RESUMEN
Megakaryocytes are highly specialized cells that arise from a bipotent megakaryocytic-erythroid progenitor (MEP). This developmental leap requires coordinated activation of megakaryocyte-specific genes, radical changes in cell cycle properties, and active prevention of erythroid differentiation. These programs result from upregulation of megakaryocyte-selective transcription factors, downregulation of erythroid-selective transcription factors and ongoing mediation of common erythro-megakaryocytic transcription factors. Unlike most developmental programs, no single lineage-unique family of master regulators exerts executive control over the megakaryocytic plan. Rather, an assemblage of non-unique factors and signals converge to determine lineage and differentiation. In human megakaryopoiesis, hereditary disorders of platelet production have confirmed contributions from three distinct transcription factor families. Murine models have extended this repertoire to include multiple additional factors. At a mechanistic level, the means by which these non-unique factors collaborate in the establishment of a perfectly unique cell type remains a central question.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Megacariocitos/citología , Megacariocitos/metabolismo , Trombopoyesis/genética , Transcripción Genética/fisiología , Animales , Diferenciación Celular/genética , Humanos , Megacariocitos/patologíaRESUMEN
Contact with bone marrow stromal cells maintains normal and leukemic hematopoietic progenitors in an undifferentiated state. Recently, stromal contact has been shown to diminish the yield of megakaryocytes in cultures of primary human hematopoietic stem cells. This inhibition may explain the poor megakaryocytic engraftment frequently observed after bone marrow transplantation. In the current study, stromal co-culture is shown to render K562 cells refractory to megakaryocytic induction. This stromal inhibition correlated with the selective down-regulation in K562 cells of protein kinase C-epsilon (PKC-epsilon), which has recently been implicated in regulation of megakaryocytic lineage commitment. In addition, the stromal inhibition correlated with inactivation of the ERK/MAPK pathway, which has also been implicated in promoting megakaryocytic development. Forced expression of PKC-epsilon by retroviral transduction was insufficient to reverse the stromal blockade of ERK/MAPK signaling or of megakaryocytic induction. Thus stromal interruption of ERK/MAPK signaling occurred independently of PKC-epsilon levels and correlated more closely with megakaryocytic blockade. These findings provide potential mechanisms for stromal inhibition of hematopoietic differentiation and possibly for the poor megakaryocytic engraftment seen after bone marrow transplantation.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Isoenzimas/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Megacariocitos/citología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Células del Estroma/fisiología , Células de la Médula Ósea/citología , Línea Celular , Técnicas de Cocultivo , Citometría de Flujo , Genes Reporteros , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas/citología , Humanos , Inmunofenotipificación , Isoenzimas/genética , Células K562 , Proteínas Luminiscentes/genética , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C-epsilon , Proteínas Recombinantes/metabolismo , Células del Estroma/citología , TransfecciónRESUMEN
In myelodysplasias and acute myeloid leukemias, abnormalities in erythroid development often parallel abnormalities in megakaryocytic development. Erythroleukemic cells in particular have been shown to possess the potential to undergo megakaryocytic differentiation in response to a variety of stimuli. Whether or not such lineage plasticity occurs as a consequence of the leukemic phenotype has not previously been addressed. In this study, highly purified primary human erythroid progenitors were subjected to stimuli known to induce megakaryocytic differentiation in erythroleukemic cells. Remarkably, the primary erythroid progenitors rapidly responded with morphological and immunophenotypic evidence of megakaryocytic differentiation, equivalent to that seen in erythroleukemic cells. Even erythroblasts expressing high levels of hemoglobin manifested partial megakaryocytic differentiation. These results indicate that the lineage plasticity observed in erythroleukemic cells reflects an intrinsic property of cells in the erythroid lineage rather than an epiphenomenon of leukemic transformation.
Asunto(s)
Eritroblastos/citología , Megacariocitos/citología , Diferenciación Celular , Separación Celular , Sangre Fetal/citología , Humanos , Leucemia/patología , Células Tumorales CultivadasRESUMEN
Multiple studies have shown that intracellular signal transduction by the protein kinase C (PKC) family participates in the initiation of megakaryocyte differentiation. In this study, multiple approaches addressed the functional contributions by specific PKC isozymes to megakaryocytic lineage commitment of two independent cell lines, K562 and human erythroleukemia (HEL). Pharmacologic profiles of induction and inhibition of megakaryocytic differentiation in both cell lines suggested a role for the calcium-independent novel PKCs, in particular PKC-epsilon. In transfection studies, the isolated variable domain of PKC-epsilon selectively blocked exogenous activation of the megakaryocyte-specific alpha IIb promoter. Constitutively active mutants of PKC-epsilon, but not of other PKC isozymes, cooperated with the transcription factor GATA-1 in the activation of the alpha IIb promoter. The functional cooperation between GATA-1 and PKC-epsilon displayed dependence on cellular milieu, as well as on the promoter context of GATA binding sites. In aggregate, the data suggest that PKC-epsilon specifically participates in megakaryocytic lineage commitment through functional cooperation with GATA-1 in the activation of megakaryocytic promoters.
Asunto(s)
Diferenciación Celular , Isoenzimas/metabolismo , Megacariocitos/citología , Megacariocitos/enzimología , Proteína Quinasa C/metabolismo , Carbazoles/farmacología , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Proteínas de Unión al ADN/metabolismo , Diterpenos/farmacología , Factores de Unión al ADN Específico de las Células Eritroides , Técnica del Anticuerpo Fluorescente , Factor de Transcripción GATA1 , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Células K562 , Maleimidas/farmacología , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Mutación , Regiones Promotoras Genéticas/genética , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Proteína Quinasa C/genética , Proteína Quinasa C-epsilon , Transporte de Proteínas/efectos de los fármacos , Elementos de Respuesta/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Basic helix-loop-helix (bHLH) transcription factors often function as heterodimeric complexes consisting of a tissue-specific factor such as SCL/tal or MyoD bound to a broadly expressed E protein. bHLH dimerization therefore appears to represent a key regulatory step in cell lineage determination and oncogenesis. Previous functional and structural studies have indicated that the well defined HLH domain is both necessary and sufficient for dimerization. Most of these studies, however, have employed in vitro systems for analysis of HLH dimerization, and their implications for the requirements for in vivo dimerization remain unclear. Using multiple approaches, we have analyzed bHLH dimerization in intact, living cells and have identified a novel domain in E proteins, domain C, which is required for in vivo dimerization. Domain C, which lies just carboxyl-terminal to helix 2 of the HLH domain, represents the most highly conserved region within E proteins and appears to influence the in vivo conformation of the adjacent HLH domain. These results suggest that HLH dimerization in vivo may represent a complex, regulated process that is distinct from HLH dimerization in vitro.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Proteínas Proto-Oncogénicas , Transactivadores/metabolismo , Secuencia de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Secuencia Conservada , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/genética , Dimerización , Evolución Molecular , Proteína MioD/genética , Proteína MioD/metabolismo , Filogenia , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Factores de Transcripción TCF , Transactivadores/clasificación , Transactivadores/genética , Proteína 1 Similar al Factor de Transcripción 7 , Proteína 2 Similar al Factor de Transcripción 7 , Factores de Transcripción/clasificaciónRESUMEN
The extracellular signal-regulated kinase (ERK), originally identified as a participant in mitogenic signaling, has recently been implicated in the signaling of cellular differentiation. To examine the role of the ERK/MAP kinase pathway in megakaryocytic differentiation of K562 cells, the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin on ERK activation were determined. Both TPA and bryostatin are known to activate PKC but paradoxically have opposing effects on megakaryocytic differentiation. TPA, a differentiation inducer, caused sustained activation of ERK (>24 h), whereas bryostatin, a differentiation blocker, only transiently activated ERK ( approximately 6 h) and attenuated the activation of ERK by TPA. To confirm a requirement for sustained ERK activation for megakaryocytic differentiation, PD098059, a synthetic inhibitor of the MAP kinase kinase 1 (MEK1) was employed. Introduction of PD098059 at any time during the first 18 h of TPA treatment completely abrogated megakaryocytic differentiation of K562 cells. After 24 h of TPA treatment, introduction of PD098059 failed to block differentiation. Differentiation blockade by PD098059 occurred via inhibition of MEK because transfection of a constitutively active mutant of MEK2 could override the PD098059 blockade. Experiments with conditioned media suggested that sustained activation of the ERK/MAP kinase pathway promoted the autocrine secretion of megakaryocytic lineage determination factors.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Megacariocitos/fisiología , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Diferenciación Celular , Células Cultivadas , Medios de Cultivo Condicionados , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 2 , Megacariocitos/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Transducción de Señal , Acetato de TetradecanoilforbolRESUMEN
The basic helix-loop-helix (bHLH) transcriptional factor E2A has previously been shown to play a critical role in early B cell development, with E2A knockout mice and Id1 transgenic mice showing an arrest at the pro-B cell stage of development. More recent data suggest that E2A, through an interaction with the immunoglobulin heavy chain 3' enhancer, might also regulate later events in B cell development such as heavy chain class switching. The patterns of E2A protein expression in secondary lymphoid tissues support a role in later stages of B cell maturation. In particular, immunostaining reveals upregulation of E2A protein in cells of the dark zone of the germinal center, the site of immunoglobulin heavy chain class switching. To examine the role of E2A in class switching, the inhibitory HLH protein Id1 was expressed in B cell lines which normally undergo spontaneous and inducible switching from IgM to IgA. The forced expression of Id1 in these cell lines effectively blocked class switching. This Id1 blockade of class switching did not occur via downregulation of immunoglobulin heavy chain germline transcription or through inhibition of cell cycling. Furthermore, Id1 inhibited spontaneous and, to a lesser degree, cytokine inducible class switching. From these data, we conclude that E2A plays an important role in the class switching process.
Asunto(s)
Proteínas de Unión al ADN/inmunología , Reordenamiento Génico de Linfocito B/genética , Genes de Inmunoglobulinas/genética , Secuencias Hélice-Asa-Hélice/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Factores de Transcripción/inmunología , Animales , Ratones , Ratones Noqueados , Ratones TransgénicosRESUMEN
Dimerization represents a key regulatory step in the function of basic helix-loop-helix transcriptional factors. In many instances tissue-specific basic helix-loop-helix proteins, such as the hematopoietic factor SCL/tal or the myogenic factor MyoD, interact with ubiquitously expressed basic helix-loop-helix proteins, such as E2A or E2-2. Such dimerization is necessary for high affinity, sequence-specific DNA binding. Previous biochemical and structural studies have shown the helix-loop-helix region to be necessary and sufficient for this interaction. In the present study, we analyzed the relative affinities of various helix-loop-helix interactions using the yeast two-hybrid system. The relative affinities of selected helix-loop-helix species for the partner protein E2-2 were as follows: Id2 > MyoD > SCL/tal. Mutants of SCL/tal with increased affinity for E2-2 were selected from a library of randomly mutated basic helix-loop-helix domains. The amino acid changes in these high affinity versions of SCL/tal introduced residues that resembled those in the corresponding positions of the Id proteins and MyoD. One of the mutants, SCL 12, also contained mutations in highly conserved residues previously thought to be necessary for dimerization. This mutant of SCL demonstrated diminished temperature sensitivity in in vitro interaction assays as compared with the wild type protein. Computational modeling of helix-loop-helix dimers provides an explanation for the increased dimerization affinity of SCL mutant 12.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Proteínas Proto-Oncogénicas , Factores de Transcripción , Secuencia de Aminoácidos , Secuencia de Bases , Biopolímeros , Análisis Mutacional de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Datos de Secuencia Molecular , Mutagénesis , Proteína MioD/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
In cases of T-cell acute lymphoblastic leukemia (T-ALL), the basic helix-loop-helix (bHLH) oncogene SCL/tal undergoes frequent rearrangements activating ectopic expression. Despite the compelling epidemiological association of SCL/tal expression with T-ALL, no specific transforming function has been attributable to the protein product. However, investigators have recently demonstrated that forced overexpression of SCL/tal can block monocytic differentiation of M1 murine myeloid leukemia cells. Thus, inappropriate expression of wild-type SCL/tal protein may in part account for the maturation arrest phenotype observed in T-ALL cells. In this study, ectopic expression of the SCL/tal gene blocked the differentiation of C2C12 muscle precursor cells. Characterization of the mechanism of differentiation blockade showed that the SCL/tal protein repressed transcriptional activation by the myogenic bHLH factor MyoD. Protein interaction analysis showed that SCL/tal and MyoD compete for common partners (E bHLH proteins) but do not directly bind one other. A model is thus proposed in which ectopic SCL/tal protein, by its ability to titrate out E proteins, prevents the formation of bHLH complexes that drive cellular differentiation: the "Id-like" mechanism.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación Leucémica de la Expresión Génica , Secuencias Hélice-Asa-Hélice/fisiología , Leucemia-Linfoma de Células T del Adulto/patología , Células Madre Neoplásicas/patología , Proteínas del Tejido Nervioso , Proteínas Proto-Oncogénicas , Factores de Transcripción , Transcripción Genética/fisiología , Células 3T3 , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Fibroblastos/metabolismo , Reordenamiento Génico de Linfocito T , Genes Sintéticos , Leucemia Experimental/patología , Leucemia Mieloide/patología , Leucemia-Linfoma de Células T del Adulto/genética , Ratones , Ratones Endogámicos C3H , Músculos/citología , Proteína MioD/antagonistas & inhibidores , Proteína MioD/química , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína 1 de la Leucemia Linfocítica T Aguda , Factores de Transcripción TCF , Transactivadores/antagonistas & inhibidores , Transactivadores/química , Factor de Transcripción 4RESUMEN
The DNA-binding basic domain of helix-loop-helix transcriptional (HLH) factors in several instances also serves as a nuclear localization signal (NLS). Interestingly, some members of the HLH family of proteins lack a basic domain or any other recognizable NLS and yet still display efficient nuclear localization. To study this apparent paradox, we used the hematopoietic HLH protein SCL/tal as a model. Deletion of the basic domain converted SCL/tal from nuclear to a cytoplasmic protein. However, the basic domain deficient SCL/tal protein could be redirected to the nucleus by coexpression of E2-5, an SCL/tal-binding HLH protein with an intact basic domain. These results indicate that heterodimerization of HLH proteins may take place in the cytoplasm prior to nuclear localization and that nuclear localization for HLH complexes is a dominant process requiring only a single NLS per complex.
Asunto(s)
Compartimento Celular/fisiología , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Proteínas Proto-Oncogénicas , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células Cultivadas , Proteínas de Unión al ADN/aislamiento & purificación , Técnica del Anticuerpo Fluorescente , Datos de Secuencia Molecular , Factores de Transcripción/aislamiento & purificaciónRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is a relatively uncommon disease, constituting only approximately 15% of newly diagnosed acute lymphoblastic leukemias (ALL) in the United States, or roughly 300 cases per year. Outside of the United States, in countries such as Egypt and India, T-ALL may represent as much as 50% of all ALL's but still remains an overall rare disease. The clinical importance of T-ALL lies in its poor responsiveness to therapy that has proved highly effective with standard B-cell precursor ALL (BCP-ALL). The scientific importance of human T-ALL has resided in its role as a cancer prototype, permitting the identification of novel genes centrally involved in both neoplastic change and normal cellular differentiation. One of these genes, SCL/tal, has received significant attention due to its intimate involvement in T-ALL, as well as in normal hematopoiesis. Although a tremendous amount has been recently discovered about SCL/tal, its exact roles in leukemogenesis and normal hematopoiesis remain obscure.
Asunto(s)
Proteínas de Unión al ADN/genética , Secuencias Hélice-Asa-Hélice/genética , Leucemia-Linfoma de Células T del Adulto/genética , Proto-Oncogenes , Factores de Transcripción , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cromosomas Humanos Par 1 , Egipto/epidemiología , Expresión Génica , Humanos , Incidencia , India/epidemiología , Leucemia-Linfoma de Células T del Adulto/epidemiología , Familia de Multigenes , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiología , Proteínas Proto-Oncogénicas/genética , Proteína 1 de la Leucemia Linfocítica T Aguda , Transcripción Genética , Translocación Genética , Estados Unidos/epidemiologíaRESUMEN
We have studied eight different Epstein-Barr virus transformed cell lines (EBV-LCL) with respect to genomic methylation at a single locus, M-bcr (the major breakpoint cluster region of chronic myelogenous leukemia). Restriction digests with the methylation sensitive enzyme, Hpall, illustrated marked differences in M-bcr methylation patterns between the various cell lines. Some of the cell lines displayed prominent allelic heterogeneity: within each of these samples there were numerous different M-bcr/Hpall allelic fragments on Southern analysis. Other cell lines displayed allelic predominance: within each of these samples a limited number of M-bcr/Hpall allelic fragments predominated. Analysis of Immunoglobulin JH rearrangements demonstrated a close correlation between clonal complexity (i.e. the number of rearranged JH fragments) and allelic heterogeneity of M-bcr methylation (i.e. the number of M-bcr/Hpall fragments). We conclude that analysis of genomic methylation in the M-bcr locus may offer a novel approach to clonality determination in EBV transformed lymphocytes.
Asunto(s)
ADN/metabolismo , Genoma , Herpesvirus Humano 4/fisiología , Activación de Linfocitos , Linfocitos/metabolismo , Alelos , Línea Celular Transformada , Mapeo Cromosómico , Células Clonales , Humanos , MetilaciónRESUMEN
SCL/tal is a putative oncogene originally identified through its involvement in the translocation t(1;14)(p32;q11) present in the leukemic cell line DU.528. Subsequent studies have shown an upstream deletion activating expression of SCL/tal to be one of the most common genetic lesions in T-cell acute lymphoblastic leukemia (T-ALL). The cDNA sequence of SCL/tal encodes a basic helix-loop-helix (bHLH) protein with regions of marked homology to lyl-1 and tal-2, two other bHLH proteins involved in T-ALL chromosomal translocations. The bHLH motif suggests that the SCL/tal product localizes to the nucleus, binds to specific DNA sequences, and regulates transcription of a specific array of target genes. Our studies directly identify the SCL/tal product as a 42-Kd phosphoprotein that efficiently localizes to the nucleus. Deletion mutagenesis has allowed identification of a region critical for nuclear localization, a region that corresponds to the DNA-binding basic domain within the bHLH motif. Because this domain is shared by lyl-1 and tal-2, these latter putative T-cell oncoproteins probably use a nuclear localization mechanism identical to that of SCL/tal.
Asunto(s)
Cromosomas Humanos Par 14 , Cromosomas Humanos Par 1 , Proteínas de Unión al ADN/genética , Leucemia-Linfoma de Células T del Adulto/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción , Translocación Genética , Aminoácidos/análisis , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , Proteínas de Unión al ADN/análisis , Técnica del Anticuerpo Fluorescente , Humanos , Datos de Secuencia Molecular , Peso Molecular , Proteínas Nucleares/análisis , Oligodesoxirribonucleótidos , Fosfoproteínas/análisis , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/análisis , Proteína 1 de la Leucemia Linfocítica T Aguda , Transfección , Células Tumorales CultivadasRESUMEN
We have designed a chimeric gene, Id-SCL, in which the 3' helix-loop-helix encoding portion of the presumptive oncogene SCL/tal is joined to the 5' coding portion of Id, an inhibitory helix-loop-helix gene. The predicted protein product of this chimeric gene contains the helix-loop-helix dimerization domain of SCL/tal, but, lacking a basic DNA binding domain, is predicted to have the inhibitory function of the Id product. Expression of the Id-SCL fusion gene in stably transfected K562 cells reproducibly resulted in nuclear segmentation and depressed growth rates; both of these phenotypic effects demonstrated a dosage dependence on the levels of Id-SCL mRNA and protein expressed in the various clones. Electron microscopy of cells expressing high levels of Id-SCL mRNA showed a significant increase in cytoplasmic perinuclear thin filaments and diminution of marginal heterochromatin in the nuclei. No other changes in hematopoietic differentiation status were observed in association with Id-SCL expression. Expression of intact Id and SCL/tal genes, as well as deletion mutants of Id and SCL/tal, independently transfected into K562 cells, indicated that the nuclear segmentation effect is dependent on the presence of a protein possessing a helix-loop-helix domain but lacking a basic domain. Our studies suggest that the balance of transcriptional inhibitory and stimulatory helix-loop-helix proteins in cells may be important determinants of proliferation and of structural organization within cells.
Asunto(s)
Núcleo Celular/ultraestructura , Quimera/genética , Regulación Leucémica de la Expresión Génica/genética , Leucemia/genética , Leucemia/patología , Oncogenes/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Núcleo Celular/química , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Heterocromatina/ultraestructura , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Ploidias , ARN Mensajero/análisis , ARN Mensajero/genética , Transfección , Células Tumorales CultivadasRESUMEN
It has been shown that a 600 bp long cluster of cell lineage specific hypomethylated sites in the major breakpoint cluster region (M-bcr) on chromosome 22 exists in hematopoietic cells. To determine possible relationships between methylation patterns within the M-bcr and the stage of hematopoietic cell development, the M-bcr methylation status of 39 patients with leukemia and lymphoma and two patients with myelodysplastic syndrome with non-rearranged M-bcrs was examined by BgIII-HpaII digestion. In the myeloid malignancies, the presence of a hypermethylated 4.8 kb BgIII-BgIII M-bcr allele was directly proportional to the combined myeloblast and promyelocyte percentage of the specimen, whereas the presence of a 2.5 kb BgIII-HpaII allele was directly proportional to the combined percentage of monocytic cells and neutrophils. All five acute monoblastic leukemias showed a methylation pattern that closely resembled neutrophils. All of thirteen surface immunoglobulin positive B-cell malignancies showed a distinct methylation pattern consisting of three or more BgIII-HpaII restriction fragments of 2.5 kb or less in length. The B-cell precursor leukemias showed heterogeneous M-bcr methylation patterns, with four of seven showing a B-cell pattern and three showing a hypermethylated pattern with 4.8, 3.1/3.0 and/or 2.5 kb BgIII-HpaII M-bcr alleles. It is concluded that the M-bcr methylation status is related to the maturation of the neutrophil series; the surface immunoglobulin positive B-cell malignancies are characterized by a distinct, extreme hypomethylation pattern of the M-bcr; and the B-cell precursor malignancies appear to have a heterogeneous M-bcr methylation pattern.
Asunto(s)
Alelos , Anemia Refractaria/genética , Cromosomas Humanos Par 22 , ADN de Neoplasias/análisis , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Mieloide/genética , Linfoma no Hodgkin/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Enfermedad Aguda , Anemia Refractaria/patología , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Mieloide/patología , Linfoma no Hodgkin/patología , Metilación , Neutrófilos/patología , Polimorfismo de Longitud del Fragmento de Restricción , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
To examine the role of DNA methylation in breakpoint location of chromosomal translocation, HpaII sites in and flanking the M-bcr on chromosome 22 were mapped in DNA from blood granulocytes and lymphocytes, bone marrow cells, thymic tissue, and spermatozoa from normal individuals. Allelic HpaII sites were identified clustered in a 600-base pair genomic area of the M-bcr. Bone marrow cells and blood granulocyte DNA showed identical allelic patterns. Thymic tissue and blood lymphocytes showed identical allelic patterns distinct from bone marrow cells and blood granulocytes. Spermatozoa showed a third methylation pattern. In all individuals, the HpaII sites were present within the BamHI/BglII fragment of the M-bcr, the same area associated with high breakpoint frequency in chronic myelogenous leukemia (CML). Three of 15 patients with chronic phase CML showed fully methylated rearranged BglII/BglII M-bcr restriction fragments not seen in normal bone marrow cells. These methylation patterns of the M-bcr may be important in CML breakpoint location and may be a marker for tissue differentiation.
Asunto(s)
ADN/genética , Leucocitos/análisis , Southern Blotting , ADN/sangre , ADN/aislamiento & purificación , Granulocitos/análisis , Humanos , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/genética , Linfocitos/análisis , Metilación , Hibridación de Ácido Nucleico , Valores de Referencia , Mapeo RestrictivoRESUMEN
Patients afflicted by homozygous beta-thalassemia suffer from severe anemia and hypersplenism and are dependent on blood transfusions. The consequent hypoxia and massive tissue iron deposition lead to concomitant cardiac, hepatic, and endocrine system failure. Hemodynamic changes related to gestation may aggravate the underlying multiorgan damage of the pregnant mother and lead to high fetal wastage. These entanglements may be prevented by performing thorough maternal and fetal surveillance and by maintaining the hemoglobin concentration above 10 g/dL. We describe a successful full-term pregnancy in a patient with transfusion-dependent homozygous beta-thalassemia major.
Asunto(s)
Homocigoto , Complicaciones Hematológicas del Embarazo , Talasemia , Adulto , Transfusión Sanguínea , Clomifeno , Transfusión de Eritrocitos , Femenino , Monitoreo Fetal , Hemoglobinas/análisis , Humanos , Inducción de la Ovulación , Embarazo , Complicaciones Hematológicas del Embarazo/sangre , Complicaciones Hematológicas del Embarazo/terapia , Talasemia/sangre , Talasemia/genética , Talasemia/terapiaRESUMEN
Peritoneovenous shunting has been successfully employed for several years in the treatment of severe ascites resistant to medical therapy. Many short and long term complications of this procedure have been documented, including thrombotic events and consumptive coagulopathy. Reported below is the finding of a single, continuous thrombus extending from the right atrium to the distal pulmonary arterial tree, a novel complication of peritoneovenous shunting with multiple possible causes.