摘要:

AbstractErythropoietin (Epo), the hormone that stimulates red blood cell production, is induced by hypoxia. We have utilized the human hepatoma cell line, Hep3B, to investigate the regulation of the Epo gene. We present evidence that the oxygen sensor in Hep3B cells is a heme protein. Hypoxic and cobalt induction of Epo protein is paralleled by a 50‐ to 100‐fold increase in Epo mRNA which we have accurately quantified by means of an assay based on competitive polymerase chain reaction. This increase in Epo mRNA is due primarily to increased transcription. Transfection experiments utilizing the sensitive luciferase reporter gene show that the minimal portions of the Epo gene required for hypoxic induction include a 53 bp promoter element and a 43 bp enhancer located downstream from the polyade‐nylation site. Gel shift experiments show that these two regions cross‐compete for specific DNA binding proteins. The enhancer contains a hexanudeotide direct repeat with a two bp insert which footprints with nuclear extracts from Hep3B cells and, when mutated, results in loss of hypoxic induction. This sequence is likely to bind to a member of the steroid/ thyroid hormone receptor family of DNA binding proteins. These enhancer and promoter elements appear to cooperate in enabling the Epo gene to respond to hypoxia in a physiologically appropriate

ISSN:1066-5099    

DOI:10.1002/stem.5530110604

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractTo study the biogenesis of red ceil membrane skeleton at various stages of erythroid differentiation, we have chosen the following model systems: a) Rauscher erythroleukemia cell line representing the early stages of differentiation, b) Friend erythroleukemia cells, and c) in vitro cultured human erythroblasts. The latter two systems represent terminally differentiated erythroblasts. Using these model systems, we have shown asynchronous synthesis of membrane proteins during erythroid differentiation. At the early stages of erythroid development, the synthesis of spectrin, ankyrin and band 4.1 proteins is initiated before that of the band 3 protein. Following erythroid induction with erythropoietin and dimethylsulfoxide (DMSO), there is a dramatic increase in the synthesis of the band 3 protein without noticeable changes in the synthesis of other membrane proteins. This increase in band 3 synthesis is accompanied by increased stability and recruitment of the skeletal proteins into the membrane skeleton, leading to increased steady state levels. The progressive increase in band 3 synthesis continues during terminal maturation of erythroblasts. This is accompanied by increased stability and assembly of spectrin and ankyrin on the membrane, despite their reduced synthesis. These results point to a key role for the band 3 protein in anchoring and stabilizing these proteins into the permanent skeletal network. Finally, to detect defects of skeletal biosynthesis, we have extended these studies to a patient with severe hereditary spherocytosis characterized by a combined deficiency of spectrin and ankyrin. We have shown that this combined deficiency is a consequence of reduced ankyrin synthesis and mRNA content representing a thalassemia‐like membrane protein mutatio

ISSN:1066-5099    

DOI:10.1002/stem.5530110605

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractAbstract Mouse erythroleukemia Friend cells induced to undergo erythroid differentiation by treatment with hexamethylenebisacetamide (HMBA) were shown to increase cytoplasmic ferrochelatase mRNA, transferrin receptor (TfR) mRNA and glutathione peroxidase (GSHPx) mRNA levels. Inhibition of heme synthesis at the level of 5‐aminolevulinic acid syn‐thase by isonicotinic acid hydrazide (INH) and D,L‐penicillamine (PA) or at the level of 5‐aminolevulinic acid dehydratase by succinylacetone (SA) decreased the expression of ferrochelatase mRNA and TfR mRNA. In contrast with these mRNAs, the synthesis and the levels of glutathione peroxidase mRNA increased by the addition of these inhibitors of heme synthesis. The amount of iron in the intracellular low molecular mass iron pool detected in the post‐mitochondrial supernatant of Friend cells treated with heme synthesis inhibitors was increased. On the other hand, iron levels in this pool declined with the preincubation of Friend cells with iron chelator pyridoxal isonicotinoylhydrazone (PIH). Further treatment with PIH or desferrioxamine (Desferal) increased the synthesis of TfR mRNA in induced Friend cells. The synthesis of ferrochelatase mRNA declined by the same treatment. The opposite was observed when the iron level in the low molecular mass intracellular nonheme iron pool was elevated by treatment with either diferric transferrin (Fe‐Tf) or ferric pyridoxal isonicotinoylhydrazone (Fe‐PIH). Exogenously supplied hemin stimulated the synthesis of ferrochelatase mRNA in uninduced Friend cells, while the synthesis of this mRNA in Friend cells taken on the fifth day after induction was inhibited by the addi

ISSN:1066-5099    

DOI:10.1002/stem.5530110606

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractOur previous research has demonstrated that in hemoglobin‐synthesizing cells, as compared with nonerythroid cells, a step in iron transport from transferrin localized between the transferrin receptor and ferrochelatase is rate‐limiting for the synthesis of heme. In this communication we report our more recent studies on the mechanisms involved in the regulation of the transferrin receptors and ferrochelatase in differentiating erythroid cells. Our studies indicate that transferrin receptor gene expression is regulated differently in hemoglobin synthesizing as compared with uninduced murine erythroleukemia (MEL) cells: 1) With nuclear run‐on assays our experiments showed increased transferrin receptor mRNA transcription cells of MEL following induction of erythroid differentiation with dimethj Isulfoxide (DMSO). 2) DMSO treatment of MEL cells does not increase iron‐responsive element binding protein (ERE‐BP) activity which is, however, increased in uninduced MEL cells by Fe chelators. 3) Following induction of MEL cells there is an increase in the stability of transferrin receptor mRNA whose level is only slightly affected by iron excess. Using murine ferrochelatase cDNA as a probe, two ferrochelatase transcripts having lengths of 2.9 kb and 2.2 kb were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in nonerythroid tissues and the 2.2 more predominant in spleen. In MEL cells, the 2.9 ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by DMSO there is a preferential increase in the 2.2 kb transcript which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript Our further experiments indicate that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal and suggest that the preferential utilization of the upstream polyadenylation signal may be an erythroid‐specific characteristic of ferrochelatase gene expression. These results provide further evidence for the idea that iron metabolism and heme synthesis are controlled by distinct mechanisms in erythroid versus noneryt

ISSN:1066-5099    

DOI:10.1002/stem.5530110607

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have recently described a novel two‐phase liquid culture procedure for growing human erythroid cells in vitro. The two phases are 1) an erythropoietin (EPO)‐independent phase, in which the cells are first cultured in the presence of a combination of growth factors excluding EPO; during this phase, early erythroid committed progenitors, burst forming units (BFU‐e), proliferate and differentiate into colony forming unit (CFU‐e)‐like progenitors; 2) a second phase, in which the latter cells are cultured in an EPO‐supplemented medium, in which the CFU‐e‐like progenitors continue to proliferate and mature into orthochromatic normoblasts and then enucleated erythrocytes. This procedure yields large (up to 5 × 102) and pure (95‐98%) populations of erythroid cells, which allow detailed study of normal and pathologic erythroid maturation, including 1) the effects of growth factors on proliferation and differentiation at various erythroid developmental stages, 2) intracellular iron metabolism in normal and thalassemic erythroid cells and the role of ferritin as an iron donor for heme synthesis, 3) the expression of surface antigens: transferrin receptor, glycophorin, A, B, H, D and I/i antigens, 4) synthesis of erythroid‐specific membrane proteins, 5) the kinetics of globin mRNA accumulation during erythroid maturation, 6) the expression of exogenous human β globin gene in β‐thalassemic cells as a model for gene therapy, and 7) the enhancement of γ globin chain syn

ISSN:1066-5099    

DOI:10.1002/stem.5530110608

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractAbstract The follow‐up of patients after bone marrow transplantation (BMT) revealed some discrepancies between red blood cell and white blood cell origin. In all six patients under study, the DNA analysis showed full engraftment, while red blood cells in some of them indicated persistence of recipient bone marrow activity. Abnormalities detected by the probe p362A (XY homologous region) in electrophoretic patterns observed during the period of graft versus host disease (GVHD) are discusse

ISSN:1066-5099    

DOI:10.1002/stem.5530110609

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractThe effect of recombinant human granulocyte colony‐stimulating factor (G‐CSF) administration to mice on the retroviral‐mediated transfer of the selectable marker geneneoto hematopoietic cells was studied. After G‐CSF treatment, blood mononuclear cells and bone marrow cells were infected with the retrovirus, and the efficiency of gene transfer into myeloid progenitors was increased in peripheral blood and decreased in bone marrow cells. Bone marrow four to six months after transplantation from G‐CSF treated mice revealed the presence of the transferredneogene in 4 out of 104 mice, which is an eightfold lower proportion than in the control group. These data suggest that G‐CSF in vivo treatment decreases bone marrow sensitivity and increases the sensitivity of peripheral blood cells to retrovira

ISSN:1066-5099    

DOI:10.1002/stem.5530110610

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractRecent evidence from molecular biology studies of the cell cycle machinery suggests that, apart from oncogenes and tumor suppressor genes, the genes encoding the key cell cycle regulatory proteins could serve as additional targets for oncogenic mutations involved in the multistep process of car‐cinogenesis. In an attempt to identify such potential cancer‐associated aberrations of the cell cycle regulators, the expression of cdc2 and cdk2 kinases, as well as cyclins A, Bl and Dl, was analyzed by immunoblotting in a panel of more than 40 human cancer cell lines derived from 17 different tumor types. The expression of cdc2, cdk2, cyclin Bl and cyclin A polypeptides was detectable in all lines examined, and moderate variation in protein level does not provide evidence for any obvious abnormalities in the cancer cell lines studied. The application of a series of novel monoclonal antibodies (Mab) to human cdc2 revealed the existence of an intriguing protein, designated p37, immunologically and structurally related to cdc2, which is strongly and selectively expressed in about 50% of the cancer cell lines. In contrast to cyclin A, which has also been implicated in tumorigenesis, we found pronounced variation in abundance of the cyclin Dl protein. Our data suggest that dysregulation of cyclin Dl (a candidatebcl‐1, PRAD1 oncogene) can be involved in the path‐ogenesis of some additional tumor types (e.g., sarcomas and neuroblastomas) besides those reported for amplification and/or mRNA overexpression of this oncogene. The cancer cell lines with high and extremely low levels of cyclin Dl identified in the present study could represent suitable in vitro models to further elucidate the role of this cyclin in the process of neoplastic transfo

ISSN:1066-5099    

DOI:10.1002/stem.5530110611

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractBone marrow cells from patients with chronic myeloid leukemia (CML) in in vitro long‐term bone marrow culture (LTBMC) show an impaired survival of Philadelphia (Ph) positive cells and, in a proportion of patients, the emergence of Philadelphia negative hemopoiesis. The standard conditions of in vitro cultures provide optimal purging effect Selected patients can now have autologous bone marrow transplants (ABMT) with in vitro purged cell

ISSN:1066-5099    

DOI:10.1002/stem.5530110612

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractAbstract The clonal origin of malignancy and he‐matopoiesis is a principal tenet of modern biology and medicine. This paper describes a highly specific and sensitive assay for the detection of clonality in cells and cell lineages suitable for studies in a large proportion of females. The specific ligase chain and/ or ligase detection reactions (LCR/LDR) are utilized at a polymorphic glucose‐6‐phosphate dehydrogen‐ase (G‐6‐PD) locus for discrimination of the mRNA transcripts of the active X chromosome. This combination approach circumvents problems encountered with other currently used assays of clonality based either on peptide G‐6‐PD polymorphism or on DNA methylation differences between the active and inactive X chromosomes. The veracity of this assay was verified by analysis of 19 random healthy females as well as by the study of hemopoietic and nonhemopo‐ietic tissues from a patient with clonal hemopoiesis/ polycythemia vera. Furthermore, we demonstrate that the G‐6‐PD locus used in our clonal assay does not display marked differences in methylation between the active and the i

ISSN:1066-5099    

DOI:10.1002/stem.5530110613

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY