda/patologia , Fator de Necrose Tumoral alfa/biossíntese , Anticorpos , Divisão Celular , Sinergismo Farmacológico , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Fatores de Crescimento de Células Hematopoéticas , Humanos , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Ensaio Tumoral de Célula-TroncoRESUMO
In this study we demonstrate that tumor necrosis factors (TNF alpha and TNF beta) are potent modulators of the in vitro proliferation of human AML cells. Blast cells from 11 cases of acute myeloblastic leukemia (AML) were incubated with recombinant TNF alpha or TNF beta in serum-free 3H-TdR uptake and colony culture systems in the presence or absence of recombinant interleukin-3 (IL-3), granulocyte macrophage colony-stimulating factor (GM-CSF), G-CSF, or M-CSF. Depending on the supplemented CSF, TNF could upregulate or suppress AML blast proliferation. Enhancement of AML growth by TNF was observed in the presence of IL-3 (in 9 of 11 cases in 3H-TdR assay; 6 of 9 cases in colony assay) and GM-CSF (in 8 of 11 cases in 3H-TdR assay; 4 of 9 cases in colony assay). In certain cases in which IL-3 or GM-CSF alone was unable to induce proliferative responses of AML cells, the simultaneous addition of TNF elicited colony growth and DNA synthesis suggesting a synergistic action between TNF and IL-3 or GM-CSF. In contrast, TNF suppressed G-CSF-induced growth (9 of 10 cases in 3H-TdR assay; 5 of 6 cases in colony assay). TNF could also stimulate DNA synthesis (in 2 of 11 cases) or colony formation (in 2 of 9 cases) in AML cultures without the addition of other growth factors. Experiments with neutralizing antibodies and specific radioimmunoassays for individual CSFs showed that the synergistic and antagonistic effects of TNF on AML growth could not be attributed to a release of one of these CSFs by the AML cells. The opposing consequence of exposure of AML blasts to TNF are of interest in view of our understanding of the pathophysiology of AML growth and the in vivo application of recombinant cytokines in AML patients.
Assuntos
Fatores Estimuladores de Colônias/farmacologia , Leucemia Mieloide Aguda/patologia , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Divisão Celular/efeitos dos fármacos , Fatores Estimuladores de Colônias/biossíntese , Fator Estimulador de Colônias de Granulócitos , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Substâncias de Crescimento/farmacologia , Humanos , Interleucina-3/farmacologia , Células Tumorais CultivadasRESUMO
Among 52 patients diagnosed as acute myeloid leukemia (AML), nine cases were found in which interleukin-5 (IL-5) induced a proliferative response in the leukemic cells, as measured by the stimulation of DNA synthesis or colony formation in vitro. All cases (n = 7) with the cytogenetic abnormality t(8;21)(q22;q22) belonged to this group of IL-5 responders. Of the additional two cases, one had an apparently normal karyotype, but the other expressed a dicentric chromosome 21, an abnormality also involving the breakpoint region 21q22. The leukemic cells of the IL-5 responsive patients could also be stimulated to proliferate by IL-3, GM-CSF and G-CSF, and in some cases by IL-6 or M-CSF. Immunophenotypic analysis revealed the presence of the immature hematopoietic cell antigen CD34, the myelomonocytic maturation antigens CD13 and CD33, in association with the B-cell related surface marker CD19 on the leukemic cells. Immunoglobulin mu and T-cell receptor beta-genes in the leukemic cells were in germline configuration. Upon incubation in colony culture, clonogenic cells were capable of producing progeny showing eosinophilic or neutrophilic maturation following stimulation with IL-5 or G-CSF, respectively. It is concluded that IL-5 responsive AML represents a subgroup of leukemia with distinct immunotypic and cytogenetic features.
Assuntos
Interleucina-5/farmacologia , Leucemia Mieloide/genética , Antígenos CD/análise , Divisão Celular/efeitos dos fármacos , Aberrações Cromossômicas/genética , Aberrações Cromossômicas/imunologia , Aberrações Cromossômicas/patologia , Transtornos Cromossômicos , Cromossomos Humanos Par 21 , Feminino , Rearranjo Gênico do Linfócito B , Rearranjo Gênico do Linfócito T , Genes de Imunoglobulinas , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Técnicas In Vitro , Interleucina-3/farmacologia , Leucemia Mieloide/imunologia , Leucemia Mieloide/patologia , Masculino , Receptores de Antígenos de Linfócitos T/genética , Células Tumorais CultivadasRESUMO
Aberrant expression of the Evi1 (ecotropic virus integration site 1) proto-oncogene has been associated with hematopoietic malignancies in both mice and man. To determine the effect of enforced expression of Evi1 in vivo, we developed a transgenic mouse model utilizing the murine Sca-1 (Ly-6E.1) promoter. Here, we describe the generation and analysis of three independent lines of Evi1 transgenic mice. Transgenic animals of two founder lines developed normally. These mice did not show any obvious hematological abnormalities but showed a significant reduction in the number of bone marrow colony-forming unit erythroid (CFU-E)-derived colonies. This implies a defect of normal erythroid hematopoiesis affecting relatively late erythroid progenitor cells. We also show that when newborn Evi1 transgenic mice of these two lines were infected with Cas-Br-M MuLV, tumor incidence was greatly enhanced in comparison with nontransgenic littermates, indicating an increased susceptibility for leukemia development. Interestingly, analysis of a third founder line revealed that all male progeny consistently displayed severely impaired erythropoiesis with major defects in the bone marrow, spleen and peripheral blood. Taken together, our results present the first evidence of Evi1 disturbing normal erythropoiesis in vivo and provides evidence for cooperative potential of Evi1 in tumor progression.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Leucemia Experimental/etiologia , Fatores de Transcrição/fisiologia , Dedos de Zinco/fisiologia , Animais , Antígenos Ly/genética , Células Sanguíneas/patologia , Medula Óssea/patologia , Proteínas de Ligação a DNA/genética , Células Precursoras Eritroides/patologia , Eritropoese/genética , Predisposição Genética para Doença , Vírus da Leucemia Murina/patogenicidade , Leucemia Experimental/genética , Leucemia Experimental/virologia , Proteína do Locus do Complexo MDS1 e EVI1 , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Modelos Animais , Regiões Promotoras Genéticas , Proto-Oncogenes , Proteínas Recombinantes de Fusão/genética , Infecções por Retroviridae/genética , Baço/patologia , Fatores de Transcrição/genética , Infecções Tumorais por Vírus/genética , Dedos de Zinco/genéticaRESUMO
Blood-cell production is regulated by hemopoietic growth factors, which act at specific stages of hemopoietic cell differentiation. Murine interleukin-3 (mIL-3)/multilineage colony-stimulating factor (multi-CSF) has been shown to stimulate colony formation in vitro by multipotent hemopoietic cells and production of spleen colony-forming units (CFU-S) in suspension cultures. The molecular cloning of the human counterpart of mIL-3 is described here. Hybridization of radiolabeled mIL-3 cDNA with a cDNA library obtained from mRNA of stimulated human lymphocytes resulted in the identification of a human (h)multi-CSF cDNA clone. Sequence homology (73%) in the 3'-noncoding region of mIL-3 enabled the detection of the hmulti-CSF cDNA clone. Whereas only 45% sequence homology was found in the coding region, specific A + T-rich domains in the 3'-noncoding region were highly conserved (93%). As far as we know, this is the first example of gene identification by sequence homology occurring only within the 3'-noncoding region. The protein encoded by this hmulti-CSF cDNA stimulates in vitro colony formation by multipotent human hemopoietic stem cells. In addition, the growth factor strongly stimulates the in vitro proliferation of human leukemic blast cells.
Assuntos
Cromossomos Humanos Par 5 , Interleucina-3/genética , Animais , Sequência de Bases , Bioensaio , Células Cultivadas , Mapeamento Cromossômico , Clonagem Molecular , DNA/genética , Humanos , Camundongos , Homologia de Sequência do Ácido NucleicoRESUMO
Chemosensitivity of purified AML blasts to daunomycin was examined under conditions of CSF stimulation and compared with clinical outcome of DNR combination chemotherapy. AML blasts from 16 patients were purified, incubated serum-free in the presence of optimal concentrations of a complete cocktail of IL-3, GM-CSF and G-CSF to provoke cell proliferation maximally, and DNR drug sensitivity in vitro was assessed by inhibition of DNA synthesis in response to titrated DNR concentrations. The sensitivity of proliferating AML cells to DNR did not correlate with the clinical response of the patients as identical dose-response curves were obtained for complete responders (n = 6), partial responders (n = 5) and resistant cases (n = 5). As a major part of the AML population was induced to enter DNA synthesis in vitro, these data suggest that the manoeuvre of cell cycle stimulation has abrogated cellular resistance to daunomycin and rendered in vivo, apparently refractory cells susceptible to the anthracycline.
Assuntos
Fatores Estimuladores de Colônias/farmacologia , Daunorrubicina/farmacologia , Leucemia Mieloide Aguda/patologia , Resistência a Medicamentos , Humanos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Here we report experiments dealing with the development of a fully serum-free culture system for AML cell proliferation. A number of compounds were tested for the requirements of AML colony formation and DNA synthesis in vitro in an attempt to replace serum with an artificial mixture. The results indicate that bovine serum albumin (BSA, 15 mg/ml), cholesterol (7.8 micrograms/ml), transferrin (7.7 X 10(-6) M) and insulin (1 microgram/ml) were essential for AML cell proliferation. Linoleic acid, 2-mercaptoethanol and selenite contributed only moderately beneficial effects. This mixture obviated the need for adding exogenous fetal calf or horse serum to the cultures and permitted equivalent and frequently superior AML colony formation and DNA synthesis. This is the first serum-free culture method for human AML that may allow for in-vitro studies of the growth regulation by recombinant growth factors under exactly defined and standardized conditions.
Assuntos
Meios de Cultura , Leucemia Mieloide Aguda/patologia , Células Tumorais Cultivadas/patologia , Divisão Celular , Técnicas de Cultura/métodos , Replicação do DNA , Humanos , Ensaio Tumoral de Célula-TroncoRESUMO
Acute myeloblastic leukemia (AML) cells were cultured under conditions facilitating or preventing cell to cell contact. Proliferation of AML blasts of 24 patients was assessed in semisolid (containing 0.9% methylcellulose) and in liquid cultures, in which identical concentrations of colony stimulating factors (CSFs) had been provided. In all but one of the cases, significant DNA synthesis (evaluated by 3H-thymidine uptake) was observed when the cells were incubated in the liquid system, whereas in only 14 cases (58%), the cells were able to form clusters or colonies in the semisolid system. These findings suggest that AML cells from a large proportion of patients can proliferate only after stimulation with CSFs in a liquid system, i.e. when cultured under conditions permitting reciprocal contact between the cells. To establish further the importance of cell to cell contact for AML cell proliferation, cells from 19 patients were cultured in liquid medium concurrently in flat bottom microwells (in which a majority of the cells lie separate) and in round bottom microwells (in which cells show a tendency to aggregate). A significantly higher 3H-thymidine (TdR) incorporation in the round bottom cultures was observed in 15 out of 19 cases. The role of the leukocyte function antigens (LFA) LFA1, Mac1, P150-95 in this phenomenon was then analyzed, as these structurally related glycoproteins are involved in reactions requiring contact between hematopoietic cells. Membrane expression of the three antigens was first examined in 16 patients. LFA1, Mac1 and P150-95 were expressed on the AML cells of 15, 6 and 8 patients, respectively. Expression of these antigens did not change following short term incubation of the AML cells in the presence of CSFs. AML cells cultured in presence of saturating concentrations of monoclonal antibodies reacting with structures of these 3 antigens in order to abrogate their function did not suppress 3H-TdR uptake. Thus, no direct role for LFA1 and/or Mac1 and/or P150-95 antigens in mediating contact-induced AML proliferation could be demonstrated. It remains to be established which components are involved in the cell-cell contact-mediated upregulation of AML cell proliferation.
Assuntos
Antígenos CD/fisiologia , Moléculas de Adesão Celular/fisiologia , Adesão Celular , Divisão Celular , Leucemia Mieloide Aguda/patologia , Antígenos de Diferenciação/fisiologia , DNA/biossíntese , Humanos , Integrina alfaXbeta2 , Leucemia Mieloide Aguda/imunologia , Antígeno-1 Associado à Função Linfocitária , Antígeno de Macrófago 1 , Receptores de Adesão de Leucócito/fisiologiaRESUMO
AML cells were cultured free of serum with G-CSF in combination with all-trans-retinoic acid (RA), prostaglandin E2 or 8-bromocyclic AMP to see whether the maturation blockade of these cells could be overcome. The combination G-CSF + RA was most effective in inducing morphologic maturation, i.e. in 7/10 cases. Morphological alterations in response to G-CSF + RA indicated progression of the cells along the granulocytic pathway towards metamyelocytes and granulocytes. However, morphologically mature AML cells remained negative for myeloperoxidase and Sudan black stainings, indicators of granulocytic maturation. Chloracetate esterase positivity and CD15 membrane antigens became expressed on cultured AML cells, i.e. on unstimulated and G-CSF/RA exposed blasts. Ingestion of latex beads and reduction of nitroblue tetrazolium salt occurred in cultured AML cells regardless of the presence of inducers. In almost all cases clonogenic cells persisted after exposure to G-CSF + RA suggesting that subpopulations of immature cells escaped the action of these inducers. Thus although G-CSF + RA were capable of inducing maturation of AML cells along the granulocytic lineage, maturation was incomplete and the effect was evident in a subfraction of the cells only.
Assuntos
Medula Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Granulócitos/patologia , Leucemia Mieloide/sangue , Tretinoína/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Doença Aguda , Células Cultivadas , Dinoprostona/farmacologia , Citometria de Fluxo , Imunofluorescência , Granulócitos/efeitos dos fármacos , Humanos , Cinética , Leucemia Mieloide/patologia , Coloração e RotulagemRESUMO
The proliferative and maturation abilities of bone marrow progenitors in patients with refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T) have previously been investigated in vitro using impure sources of colony stimulating activity. Here we report studies that were concerned with defining growth factor responses of RAEB progenitors (RAEB-CFU) in colony culture using pure hematopoietic growth factors. Marrow cells of 10 RAEB patients were cultured with recombinant IL3, GM-CSF, G-CSF, M-CSF and EPO. Factor dependent colony growth of four patients was examined in detail cytologically. The analysis revealed notable deficiencies in the colony forming spectrum as compared with normal marrow: although granulocytic colonies were formed in all of these four RAEB cases, macrophage colonies could not be induced in 1/4 cases and eosinophilic and erythroid colony formation could not be propagated in 2/4 cases with the proper stimuli. These findings are indicative of the intrinsic incapabilities of RAEB-CFU to mature along certain differentiation pathways in response to the growth factors. We then determined the surface phenotypes of RAEB-CFU using MoAbs Vim-2 (myelomonocytic) and B13C5 (CD34) following dual labeling and fluorescence activated cell sorting and subsequent culture of the separately sorted BI3C5+/Vim-2+, BIC5+/Vim-2-, BI3C5-/Vim-2+ and BIC5-/Vim-2- cells. In normal marrow most clonogenic cells were recovered from the BI3C5+/Vim-2- fraction. In contrast, in RAEB marrow increased proportions of the colony forming cells were BI3C5+/Vim-2+, BI3C5-/Vim-2+, or BI3C5-. The altered distribution of surface immunophenotypes of RAEB-CFU provides further evidence for the imbalance of maturation in the progenitor cell compartment. The results are discussed in view of the concept that the inabilities of the RAEB hematopoietic precursors to mature in response to the hematopoietic growth factors are partial and variable, but may culminate in a progressive loss of the differentiation competence of the progenitors when leukemia evolves.
Assuntos
Anemia Refratária com Excesso de Blastos/patologia , Células-Tronco/citologia , Adulto , Anemia Refratária com Excesso de Blastos/genética , Anemia Refratária com Excesso de Blastos/imunologia , Células da Medula Óssea , Diferenciação Celular , Separação Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Fatores Estimuladores de Colônias/farmacologia , Citometria de Fluxo , Humanos , Interleucina-3/farmacologia , Fenótipo , Proteínas Recombinantes/farmacologiaRESUMO
In this report we present a method for purging acute myeloid leukaemia progenitors (AML-CFU) from autologous marrow grafts. Previous studies using flow cytometry and cell sorting had indicated that the monoclonal antibody (MCA) Vim-2 reacts strongly with AML-CFU in a significant number of cases, whereas the majority of normal bone marrow precursors are Vim-2 negative. To investigate the applicability of Vim-2 for selective purging, we performed complement-dependent cytotoxicity experiments and determined the specific lysis of AML-CFU. In 11 of 17 patients, 97.5 +/- 4% (mean +/- SD) of AML-CFU were killed following immune-mediated lysis. Multi-lineage normal haematopoietic progenitors (CFU-GEMM) were not significantly affected by the MCA Vim-2-mediated complement lysis. These data suggest that the MCA Vim-2 is a useful reagent for the elimination of residual AML progenitor cells from remission bone marrow in patients subjected to autologous marrow transplantation.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos de Diferenciação/imunologia , Transplante de Medula Óssea , Leucemia Mieloide Aguda/terapia , Células-Tronco Neoplásicas/imunologia , Antígenos de Neoplasias/imunologia , Células da Medula Óssea , Proteínas do Sistema Complemento , Citotoxicidade Imunológica , Citometria de Fluxo , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologiaRESUMO
INTRODUCTION: Anandamide (ANA) is an endogenous ligand for the cannabinoid receptors Cb1 and Cb2 that is able to synergistically stimulate the proliferation of hematopoietic growth factor-dependent blood cells in serum-free culture. To elucidate the mechanisms by which ANA enhances the proliferative responses of hematopoietic cells, we investigated the ANA-mediated effects on proliferation, cell cycling, apoptosis and intracellular signaling of erythropoietin-stimulated 32D/EPO cells. MATERIALS AND METHODS: 32D/EPO cells were cultured serum free to determine the effects of EPO and anandamide on these cells. Proliferation was analyzed by tritiated thymidine incorporation. Apoptosis as well as cell cycle analysis was carried out by flow cytometry. MAPKinase activation was determined by Western blotting, using phospho-specific MAPK antibodies. RESULTS: Simultaneous addition of erythropoietin (EPO) and ANA enhanced DNA synthesis and increased 32D/EPO cell numbers in serum-free culture. Interestingly, ANA did not alter the G1/S transition but it accelerated each of the successive cell cycle phases of EPO-stimulated 32D/EPO cells. Percentages of apoptotic 32D/EPO cells were equally low in cultures supplemented with EPO alone or a combination of EPO and ANA. Both cultures showed enhanced activation of two mitogen-activated protein kinases, namely, extracellular factor responsive kinases 1 and 2 (ERK1/2), as well as the MAPK-target gene protein c-Fos. This fully correlated with the synergistic stimulation of proliferation of 32D/EPO cells by EPO and ANA. ANA had no effect on EPO-induced STAT-5 activation of 32D/EPO cells. Experiments with the Cb2 receptor-specific antagonist SR144528 demonstrated that the synergistic stimulation of proliferation by ANA was partially Cb2 receptor-mediated. CONCLUSION: These data suggest that the positive effects of ANA on the erythropoietin-induced proliferation of 32D/EPO cells are mediated by receptor-dependent as well as receptor-independent mechanisms, both of which involve activation of the mitogen-activated protein kinases, ERK1/2.