How do retroviral oncogenes induce transformation in avian erythroid cells?

1985 ◽  
Vol 226 (1242) ◽  
pp. 121-126 ◽  
Keyword(s):  
Early Stage ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Self Renewal ◽  
Erythroid Precursor ◽  
Erythroid Precursors ◽  
Leukaemic Cells ◽  
Transforming Proteins

The v-erb B oncogene, as well as other oncogenes of the src -gene family transform immature erythroid cells from chick bone marrow in vivo and in vitro . The erb B-transformed erythroid cells differ from normal late erythroid precursors (CFU-E) in that they have acquired the capacity to undergo self-renewal as well as to differentiate terminally. They also do not require the normal erythroid differentiation hormone, erythro-poietin, for either process. Cooperation of v-erb B with a second oncogene, v-erb A, results in a differentiation arrest of the transformed cells, which now only use the self-renewal pathway. Studies with conditional and non-conditional mutants in both v-erb B and v-erb A will be presented to elucidate further how the transforming proteins encoded by these oncogenes, gp74 erb B and gp75 gag-erb A , affect the differentiation programme of the infected erythroid precursor with the outcome of hormone-independent leukaemic cells arrested at an early stage of erythroid differentiation.

scholarly journals Functional Investigation of the Argonaute Proteins in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Gordon G. L. Wong ◽  
Gabriela Krivdova ◽  
Olga I. Gan ◽  
Jessica L. McLeod ◽  
John E. Dick ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Lineage Commitment ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Erythroid Precursor ◽  
Lineage Differentiation ◽  
Erythroid Precursors

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

b1 Integrin Deficiency in Both Erythroid Cells and Their Microenvironment Does Not Affect Basal Erythropoiesis but Critically Impairs Survival and Erythroid Response to Phenylhydrazine-Induced Stress in Adult Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3567-3567
Author(s):  
Tatiana Ulyanova ◽  
Gregory V. Priestley ◽  
Yi Jiang ◽  
Stephen Padilla ◽  
Thalia Papayannopoulou
Keyword(s):  
Critical Role ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
In Vivo Experiments ◽  
Adult Mice ◽  
Erythroid Precursors ◽  
And Control ◽  
Deficient Mice

Abstract Previous experiments in vitro have emphasized the important role of a5b1 integrin/fibronectin interactions in terminal stages of erythroid differentiation (JCB1987, 105:3105), whereas in vivo experiments with genetically deficient mice (JI2000, 165:4667) and recent in vitro ones emphasized the important contribution of a4b1 integrin in the expansion of fetal erythroid progenitors (JCB2007, 177:871) or for optimal responses post stress in adult animals (MCB2003, 23:9349). However, no abnormalities in erythropoiesis were reported in a model of conditional ablation of b1 integrins post-transplantation (Blood2006, 108:1857). Therefore, it has not been clear to what extent each of the two major b1 integrins (a4b1 and a5b1) alone or in combination is critical for expansion and/or terminal erythroid differentiation of adult cells at homeostasis and/or after stress. We have made detailed and parallel observations comparing erythropoiesis in two genetic models with conditional ablation of b1 or a4 integrins at homeostasis and after phenylhydrazine (PHZ)-mediated stress. Basal erythropoiesis in b1-, a4-deficient and control mice as assessed by hematocrit levels and total nucleated erythroid cells (Ter119+) in BM and spleen was similar. Furthermore, both b1 and a4-deficient mice showed an increase in circulating progenitors (1275±230 CFC/ml PB, 2446±256 CFC/ml PB, respectively) over controls (338±113 CFC/ml PB). However, post PHZ-induced hemolytic stress there was a dramatic difference in outcomes of b1-deficient, but modest differences in a4-deficient mice compared to controls. Survival of b1-deficient mice by day 6 post PHZ was 33% compared to 100% in a4-deficient and control groups. In b1-deficient animals, no significant increase in spleen cellularity (153±26×106 and194±64×106 cells/spleen at day 0 and 6 post PHZ, respectively) was detected and the expansion of total erythroid precursors (CD71hi,Ter119+) in the spleen was minimal (from 2.08×106 to 10.8×106 cells/spleen at day 6). In contrast, in a4-deficient and control mice by the same time spleen cellularity increased respectively by 3 and 8 fold, and erythroid precursors expanded by 400 and 2,500 fold. Of interest, BM response to PHZ was not significantly different among all groups. To test whether the splenic response was cell-autonomous or environmentally controlled we compared PHZ response in wild type recipients reconstituted with b1-ablated (Cre+b1D/D) or with control (Cre-b1f/f) BM cells. Recipients of b1-ablated cells had an impaired response compared to recipients of control cells, which was somewhat intermediate to that seen in non-transplanted b1-deficient animals; by day 6 post PHZ, spleen cellularity was 300±24×106 cells/spleen and erythroid precursors expanded by 130 fold in recipients of b1-ablated BM cells compared to 859±159×106 cells/spleen and 900 fold precursor increase in control recipients. These data suggest that both erythroid and their environmental cells were responsible for the reduced survival and poor spleen response in b1-deficient mice. The target environmental cells (fibroblasts, endothelial cells, macrophages) and/or matrix involved will be the focus of future studies. It is of interest that in contrast to splenic response, the increased release of progenitors from BM seen in animals reconstituted with b1D/D cells was as high as that seen in non-transplanted b1- deficient animals and with the same qualitative characteristics, suggesting this alteration in biodistribution of progenitors is cell autonomous. Taken together, our data suggest that a combined expression of b1 integrins in erythroid and cells in their microenvironment is critical for survival and optimal splenic response to a PHZ-induced stress in adult mice; release of progenitors seen at homeostasis in both b1 and a4 models is cell autonomous with a preferential erythroid progenitor release from BM seen only in b1-deficient but not in a4-deficient mice; in contrast to results with fetal liver cells showing a critical role of a4b1 but not a5b1 integrin for proliferative expansion of erythroid cells, in adults a5b1 expression in erythroid and environmental cells in the spleen assumes a more critical role. Our data expand the current knowledge on the distinct dependency of a4b1 vs a5b1 integrins in basal vs stress erythropoiesis and bridge previously divergent information from in vitro and in vivo experiments.

scholarly journals Isolation of erythropoietin-sensitive cells from Friend virus-infected marrow cultures: characteristics of the erythropoietin response

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 751-758 ◽  
Author(s):  
M Bondurant ◽  
M Koury ◽  
SB Krantz ◽  
T Blevins ◽  
DT Duncan
Keyword(s):  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Precursor Cells ◽  
Erythroid Cells ◽  
Friend Virus ◽  
Isolated Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Erythroid Precursor ◽  
Short Time

Abstract Murine erythroid precursor cells, stimulated to proliferate in vitro in the absence of added erythropoietin (EP) by the anemia strain of Friend virus (FVA), will subsequently respond to EP by complete erythrocyte differentiation. If not exposed to EP, the erythroid cells divide for about 120 hr in culture, and they maintain the potential for full differentiation in response to EP added at any time during the period from 72 to 120 hr. Between 96 and 120 hr of culture without added EP, the EP-sensitive erythroid precursor cells that have formed discrete erythroid bursts can be isolated in relatively large numbers from such cultures by plucking with a Pasteur pipette. The addition of EP initiates the final stages of erythroid differentiation, including heme synthesis in 70%-80% of these isolated cells. With respect to homogeneity of the precursor cells, quantity of EP-responsive cells obtainable, and uniformity of EP responsiveness, this system is uniquely favorable for biochemical studies of the late differentiation effects of EP. The overall changes in gene expression accompanying EP- induced terminal differentiation were examined by two-dimensional gel electrophoresis of proteins labeled for a short time with radioactive amino acids. Several new proteins are synthesized in these erythroid cells during terminal differentiation, but the number is a very small percentage of the total number of proteins being made. Thus, in this system, the effect of EP is to initiate expression of a small group of genes, including those for globins, spectrin, and other proteins involved in the final stages of erythroid differentiation.

TIF1γ Knockdown Enhances Hematopoietic Stem Cell Self Renewal with Preferential Myeloid Differentiation and Delayed Erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4829-4829
Author(s):  
David C Dorn ◽  
Wei He ◽  
Joan Massague ◽  
Malcolm A.S. Moore
Keyword(s):  
Transforming Growth Factor ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Transforming Growth Factor Β ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 4829 The role of TIF1γ in hematopoiesis is still incompletely understood. We previously identified TIF1γ as a novel binding factor for Smad2/3 in the Transforming Growth Factor-β (TFGβ)-inducible signaling pathway implicated in the enhancement of erythropoiesis. To investigate the function of TIF1γ in regulation of hematopoietic stem cells we abrogated TIF1γ signaling by shRNA gamma-retroviral gene transfer in human umbilical cord blood-derived CD34+ hematopoietic stem/ progenitor cells (HCS/ HPCs). Upon blocking TIF1γ the self-renewal capacity of HSCs was enhanced two-fold in vitro as measured by week 5 CAFC assay and three-fold in vivo as measured by competitive engraftment in NOD/ SCID mice over controls. This was associated with a delay in erythroid differentiation and enhanced myelopoiesis. These changes were predominantly observed after TIF1γ knockdown and only mildly after Smad2 depletion but not after Smad3 or 4 reduction. Our data reveal a role for TIF1γ-mediated signaling in the regulation of HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

GATA-1 Forms Distinct Activating and Repressive Complexes in Erythroid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 356-356
Author(s):  
John Strouboulis ◽  
Patrick Rodriguez ◽  
Edgar Bonte ◽  
Jeroen Krijgsveld ◽  
Katarzyna Kolodziej ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Chromatin Remodeling ◽  
Gene Activation ◽  
Erythroid Differentiation ◽  
Specific Gene ◽  
Erythroid Cells ◽  
In Erythroid Cells

Abstract GATA-1 is a key transcription factor essential for the differentiation of the erythroid, megakaryocytic and eosinophilic lineages. GATA-1 functions in erythropoiesis involve lineage-specific gene activation and repression of early hematopoietic transcription programs. GATA-1 is known to interact with other transcription factors, such as FOG-1, TAL-1 and Sp1 and also with CBP/p300 and the SWI/SNF chromatin remodeling complex in vitro. Despite this information the molecular basis of its essential functions in erythropoiesis remains unclear. We show here that GATA-1 is mostly present in a high (> 670kDa) molecular weight complex that appears to be dynamic during erythroid differentiation. In order to characterize the GATA-1 complex(es) from erythroid cells, we employed an in vivo biotinylation tagging approach in mouse erythroleukemic (MEL) cells1. Briefly, this involved the fusion of a small (23aa) peptide tag to GATA-1 and its specific, efficient biotinylation by the bacterial BirA biotin ligase which is co-expressed with tagged GATA-1 in MEL cells. Nuclear extracts expressing biotinylated tagged GATA-1 were bound directly to streptavidin beads and co-purifying proteins were identified by mass spectrometry. In addition to the known GATA-1-interacting transcription factors FOG-1, TAL-1 and Ldb-1, we describe novel interactions with the essential hematopoietic transcription factor Gfi-1b and the chromatin remodeling complexes MeCP1 and ACF/WCRF. Significantly, GATA-1 interaction with the repressive MeCP1 complex requires FOG-1. We also show in erythroid cells that GATA-1, FOG-1 and MeCP1 are stably bound to repressed genes representing early hematopoietic (e.g. GATA-2) or alternative lineage-specific (e.g. eosinophilic) transcription programs, whereas the GATA-1/Gfi1b complex is bound to repressed genes involved in cell proliferation. In contrast, GATA-1 and TAL-1 are bound to the active erythroid-specific EKLF gene. Our findings on GATA-1 complexes provide novel insight as to the critical roles that GATA-1 plays in many aspects of erythropoiesis by revealing the GATA-1 partners in the execution of specific functions.

scholarly journals Isolation of erythropoietin-sensitive cells from Friend virus-infected marrow cultures: characteristics of the erythropoietin response

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 751-758 ◽  
Author(s):  
M Bondurant ◽  
M Koury ◽  
SB Krantz ◽  
T Blevins ◽  
DT Duncan
Keyword(s):  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Precursor Cells ◽  
Erythroid Cells ◽  
Friend Virus ◽  
Isolated Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Erythroid Precursor ◽  
Short Time

Murine erythroid precursor cells, stimulated to proliferate in vitro in the absence of added erythropoietin (EP) by the anemia strain of Friend virus (FVA), will subsequently respond to EP by complete erythrocyte differentiation. If not exposed to EP, the erythroid cells divide for about 120 hr in culture, and they maintain the potential for full differentiation in response to EP added at any time during the period from 72 to 120 hr. Between 96 and 120 hr of culture without added EP, the EP-sensitive erythroid precursor cells that have formed discrete erythroid bursts can be isolated in relatively large numbers from such cultures by plucking with a Pasteur pipette. The addition of EP initiates the final stages of erythroid differentiation, including heme synthesis in 70%-80% of these isolated cells. With respect to homogeneity of the precursor cells, quantity of EP-responsive cells obtainable, and uniformity of EP responsiveness, this system is uniquely favorable for biochemical studies of the late differentiation effects of EP. The overall changes in gene expression accompanying EP- induced terminal differentiation were examined by two-dimensional gel electrophoresis of proteins labeled for a short time with radioactive amino acids. Several new proteins are synthesized in these erythroid cells during terminal differentiation, but the number is a very small percentage of the total number of proteins being made. Thus, in this system, the effect of EP is to initiate expression of a small group of genes, including those for globins, spectrin, and other proteins involved in the final stages of erythroid differentiation.

scholarly journals A murine recombinant retrovirus containing the src oncogene transforms erythroid precursor cells in vitro.

1985 ◽  
Vol 5 (12) ◽  
pp. 3369-3375 ◽  
Author(s):  
S M Anderson ◽  
S P Klinken ◽  
W D Hankins
Keyword(s):  
Time Course ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Kinase Assay ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Erythroid Precursor

A murine retrovirus (MRSV) containing the src gene of Rous sarcoma virus has been shown to cause an erythroproliferative disease in mice (S. M. Anderson and E. M. Scolnick, J. Virol. 46:594-605, 1983). We now demonstrate that this same virus can transform erythroid progenitor cells in vitro. Infection of fetal liver cells or spleen and bone marrow cells from phenylhydrazine-treated adult mice gave rise to colonies of erythroid cells which grew in methylcellulose under conditions not favorable for the growth of normal erythroid cells. The presence of pp60src in the transformed erythroid cells was demonstrated by an immune complex protein kinase assay. The time course of appearance and subsequent differentiation of erythroid colonies indicated that the target cell for MRSV was a 6- to 8-day burst-forming unit. Differentiation of the erythroid progenitors was not blocked by the presence of pp60src, and the cells retained sensitivity to the hormone erythropoietin. In fact, the transformed cells exhibited increased hormone sensitivity since the number, the size, and the extent of hemoglobinization of the colonies were all increased by the addition of small amounts of erythropoietin. MRSV was not susceptible to restriction by the Fv-2 locus, as MRSV could transform hematopoietic cells from C57BL/6 mice. These results indicate that (i) the erythroid proliferation observed in vivo is caused by a direct effect of MRSV on erythroid progenitors and (ii) the transformed erythroid precursors acquire a growth advantage over uninfected cells without losing the ability to differentiate and respond to physiologic regulators.

An EVI1 Point Mutant Unable To Bind GATA1 Rescues EVI1−Induced Defects in Erythroid Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 775-775
Author(s):  
Leopoldo Laricchia-Robbio ◽  
Ciro R. Rinaldi ◽  
Raffaella Fazzina ◽  
Giuseppina Nucifora
Keyword(s):  
Bone Marrow ◽  
Target Genes ◽  
Chromosomal Abnormalities ◽  
Direct Interaction ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Murine Bone Marrow ◽  
Differentiation Block

Abstract EVI1 is a nuclear oncoprotein deregulated by recurring chromosomal abnormalities in MDS. The expression of this gene in MDS patients represents a very poor prognostic marker and is associated with erythroid and megakaryocytic defects. We have previously shown that the forced expression of EVI1 in mice results in a fatal disease with features characteristic of MDS including dyserythropoiesis, dysmegakaryopoiesis, and anemia. More recently we have shown that EVI1 directly interacts with GATA1 and disrupts GATA1−binding to DNA leading to deregulation of GATA1−dependent genes. Here we describe the effects of an EVI1 mutant unable to bind GATA1, on the regulation of GATA1 target genes and on the erythroid differentiation of murine bone marrow progenitors. The structure of two zinc finger motifs of this mutant, EVI1(1+6Mut), were destroyed by His to Ala and Cys to Ala changes. Semi−quantitative RT−PCR showed that most of the analysed genes were down−regulated in 32Dcl3 cells expressing EVI1 but not EVI1(1+6Mut). Bone marrow lineage negative cells infected with EVI1, EVI1(1+6Mut), or the empty vector as control, selected in G418, and plated in presence of Epo were utilized to determine the progenitors’ potential to differentiate in response to this cytokine. The EVI1−expressing cells were virtually unable to generate erythroid colonies after Epo stimulation and only scarce small colonies were observed. In contrast, the EVI1(1+6Mut)−cells produced about 65% of the colonies formed by the control cells. The cells were recovered and their morphology was analysed after Wright−Giemsa staining. All EVI1−positive erythroid cells showed an impaired differentiation that was arrested at the basophilic−erythroblast stage. These cells were bi− and tetra−nucleated with chromatin bridges, budding nuclei and nuclear−cytoplasmatic maturative asynchronizations. All of these features are known as dysplastic erythroid aspects in MDS patients. These dysplastic characteristics were less prominent in EVI1(1+6Mut)−positive cells and were observed only in a minority of cells rather than in the entire population. As expected, the control cells had the appearance of normal erythroblasts and none of the aberrant features were observed. These results parallel the morphology observation of peripheral blood smears obtained from EVI1−mice and EVI1(1+6Mut)−mice in that erythropoiesis defects such as aniso−poikilocytosis and orthochromatic erythroblasts were less striking and observed only in a limited number of erythroid cells in EVI1(1+6Mut)−mice. Based on these results, we propose that EVI1 blocks erythroid differentiation by direct interaction with GATA1 leading to impairment of GATA1 regulation. This differentiation block is significantly reduced in vitro and in vivo with the EVI1(1+6Mut) mutant, which re−establishes to a high degree the normal functions of GATA1 and diminishes the erythroid dysplastic aspects observed in EVI1 cells.

Rescue of early-stage myelodysplastic syndrome-deriving erythroid precursors by the ectopic expression of a dominant-negative form of FADD

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 4035-4042 ◽  
Author(s):  
Yann-Erick Claessens ◽  
Sophie Park ◽  
Anne Dubart-Kupperschmitt ◽  
Virginie Mariot ◽  
Carmen Garrido ◽  
...  
Keyword(s):  
Early Stage ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Caspase 8 ◽  
Death Domain ◽  
Erythroid Cells ◽  
Ineffective Erythropoiesis ◽  
Extrinsic Pathway ◽  
Erythroid Precursors

AbstractMyelodysplastic syndromes (MDSs) are characterized by peripheral blood cytopenia including anemia. We have investigated the implication of the extrinsic pathway of apoptosis in MDS-ineffective erythropoiesis by in vitro expansion of erythroid precursors from early stage (low and intermediate-1 International Prognosis Scoring System [IPSS]) MDS, advanced stage (intermediate-2 IPSS) MDS, and control bone marrow samples. We have previously shown that Fas and its ligand were overexpressed in early stage MDS erythroid cells. Here, we show that caspase-8 activity is significantly increased, whereas the expression of death receptors other than Fas, including the type 1 receptor for tumor necrosis factor α (TNF-α) and the receptors for the TNF-related apoptosis-inducing ligand (TRAIL), DR4 and DR5, was normal. We also observed that the adapter Fas-associated death domain (FADD) was overexpressed in early stage MDS erythroid cells. Transduction of early stage MDS-derived CD34+ progenitors with a FADD-encoding construct increased apoptosis of erythroid cells and dramatically reduced erythroid burst-forming unit (BFU-E) growth. Transduction of a dominant-negative (dn) mutant of FADD inhibited caspase-8 activity and cell death and rescued BFU-E growth without abrogating erythroid differentiation. These results extend the observation that Fas-dependent activation of caspase-8 accounts for apoptosis of early stage MDS erythroid cells and demonstrate for the first time that FADD is a valuable target to correct ineffective erythropoiesis in these syndromes.

A murine recombinant retrovirus containing the src oncogene transforms erythroid precursor cells in vitro

1985 ◽  
Vol 5 (12) ◽  
pp. 3369-3375
Author(s):  
S M Anderson ◽  
S P Klinken ◽  
W D Hankins
Keyword(s):  
Time Course ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Kinase Assay ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Erythroid Precursor

A murine retrovirus (MRSV) containing the src gene of Rous sarcoma virus has been shown to cause an erythroproliferative disease in mice (S. M. Anderson and E. M. Scolnick, J. Virol. 46:594-605, 1983). We now demonstrate that this same virus can transform erythroid progenitor cells in vitro. Infection of fetal liver cells or spleen and bone marrow cells from phenylhydrazine-treated adult mice gave rise to colonies of erythroid cells which grew in methylcellulose under conditions not favorable for the growth of normal erythroid cells. The presence of pp60src in the transformed erythroid cells was demonstrated by an immune complex protein kinase assay. The time course of appearance and subsequent differentiation of erythroid colonies indicated that the target cell for MRSV was a 6- to 8-day burst-forming unit. Differentiation of the erythroid progenitors was not blocked by the presence of pp60src, and the cells retained sensitivity to the hormone erythropoietin. In fact, the transformed cells exhibited increased hormone sensitivity since the number, the size, and the extent of hemoglobinization of the colonies were all increased by the addition of small amounts of erythropoietin. MRSV was not susceptible to restriction by the Fv-2 locus, as MRSV could transform hematopoietic cells from C57BL/6 mice. These results indicate that (i) the erythroid proliferation observed in vivo is caused by a direct effect of MRSV on erythroid progenitors and (ii) the transformed erythroid precursors acquire a growth advantage over uninfected cells without losing the ability to differentiate and respond to physiologic regulators.

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