scholarly journals Expression of embryonic globins by erythroid cells in juvenile chronic myelocytic leukemia

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2569-2576 ◽  
Author(s):  
T Papayannopoulou ◽  
B Nakamoto ◽  
NP Anagnou ◽  
D Chui ◽  
L Dow ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Fetal Hemoglobin ◽  
Colony Growth ◽  
Leukemic Cells ◽  
Chronic Myelocytic Leukemia ◽  
Erythroid Cells ◽  
Myelocytic Leukemia ◽  
Synthetic Profile

Juvenile chronic myelocytic leukemia (JCML) is a rare hematopoietic neoplasia of early childhood with distinct hematologic and biochemical features. We studied the biologic properties and the globin synthetic profiles of JCML erythroid cells both in vivo and in vitro from a total of 24 patients. In these cases we observed the exuberant colony-forming unit-macrophage (CFU-M) colony growth, as reported previously. Furthermore, in contrast to previous reports, we found significant erythroid colony growth in most of our cases (average: 1,182 burst- forming unit-erythroid [BFUe] per 10(5) plated cells, range: 40 to 6,927). This growth was by and large erythropoietin-dependent and was not greatly influenced by other added cytokines. By several criteria all erythroid colony growth detected in vitro was derived from JCML progenitors. The globin synthetic profile of JCML erythroid cells showed high levels of fetal hemoglobin both in vivo and in vitro (gamma/gamma + beta: 53% to 94% in reticulocytes, 62% to 98% in BFUe- derived cells). In addition (in seven cases studied) we detected embryonic globins (epsilon and zeta) at the protein and messenger RNA level, a novel finding for primary leukemic cells. We speculate that the transformed erythroid cells in JCML harbor a trans environment supporting expression of developmentally earlier genes (fetal, embryonic). However, in contrast to other acute or subacute leukemias, JCML erythroid cells also have the ability to reach full maturation to the red cell level, thus allowing detection of this primitive program in vivo.

scholarly journals Expression of embryonic globins by erythroid cells in juvenile chronic myelocytic leukemia

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2569-2576 ◽  
Author(s):  
T Papayannopoulou ◽  
B Nakamoto ◽  
NP Anagnou ◽  
D Chui ◽  
L Dow ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Fetal Hemoglobin ◽  
Colony Growth ◽  
Leukemic Cells ◽  
Chronic Myelocytic Leukemia ◽  
Erythroid Cells ◽  
Myelocytic Leukemia ◽  
Synthetic Profile

Abstract Juvenile chronic myelocytic leukemia (JCML) is a rare hematopoietic neoplasia of early childhood with distinct hematologic and biochemical features. We studied the biologic properties and the globin synthetic profiles of JCML erythroid cells both in vivo and in vitro from a total of 24 patients. In these cases we observed the exuberant colony-forming unit-macrophage (CFU-M) colony growth, as reported previously. Furthermore, in contrast to previous reports, we found significant erythroid colony growth in most of our cases (average: 1,182 burst- forming unit-erythroid [BFUe] per 10(5) plated cells, range: 40 to 6,927). This growth was by and large erythropoietin-dependent and was not greatly influenced by other added cytokines. By several criteria all erythroid colony growth detected in vitro was derived from JCML progenitors. The globin synthetic profile of JCML erythroid cells showed high levels of fetal hemoglobin both in vivo and in vitro (gamma/gamma + beta: 53% to 94% in reticulocytes, 62% to 98% in BFUe- derived cells). In addition (in seven cases studied) we detected embryonic globins (epsilon and zeta) at the protein and messenger RNA level, a novel finding for primary leukemic cells. We speculate that the transformed erythroid cells in JCML harbor a trans environment supporting expression of developmentally earlier genes (fetal, embryonic). However, in contrast to other acute or subacute leukemias, JCML erythroid cells also have the ability to reach full maturation to the red cell level, thus allowing detection of this primitive program in vivo.

scholarly journals Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2063-2063
Author(s):  
Naoya Uchida ◽  
Claire Drysdale ◽  
Morgan Yapundich ◽  
Jackson Gamer ◽  
Tina Nassehi ◽  
...  
Keyword(s):  
Rhesus Macaques ◽  
Fetal Hemoglobin ◽  
Selective Advantage ◽  
Erythroid Cells ◽  
Post Transplant ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Hbf Induction

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clonal analysis of basophil differentiation in bone marrow cultures from a Down's syndrome patient with megakaryoblastic leukemia

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1278-1283
Author(s):  
T Suda ◽  
J Suda ◽  
Y Miura ◽  
Y Hayashi ◽  
M Eguchi ◽  
...  
Keyword(s):  
Single Cells ◽  
Down's Syndrome ◽  
Calcium Ionophore ◽  
Down’S Syndrome ◽  
Colony Growth ◽  
Leukemic Cells ◽  
Cytologic Examination ◽  
Megakaryoblastic Leukemia

We present the in vitro differentiation of marrow cells from a patient with Down's syndrome accompanied by megakaryoblastic leukemia into basophils in the presence of phytohemagglutinin-stimulated leukocyte conditioned medium, using a liquid culture and methylcellulose culture system. Identification of basophils was established by metachromatic staining with toluidine blue, transmission electron microscopy, and the presence of histamine. However, these basophils did not release histamine in response to calcium ionophore or chemotactic peptide. Samples from suspension cultures that contained 90% basophils showed chromosomal markers characteristic of leukemic cells (48, XY, +11, +21, t(1;15)) in all examined mitoses. The cellular composition of leukemic colonies grown in methylcellulose culture from single cells was studied using the micromanipulation technique. High plating efficiency and extreme predominance of basophil colonies were observed. In a total 137 cultures, 79 revealed colony growth. Of 59 colonies that were analyzed by cytologic examination, 46 were pure basophil colonies. These basophil colonies showed disperse morphology, similar to that of a normal basophil colony. The clonality of the basophil colonies and skewing of lineage expression were documented from leukemic single-cell cultures. These data showed that leukemic cells have the capacity for differentiation into some lineages that are not expressed in vivo.

scholarly journals Constitutive expression of GATA-1, EPOR, alpha-globin, and gamma-globin genes in myeloid clonogenic cells from juvenile chronic myelocytic leukemia

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 323-328 ◽  
Author(s):  
E Privitera ◽  
R Schiro ◽  
D Longoni ◽  
A Ronchi ◽  
A Rambaldi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemic Cells ◽  
Globin Genes ◽  
Chronic Myelocytic Leukemia ◽  
Beta Globin ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Myelocytic Leukemia ◽  
Alpha Globin

Juvenile chronic myelocytic leukemia (JCML) is a rare disorder of early childhood. Characteristic of JCML are the progressive appearance of high levels of fetal hemoglobin (HbF), reflecting a true reversion to a fetal type of erythropoiesis, and the presence of colony-forming cells able to grow in vitro spontaneously in the absence of growth factors. To better understand the relationship between the erythroid abnormalities and the leukemic process, we analyzed the expression pattern of specific genes related to erythroid differentiation--GATA-1, EPOR, alpha-globin, beta-globin, and gamma-globin genes--in JCML peripheral blood (PB) cells and in vitro-derived colonies. Northern blot analysis of PB cells from five JCML patients indicated levels of GATA-1 transcripts much higher than those usually found in other types of leukemic cells, and S1 nuclease protection assay detected significantly increased expression of gamma-globin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of single granulocyte-macrophage colony-forming unit (CFU-GM) colonies, obtained in vitro in the absence of added growth factors from four JCML patients, detected GATA-1, EPOR, and globin (alpha and gamma) transcripts in most of the colonies tested, in contrast with control CFU-GM from normal bone marrow, which were positive only for GATA-1. Single JCML colonies were tested for the presence of two different transcripts; whereas alpha- and gamma-globin genes appeared mostly coexpressed, beta-globin mRNA was detected only in a minority of the gamma-globin-positive colonies, indicating that the leukemic pattern of hemoglobin synthesis is mainly fetal. In addition, the leukemic cells occurring during blast crisis of one of our patients displayed the typical features of a stem cell leukemia (CD34+, CD19-, CD2-, myeloperoxidase-). In this sorted CD34+ population, we detected the presence of a marker chromosome, der(12)t(3;12), previously identified in bone marrow cells at diagnosis and an expression pattern superimposable to that of the JCML colonies, consistently displaying a high gamma-globin:beta-globin mRNA ratio. The expression of erythroid markers within populations of leukemic cells, both in vivo and in vitro, supports the hypothesis that abnormal JCML erythroid cells may originate from the same mutated progenitor that sustains the growth of the leukemic cells.

In vivo and in vitro activity of neutrophil alkaline phosphatase in acute myelocytic leukemia with 8;21 translocation

Blood ◽  
1981 ◽  
Vol 58 (6) ◽  
pp. 1213-1217
Author(s):  
N Kamada ◽  
H Dohy ◽  
K Okada ◽  
N Oguma ◽  
A Kuramoto ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Genetic Alterations ◽  
Leukemic Cells ◽  
Characteristic Subgroup ◽  
Acute Myelocytic Leukemia ◽  
Myelocytic Leukemia ◽  
Fab Classification ◽  
Neutrophil Alkaline Phosphatase

Cytogenetic studies were made on 160 patients with acute nonlymphocytic leukemia (ANLL) between 1963 and 1979, of whom 115 had acute myelocytic leukemia with 67 patients showing aneuploidy (58.3%). Among these, 24 patients were found to have similar chromosome alterations that appeared to involve specifically chromosomes 8 and 21. Banding studies on at least 7 of these patients confirmed the presence of a translocation between these two chromosomes. Of 160 ANLL patients, 142 were scored for neutrophil alkaline phosphatase (neutrophil AP) at the time of diagnosis. Fifty-nine patients showed a low neutrophil AP score, 42 a normal value, and 41 a high value. All patients with 8;21 (or C/G) translocation had a low neutrophil AP score and leukemic cells with maturation (M2 of FAB classification) in the bone marrow. In vitro liquid culture for 2 wk of 8;21 translocated leukemic cells revealed no increase of neutrophil AP activity nor increase of mature granulocytes, whereas 9;22 translocated chronic myelocytic leukemia cells with a low neutrophil AP score did so. Neutrophil AP score at the time of diagnosis in acute myelocytic leukemia is very useful for detecting 8;21 translocation AML and for studying the pathophysiology and genetic alterations of the characteristic subgroup of AML with 8′21 translocation.

scholarly journals Influence of hydroxyurea on fetal hemoglobin production in vitro

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1824-1829 ◽  
Author(s):  
BA Miller ◽  
O Platt ◽  
S Hope ◽  
G Dover ◽  
DG Nathan
Keyword(s):  
Sickle Cell ◽  
Peripheral Blood ◽  
Fetal Hemoglobin ◽  
Erythroid Cells ◽  
Hemoglobin Content ◽  
Hemoglobin F ◽  
Hydroxyurea Treatment ◽  
Hemoglobin Production

Abstract Cytotoxic drugs increase circulating fetal hemoglobin levels. We examined the mechanism by measuring the fetal hemoglobin produced per BFU-E-derived erythroblast following hydroxyurea treatment in vivo and in vitro. Treatment of four sickle cell patients increased the percentage of circulating F reticulocytes. The frequencies of bone marrow or peripheral blood BFU-E or CFU-E-derived colonies and their fetal hemoglobin content were unaffected. In all cases, the number of erythroid cells/progenitor-derived colony increased. To explore further the effect of hydroxyurea on fetal hemoglobin production, we added 50 mumol/L hydroxyurea to cultures of peripheral blood BFU-E-derived erythroblasts on 1 of 9 days (day 5 through 13) to nine samples. These BFU-E were derived from the peripheral blood of normal donors, sickle trait donors, and sickle cell anemia patients and from the bone marrows of monkeys. This concentration of hydroxyurea was selected so that the frequency of BFU-E and their size was moderately decreased. Addition of hydroxyurea to these progenitor-derived erythroid cells had no effect on fetal hemoglobin content per cell. Neither did transient exposure of progenitors to hydroxyurea prior to culture in nontoxic concentrations (0 to 500 mumol/L) result in a significant increase in fetal hemoglobin content in progenitor-derived erythroblasts. These data suggest that hydroxyurea does not directly alter the HbF program expressed by progenitor-derived erythroid cells. Instead, it enhances hemoglobin F content secondarily, possibly by inducing alterations in erythropoiesis.

scholarly journals BFU-E COLONY GROWTH IN RESPONSE TO HYDROXYUREA: CORRELATION BETWEEN IN VITRO AND IN VIVO FETAL HEMOGLOBIN INDUCTION 158

1997 ◽  
Vol 41 (5) ◽  
pp. 778-778
Author(s):  
Y M Yang ◽  
B Pace ◽  
D Kitchens ◽  
S K Ballas ◽  
A Shah ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Colony Growth

scholarly journals BFU-E colony growth in response to hydroxyurea: Correlation between in vitro and in vivo fetal hemoglobin induction

1997 ◽  
Vol 56 (4) ◽  
pp. 252-258 ◽  
Author(s):  
Yih-Ming Yang ◽  
Betty Pace ◽  
David Kitchens ◽  
Samir K. Ballas ◽  
Arvind Shah ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Colony Growth

scholarly journals Clonal analysis of basophil differentiation in bone marrow cultures from a Down's syndrome patient with megakaryoblastic leukemia

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1278-1283 ◽  
Author(s):  
T Suda ◽  
J Suda ◽  
Y Miura ◽  
Y Hayashi ◽  
M Eguchi ◽  
...  
Keyword(s):  
Single Cells ◽  
Down's Syndrome ◽  
Calcium Ionophore ◽  
Down’S Syndrome ◽  
Colony Growth ◽  
Leukemic Cells ◽  
Cytologic Examination ◽  
Megakaryoblastic Leukemia

Abstract We present the in vitro differentiation of marrow cells from a patient with Down's syndrome accompanied by megakaryoblastic leukemia into basophils in the presence of phytohemagglutinin-stimulated leukocyte conditioned medium, using a liquid culture and methylcellulose culture system. Identification of basophils was established by metachromatic staining with toluidine blue, transmission electron microscopy, and the presence of histamine. However, these basophils did not release histamine in response to calcium ionophore or chemotactic peptide. Samples from suspension cultures that contained 90% basophils showed chromosomal markers characteristic of leukemic cells (48, XY, +11, +21, t(1;15)) in all examined mitoses. The cellular composition of leukemic colonies grown in methylcellulose culture from single cells was studied using the micromanipulation technique. High plating efficiency and extreme predominance of basophil colonies were observed. In a total 137 cultures, 79 revealed colony growth. Of 59 colonies that were analyzed by cytologic examination, 46 were pure basophil colonies. These basophil colonies showed disperse morphology, similar to that of a normal basophil colony. The clonality of the basophil colonies and skewing of lineage expression were documented from leukemic single-cell cultures. These data showed that leukemic cells have the capacity for differentiation into some lineages that are not expressed in vivo.

scholarly journals Prediction of response to chemotherapy in acute myelocytic leukemia

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 361-367 ◽  
Author(s):  
HD Preisler
Keyword(s):  
Cytosine Arabinoside ◽  
Chemotherapeutic Agents ◽  
Leukemic Cells ◽  
Acute Myelocytic Leukemia ◽  
Metabolic Resistance ◽  
Clear Cut ◽  
Myelocytic Leukemia ◽  
Response To Chemotherapy

Abstract Marrow specimens obtained from 23 patients with acute myelocytic leukemia were exposed to cytosine arabinoside and/or daunorubicin in vitro, and the effects of these agents on colony formation in vitro was determined. Thymidine suicide indices were determined as well, which permitted a distinction to be made between kinetic and metabolic resistance to cytosine arabinoside. The sensitivity of the colony- forming cells to the two chemotherapeutic agents did not correlate with each other, indicating that sensitivity to each was independently determined. The relationship between in vitro sensitivity to daunorubicin and cytosine arabinoside and response to 25 courses of in vivo therapy with these two agents administered to 21 patients was determined. These studies indicated a clear-cut relationship between in vitro drug sensitivity and in vivo response with patients whose leukemic cells were sensitive to both agents entering complete remission, whereas patients whose leukemic cells were insensitive to one or both drugs in vitro failed to enter remission.

Sign in / Sign up

Export Citation Format

Share Document