RESUMO
Telomere biology disorder (TBD) can present within a wide spectrum of symptoms ranging from severe congenital malformations to isolated organ dysfunction in adulthood. Diagnosing TBD can be challenging given the substantial variation in symptoms and age of onset across generations. In this report, we present two families, one with a pathogenic variant in ZCCHC8 and another with a novel variant in TERC. In the literature, only one family has previously been reported with a ZCCHC8 variant and TBD symptoms. This family had multiple occurrences of pulmonary fibrosis and one case of bone marrow failure. In this paper, we present a second family with the same ZCCHC8 variant (p.Pro186Leu) and symptoms of TBD including pulmonary fibrosis, hematological disease, and elevated liver enzymes. The suspicion of TBD was confirmed with the measurement of short telomeres in the proband. In another family, we report a novel likely pathogenic variant in TERC. Our comprehensive description encompasses hematological manifestations, as well as pulmonary and hepatic fibrosis. Notably, there are no other reports which associate this variant to disease. The families expand our understanding of the clinical implications and genetic causes of TBD.
Assuntos
Linhagem , RNA , Telomerase , Telômero , Adulto , Feminino , Humanos , Masculino , Predisposição Genética para Doença , Mutação/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/diagnóstico , Fibrose Pulmonar/patologia , RNA/genética , Telomerase/genética , Telômero/genéticaRESUMO
Differentiation of multipotent stem cells into mature cells is fundamental for development and homeostasis of mammalian tissues, and requires the coordinated induction of lineage-specific transcriptional programs and cell cycle withdrawal. To understand the underlying regulatory mechanisms of this fundamental process, we investigated how the tissue-specific transcription factors, CEBPA and CEBPE, coordinate cell cycle exit and lineage-specification in vivo during granulocytic differentiation. We demonstrate that CEBPA promotes lineage-specification by launching an enhancer-primed differentiation program and direct activation of CEBPE expression. Subsequently, CEBPE confers promoter-driven cell cycle exit by sequential repression of MYC target gene expression at the G1/S transition and E2F-meditated G2/M gene expression, as well as by the up-regulation of Cdk1/2/4 inhibitors. Following cell cycle exit, CEBPE unleashes the CEBPA-primed differentiation program to generate mature granulocytes. These findings highlight how tissue-specific transcription factors coordinate cell cycle exit with differentiation through the use of distinct gene regulatory elements.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição , Animais , Ciclo Celular , Diferenciação Celular/genética , Granulócitos/metabolismo , Mamíferos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Bone marrow specimens are the core of the diagnostic workup of patients with cytopenia. To explore whether next-generation sequencing (NGS) could be used to rule out malignancy without bone marrow specimens, we incorporated NGS in a model to predict presence of disease in the bone marrow of patients with unexplained cytopenia. We analyzed the occurrence of mutations in 508 patients with cytopenia, referred for primary workup of a suspected hematologic malignancy from 2015 to 2020. We divided patients into a discovery (n = 340) and validation (n = 168) cohort. Targeted sequencing, bone marrow biopsy, and complete blood count were performed in all patients. Mutations were identified in 267 (53%) and abnormal bone marrow morphology in 188 (37%) patients. Patients with isolated neutropenia had the lowest frequency of both mutations (21%) and abnormal bone marrow morphology (5%). The median number of mutations per patient was 2 in patients with abnormal bone marrow morphology compared with 0 in patients with a nondiagnostic bone marrow morphology (P < .001). In a multivariable logistic regression, mutations in TET2, SF3B1, U2AF1, TP53, and RUNX1 were significantly associated with abnormal bone marrow morphology. In the validation cohort, a model combining mutational status and clinical data identified 34 patients (20%) without abnormal bone marrow morphology with a sensitivity of 100% (95% confidence interval: 93%-100%). Overall, we show that NGS combined with clinical data can predict the presence of abnormal bone marrow morphology in patients with unexplained cytopenia and thus can be used to assess the need of a bone marrow biopsy.
Assuntos
Anemia , Doenças da Medula Óssea , Síndromes Mielodisplásicas , Anemia/patologia , Medula Óssea/patologia , Doenças da Medula Óssea/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Síndromes Mielodisplásicas/genéticaRESUMO
Lipocalin-2 is a constituent of the neutrophil secondary granules and is expressed de novo by macrophages and epithelium in response to inflammation. Lipocalin-2 acts in a bacteriostatic fashion by binding iron-loaded siderophores required for bacterial growth. Mycobacterium tuberculosis (M.tb) produces siderophores that can be bound by lipocalin-2. The impact of lipocalin-2 in the innate immune response toward extracellular bacteria has been established whereas the effect on intracellular bacteria, such as M.tb, is less well-described. Here we show that lipocalin-2 surprisingly confers a growth advantage on M.tb in the early stages of infection (3 weeks post-challenge). Using mixed bone marrow chimeras, we demonstrate that lipocalin-2 derived from granulocytes, but not from epithelia and macrophages, leads to increased susceptibility to M.tb infection. In contrast, lipocalin-2 is not observed to promote mycobacterial growth at later stages of M.tb infection. We demonstrate co-localization of granulocytes and mycobacteria within the nascent granulomas at week 3 post-challenge, but not in the consolidated granulomas at week 5. We hypothesize that neutrophil-derived lipocalin-2 acts to supply a source of iron to M.tb in infected macrophages within the immature granuloma, thereby facilitating mycobacterial growth.
Assuntos
Granulócitos/imunologia , Granuloma/imunologia , Imunidade Inata , Lipocalina-2/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Animais , Granulócitos/patologia , Granuloma/genética , Granuloma/microbiologia , Granuloma/patologia , Lipocalina-2/genética , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Tuberculose/genética , Tuberculose/patologiaRESUMO
BACKGROUND: Small mothers against decapentaplegic (SMAD)4 and SMAD7 are key regulatory components in the immunosuppressive transforming growth factor- (TGF-) ß signaling pathway, which is defective in inflammatory bowel disease (IBD). SMAD4 may play an important role in the pathogenesis of IBD as indicated in experimental models of colitis. AIMS: To examine the ileal expression levels of SMAD4 and to correlate these with CD disease activity. METHODS: The material comprised 29 CD patients (13 with active disease, 16 in remission) and 9 asymptomatic patients referred for ileocolonoscopy as part of an adenoma surveillance program serving as controls. Patients were examined with ileocolonoscopy. Corresponding ileal biopsies were obtained for histological analysis and assessment of SMAD4 and SMAD7 protein expression by immunohistochemistry (IHC). RESULTS: The protein expression of SMAD4 was significantly downregulated in ileal tissue sections from CD patients as compared to healthy controls (p < 0.001). Further, luminal SMAD4 expression was inversely correlated with endoscopic (rs = -0.315; p = 0.05) and histopathological activity (rs = -0.40; p = 0.013). CONCLUSIONS: The SMAD4 epithelial protein level was markedly downregulated in CD patients and inversely correlated with disease activity. This may contribute to defective mucosal TGF-ß signaling in active IBD.
RESUMO
t(8;21) acute myeloid leukemia (AML) is characterized by a translocation between chromosomes 8 and 21 and formation of a distinctive RUNX1-RUNX1T1 fusion transcript. This translocation places RUNX1T1 under control of the RUNX1 promoter leading to a pronounced upregulation of RUNX1T1 transcripts in t(8;21) AML, compared to normal hematopoietic cells. We investigated the role of highly-upregulated RUNX1T1 under the hypothesis that it acts as competing endogenous RNA (ceRNA) titrating microRNAs (miRNAs) away from their target transcripts and thus contributes to AML formation. Using publicly available t(8;21) AML RNA-Seq and miRNA-Seq data available from The Cancer Genome Atlas (TCGA) project, we obtained a network consisting of 605 genes that may act as ceRNAs competing for miRNAs with the suggested RUNX1T1 miRNA sponge. Among the 605 ceRNA candidates, 121 have previously been implied in cancer development. Players in the integrin, cadherin, and Wnt signaling pathways affected by the RUNX1T1 sponge were overrepresented. Finally, among a set of 21 high interest RUNX1T1 ceRNAs we found multiple genes that have previously been linked to AML formation. In conclusion, our study offers a novel look at the role of the RUNX1-RUNX1T1 fusion transcript in t(8;21) AML beyond previously investigated genetic and epigenetic aberrations.
Assuntos
Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Leucemia Mieloide Aguda/genética , MicroRNAs , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas , Sítios de Ligação , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação Leucêmica da Expressão Gênica , Ontologia Genética , Humanos , MicroRNAs/metabolismo , Proteínas de Fusão Oncogênica/genética , Mapas de Interação de Proteínas , Proteína 1 Parceira de Translocação de RUNX1 , Translocação Genética , Via de Sinalização Wnt/genéticaRESUMO
Jumonji Domain-Containing Protein 3 (JMJD3)/lysine demethylase 6B (KDM6B) is an epigenetic modulator that removes repressive histone marks on genes. Expression of KDM6B mRNA is elevated in leukocytes from patients with ANCA-associated vasculitis (AAV) and has been suggested to be the reason for higher proteinase 3 (PR3) mRNA expression in these cells due to derepression of PRTN3 gene transcription. MicroRNA-941 (miR-941) has been shown to target KDM6B mRNA and inhibit JMJD3 production. We therefore investigated whether polymorphonuclear granulocytes (PMNs) from patients suffering from granulomatosis with polyangiitis (GPA) have lower expression of miR-941 than healthy control donors as a biological cause for higher JMJD3 levels. We found no significant difference in the degree of maturation of PMNs from GPA patients (n = 8) and healthy controls (n = 11) as determined from cell surface expression of the neutrophil maturation marker CD16 and gene expression profile of FCGR3B. The expression of PRTN3 and KDM6B mRNAs and miR-941 was not significantly different in GPA patients and healthy controls. Transfection of pre-miR-941 into the neutrophil promyelocyte cell line PLB-985 cells did not result in reduction of the KDM6B mRNA level as shown previously in a hepatocellular carcinoma cell line. The amount of PR3 in PMNs from GPA patients and healthy controls was comparable. In conclusion, we found that PRTN3 mRNA, KDM6B mRNA, and miR-941 expression levels in PMNs do not differ between GPA patients and healthy controls, and that miR-941 does not uniformly regulate KDM6B mRNA levels by inducing degradation of the transcript. Thus, decreased miR-941 expression in PMNs cannot be part of the pathogenesis of GPA.
Assuntos
Granulomatose com Poliangiite/patologia , MicroRNAs/metabolismo , Neutrófilos/metabolismo , Adulto , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Granulomatose com Poliangiite/genética , Granulomatose com Poliangiite/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Masculino , Pessoa de Meia-Idade , Mieloblastina/genética , Mieloblastina/metabolismo , Análise de Componente Principal , RNA Mensageiro/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Transcriptoma , Adulto JovemRESUMO
Aspergillus fumigatus is an opportunistic fungal pathogen that causes severe invasive infections in immunocompromised patients. Innate immunity plays a major role in protection against A. fumigatus. The ficolins are a family of soluble pattern recognition receptors that are capable of activating the lectin pathway of complement. Previous in vitro studies reported that ficolins bind to A. fumigatus, but their part in host defense against fungal infections in vivo is unknown. In this study, we used ficolin-deficient mice to investigate the role of ficolins during lung infection with A. fumigatus. Ficolin knockout mice showed significantly higher fungal loads in the lungs 24 h postinfection compared to wild-type mice. The delayed clearance of A. fumigatus in ficolin knockout mice could not be attributed to a compromised recruitment of inflammatory cells. However, it was revealed that ficolin knockout mice exhibited a decreased production of proinflammatory cytokines in the lungs compared to wild-type mice following A. fumigatus infection. The impaired clearance and cytokine production in ficolin knockout mice was independent of complement, as shown by equivalent levels of A. fumigatus-mediated complement activation in ficolin knockout mice and wild-type mice. In conclusion, this study demonstrates that ficolins are important in initial innate host defense against A. fumigatus infections in vivo.
Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Lectina de Ligação a Manose da Via do Complemento , Imunomodulação , Inflamação/imunologia , Lectinas/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Feminino , Imunidade Inata , Lectinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Reconhecimento de Padrão/imunologia , FicolinasRESUMO
BACKGROUND: MicroRNAs (miRNAs) are important for the development and function of neutrophils. miR-130a is highly expressed during early neutrophil development and regulates target proteins important for this process. miRNA targets are often identified by validating putative targets found by in silico prediction algorithms one at a time. However, one miRNA can have many different targets, which may vary depending on the context. Here, we investigated the effect of miR-130a on the proteome of a murine and a human myeloid cell line. RESULTS: Using pulsed stable isotope labelling of amino acids in cell culture and mass spectrometry for protein identification and quantitation, we found 44 and 34 proteins that were significantly regulated following inhibition of miR-130a in a miR-130a-overexpressing 32Dcl3 clone and Kasumi-1 cells, respectively. The level of miR-130a inhibition correlated with the impact on protein levels. We used RAIN, a novel database for miRNA-protein and protein-protein interactions, to identify putative miR-130a targets. In the 32Dcl3 clone, putative targets were more up-regulated than the remaining quantified proteins following miR-130a inhibition, and three significantly derepressed proteins (NFYC, ISOC1, and CAT) are putative miR-130a targets with good RAIN scores. We also created a network including inferred, putative neutrophil miR-130a targets and identified the transcription factors Myb and CBF-ß as putative miR-130a targets, which may regulate the primary granule proteins MPO and PRTN3 and other proteins differentially expressed following miR-130a inhibition in the 32Dcl3 clone. CONCLUSION: We have experimentally identified miR-130a-regulated proteins within the neutrophil proteome. Linking these to putative miR-130a targets, we provide an association network of potential direct and indirect miR-130a targets that expands our knowledge on the role of miR-130a in neutrophil development and is a valuable platform for further experimental studies.
Assuntos
MicroRNAs/genética , Neutrófilos/metabolismo , Proteoma , Animais , Linhagem Celular , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Células Mieloides/metabolismo , Proteômica/métodosRESUMO
Smad4 is important in the TGF-ß pathway and required for transcriptional activation and inhibition of cell growth after TGF-ß1 stimulation. We demonstrate that miR-130a is differentially expressed during granulopoiesis and targets Smad4 mRNA. The transcript for Smad4 is present throughout neutrophil maturation, but Smad4 protein is undetectable in the most immature cells, where miR-130a is highly expressed. Two miR-130a binding sites were identified in the 3'-untranslated region of the Smad4 mRNA. Overexpression of miR-130a in HEK293, A549, and 32Dcl3 cells repressed synthesis of Smad4 protein without affecting Smad4 mRNA level. Repression of Smad4 synthesis in a granulocytic cell line by miR-130a reduced its sensitivity to TGF-ß1-induced growth inhibition. This effect was reversed by inhibiting the activity of miR-130a with an antisense probe or by expressing a Smad4 mRNA lacking miR-130a binding sites. High endogenous miR-130a and Smad4 mRNA levels and low expression of Smad4 protein were found in the t(8;21)(q22;q22) acute myelogenous leukemia-derived cell line Kasumi-1. When miR-130a was inhibited by an antisense RNA, the amount of Smad4 protein increased in Kasumi-1 cells and rendered it susceptible for TGF-ß1-mediated cell growth inhibition. Our data indicate that miR-130a is involved in cell cycle regulation of granulocytic cells through engagement of Smad4 in the TGF-ß pathway.
Assuntos
Regulação para Baixo , Células Precursoras de Granulócitos/efeitos dos fármacos , MicroRNAs/genética , Proteína Smad4/genética , Fator de Crescimento Transformador beta1/farmacologia , Regiões 3' não Traduzidas/genética , Doença Aguda , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Precursoras de Granulócitos/metabolismo , Células HEK293 , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad4/metabolismoRESUMO
Sequential up- and down-regulation of a handful of critical transcription factors is required for proper neutrophil differentiation. Malfunction of transcription factors may lead to diseases such as acute myeloid leukemia (AML) and specific granule deficiency. In order to understand the molecular background for normal and malignant granulopoiesis, a good model system is required that faithfully mimics the in vivo transcription factor expression profiles. The two human leukemic cell lines HL60 and NB4 have been widely used as model cell lines for these purposes. Differentiation of HL60 and NB4 cells resulted in asynchronous differentiation to morphologically mature neutrophils over a period of 5-7 days. To obtain cell populations of more even maturity, cells at different stages of in vitro differentiation were purified by immunomagnetic isolation. This resulted in three cell populations that could be classified as promyelocytes, myelocytes/metamyelocytes, and mature neutrophils, respectively. Comparison of transcription factor mRNA profiles from these cell populations with those previously seen in normal human bone marrow, demonstrated that although all of the 14 transcription factors described in vivo, could be detected during in vitro differentiation, vast differences in their expression profiles was observed. These data illustrate the limitations of cell lines as models for normal granulopoiesis.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Leucopoese , Modelos Biológicos , Neutrófilos/metabolismo , Fatores de Transcrição/biossíntese , Células Precursoras de Granulócitos/metabolismo , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , RNA Mensageiro/biossínteseRESUMO
To characterize the transcriptional program that governs terminal granulocytic differentiation in vivo, we performed comprehensive microarray analyses of human bone marrow populations highly enriched in promyelocytes (PMs), myelocytes/metamyelocytes (MYs), and neutrophils (bm-PMNs). These analyses identified 11 310 genes involved in differentiation, of which 6700 were differentially regulated, including previously unidentified effector proteins and surface receptors of neutrophils. Differentiation of PMs toward MYs was accompanied by a marked decline of proliferative and general cellular activity as defined by down-regulation of E2 promoter binding factor (E2F) target genes; cyclin dependent kinases 2, 4, and 6; and various metabolic, proteasomal, and mitochondrial genes. Expression patterns of apoptosis genes indicated death control by the p53 pathway in PMs and by death receptor pathways in bm-PMNs. Effector proteins critical for host defense were expressed successively throughout granulocytic differentiation, whereas receptors and receptor ligands essential for the activation of the host defense program were terminally up-regulated in bm-PMNs. The up-regulation of ligand-receptor pairs, which are defined inducers as well as target genes of nuclear factor-kappa B (NF-kappa B), suggests a constitutive activation of NF-kappa B in bm-PMNs by autocrine loops. Overall, these results define a granulocytic differentiation model governed by a highly coordinated fail-safe program, which promotes completion of differentiation before cells gain responsiveness toward activating stimuli that accompany infections.
Assuntos
Diferenciação Celular/genética , Granulócitos/citologia , Granulócitos/metabolismo , Transcrição Gênica , Apoptose/genética , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Separação Celular/métodos , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Perfilação da Expressão Gênica/métodos , Células Precursoras de Granulócitos/citologia , Células Precursoras de Granulócitos/metabolismo , Humanos , Ligantes , Neutrófilos/citologia , Neutrófilos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Serglycin is a major proteoglycan of hematopoietic cells. It is thought to play a role in the packaging of granule proteins in human neutrophil granulocytes. The presence of serglycin in myeloid cells has been demonstrated only at the transcriptional level. We generated a polyclonal antibody against recombinant human serglycin. Here, we show the localization of serglycin in humans during neutrophil differentiation. Immunocytochemistry revealed serglycin immunoreactivity in the Golgi area of promyelocytes (PM) and myelocytes (MC), as well as in a few band cells and mature neutrophil granulocytes. Granular staining was detected near the Golgi apparatus in some of the PM, and the major part of the cytoplasm was negative. Immunoelectron microscopy showed serglycin immunoreactivity located to the Golgi apparatus and a few immature granules of PM and MC. The decreasing level of serglycin protein during myeloid differentiation coincided with a decrease of mRNA expression, as evaluated by Northern blotting. Subcellular fractions of neutrophil granulocytes were obtained. Serglycin immunoreactivity was detected in the fraction containing Golgi apparatus, plasma membrane, and secretory vesicles by Western blotting and enzyme-linked immunosorbent assay. Serglycin was not detected in subcellular fractions containing primary, secondary, or tertiary granules. Together, these findings indicate that serglycin is located to the Golgi apparatus and a few immature granules during neutrophil differentiation. This is consistent with a function for serglycin in formation of granules in neutrophil granulocytes. Our findings contrast the view that native serglycin is present in mature granules and plays a role in packaging and regulating the activity of proteolytic enzymes there.
Assuntos
Neutrófilos/metabolismo , Proteoglicanas/metabolismo , Western Blotting , Ensaio de Imunoadsorção Enzimática , Complexo de Golgi/metabolismo , Humanos , Imuno-Histoquímica , Microscopia Imunoeletrônica , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Proteoglicanas/genética , Proteoglicanas/imunologia , RNA Mensageiro/metabolismo , Proteínas de Transporte VesicularRESUMO
The in vivo expression profiles of cell-cycle proteins regulating G1-to-S-phase transition were determined in three neutrophil precursor populations from human bone marrow: myeloblasts (MBs) and promyelocytes (PMs); myelocytes (MCs) and metamyelocytes (MMs); and band cells (BCs) and segmented neutrophil cells (SCs) and in mature polymorphonuclear neutrophils (PMNs) from peripheral blood. Complete cell-cycle arrest was observed in BCs/SCs and PMNs. Cyclins D1, D2, and D3 were found to be down-regulated during granulopoiesis, whereas a slight increase of cyclin E was seen. In contrast, cyclin-dependent kinase (CDK)2, -4, and -6 were down-regulated from the MC/MM stages and onward. The transcript levels of CDK2, -4, and -6 were concurrently down-regulated. As the only CDK inhibitor, p27kip1 protein and mRNA expression were up-regulated in MCs/MMs and reached peak levels in PMNs. Protein expression of retinoblastoma protein and the related pocket proteins p107 and p130 was down-regulated from the MC/MM stages and onward. This is the first report to describe expression levels of cell-cycle proteins during granulopoiesis in vivo, and it strongly contrasts the observations made in cell-culture systems in vitro.
Assuntos
Proteínas de Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Regulação da Expressão Gênica , Leucopoese/genética , Neutrófilos/citologia , Proteínas Proto-Oncogênicas , Proteínas Supressoras de Tumor/genética , Quinases relacionadas a CDC2 e CDC28/biossíntese , Quinases relacionadas a CDC2 e CDC28/genética , Proteínas de Ciclo Celular/biossíntese , Diferenciação Celular/genética , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/biossíntese , Ciclinas/genética , Fase G1/genética , Perfilação da Expressão Gênica , Humanos , Fase S/genética , Proteínas Supressoras de Tumor/biossínteseRESUMO
In vivo distribution of myeloid transcription factors during granulopoiesis was investigated by Northern and Western blotting in 3 neutrophil precursor populations from human bone marrow: immature (myeloblasts [MBs] and promyelocytes [PMs]); intermediate mature (myelocytes [MCs] and metamyelocytes [MMs]); and mature neutrophil cells (band cells [BCs] and segmented neutrophil cells [SCs]). Nonneutrophil cells were removed with magnetic-bead-coupled antibodies against CD2, CD3, CD14, CD19, CD56, CD61, glycophorin-A, and CD49d (BCs/SCs) before RNA and protein extraction. Polymorphonuclear neutrophils (PMNs) from peripheral blood depleted with anti-CD49d antibodies were also included. Expression of acute myeloid leukemia 1b (AML-1b), c-myb, GATA-1, and CCAAT/enhancer binding protein gamma (C/EBP-gamma) was seen primarily in MBs/PMs, and little expression was found in more mature cells. The level of C/EBP-alpha was constant in the bone marrow-derived cells and decreased in PMNs. C/EBP-epsilon was found primarily in MCs/MMs and was almost absent in more mature cells. Expression of C/EBP-beta, C/EBP-delta, and C/EBP-zeta was observed from the MC/MM stage onward, with peak levels in the most mature cells. The amount of PU.1 increased throughout maturation whereas the level of Elf-1 reached a nadir in MCs/MMs The PU.1 coactivator c-jun and c-jun's dimerization partner c-fos were both detectable in MCs/MMs and increased in amount with maturity. CCAAT displacement protein (CDP) was found at comparable levels at all stages of differentiation. This demonstrates a highly individualized expression of the transcription factors, which can form the basis for the heterogeneous expression of granule proteins during granulopoiesis and cell cycle arrest in metamyelocytes.