Bcr-Abl Efficiently Induces a Myeloproliferative Disease and Production of Excess Interleukin-3 and Granulocyte-Macrophage Colony-Stimulating Factor in Mice: A Novel Model for Chronic Myelogenous Leukemia

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3829-3840 ◽  
Author(s):  
Xiaowu Zhang ◽  
Ruibao Ren

The bcr-abl oncogene plays a critical role in causing chronic myelogenous leukemia (CML). Effective laboratory animal models of CML are needed to study the molecular mechanisms by which thebcr-abl oncogene acts in the disease progression of CML. We used a murine stem cell retroviral vector (MSCV) to transduce thebcr-abl/p210 oncogene into mouse bone marrow cells and found that expression of Bcr-Abl/p210 induced a myeloproliferative disorder that resembled the chronic phase of human CML in 100% of bone marrow transplanted mice in about 3 weeks. This CML-like disease was readily transplanted to secondary recipient mice. Multiple clones of infected cells were expanded in the primary recipients, but the leukemia was primarily monoclonal in the secondary recipient mice. Mutation analysis demonstrated that the protein tyrosine kinase activity of Bcr-Abl/p210 was essential for its leukemogenic potential in vivo. Interestingly, we found that the leukemic cells expressed excess interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the diseased mice. These studies demonstrate that expression of Bcr-Abl can induce a CML-like leukemia in mice much more efficiently and reproducibly than in previously reported mouse CML models, probably due to efficient expression in the correct target cell(s). Our first use of this model for analysis of the molecular mechanisms involved in CML raises the possibility that excess expression of hematopoietic growth factors such as IL-3 and GM-CSF may contribute to the clinical phenotype of CML.

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3829-3840 ◽  
Author(s):  
Xiaowu Zhang ◽  
Ruibao Ren

Abstract The bcr-abl oncogene plays a critical role in causing chronic myelogenous leukemia (CML). Effective laboratory animal models of CML are needed to study the molecular mechanisms by which thebcr-abl oncogene acts in the disease progression of CML. We used a murine stem cell retroviral vector (MSCV) to transduce thebcr-abl/p210 oncogene into mouse bone marrow cells and found that expression of Bcr-Abl/p210 induced a myeloproliferative disorder that resembled the chronic phase of human CML in 100% of bone marrow transplanted mice in about 3 weeks. This CML-like disease was readily transplanted to secondary recipient mice. Multiple clones of infected cells were expanded in the primary recipients, but the leukemia was primarily monoclonal in the secondary recipient mice. Mutation analysis demonstrated that the protein tyrosine kinase activity of Bcr-Abl/p210 was essential for its leukemogenic potential in vivo. Interestingly, we found that the leukemic cells expressed excess interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the diseased mice. These studies demonstrate that expression of Bcr-Abl can induce a CML-like leukemia in mice much more efficiently and reproducibly than in previously reported mouse CML models, probably due to efficient expression in the correct target cell(s). Our first use of this model for analysis of the molecular mechanisms involved in CML raises the possibility that excess expression of hematopoietic growth factors such as IL-3 and GM-CSF may contribute to the clinical phenotype of CML.


Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2652-2656 ◽  
Author(s):  
T Gesner ◽  
RA Mufson ◽  
KJ Turner ◽  
SC Clark

Abstract Granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) each bind specifically to a small number of high- affinity receptors present on the surface of the cells of the acute myelogenous leukemia line, KG-1. Through chemical cross-linking of IL-3 and GM-CSF to KG-1 cells, we identified distinct binding proteins for each of these cytokines with approximate molecular masses of 69 and 93 Kd, respectively. Although these two binding proteins are distinct, GM- CSF and IL-3 compete with each other for binding to KG-1 cells. Other cell lines, which express receptors for either factor but not for both do not display this cross-competition for binding with IL-3 and GM-CSF. These findings imply that distinct IL-3 and GM-CSF binding proteins are expressed on the cell surface and that an association exists between these proteins on KG-1 cells.


Blood ◽  
1990 ◽  
Vol 76 (3) ◽  
pp. 523-532 ◽  
Author(s):  
WP Hammond ◽  
TC Boone ◽  
RE Donahue ◽  
LM Souza ◽  
DC Dale

Cyclic hematopoiesis in gray collie dogs is a stem cell disease in which abnormal regulation of cell production in the bone marrow causes cyclic fluctuations of blood cell counts. In vitro studies demonstrated that recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and granulocyte colony stimulating factor (G-CSF) all stimulated increases in colony formation by canine bone marrow progenitor cells. Based on these results, gray collie dogs were then treated with recombinant human (rh) GM-CSF, IL-3, or G-CSF subcutaneously to test the hypothesis that pharmacologic doses of one of these hematopoietic growth factors could alter cyclic production of cells. When recombinant canine G-CSF became available, it was tested over a range of doses. In vivo rhIL-3 had no effect on the recurrent neutropenia but was associated with eosinophilia, rhGM-CSF caused neutrophilia and eosinophilia but cycling of hematopoiesis persisted. However, rhG-CSF caused neutrophilia, prevented the recurrent neutropenia and, in the two animals not developing antibodies to rhG- CSF, obliterated periodic fluctuation of monocyte, eosinophil, reticulocyte, and platelet counts. Recombinant canine G-CSF increased the nadir neutrophil counts and amplitude of fluctuations at low doses (1 micrograms/kg/d) and eliminated all cycling of cell counts at high doses (5 and 10 micrograms/kg/d). These data suggest significant differences in the actions of these growth factors and imply a critical role for G-CSF in the homeostatic regulation of hematopoiesis.


Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2479-2485 ◽  
Author(s):  
CP Stahl ◽  
EF Winton ◽  
MC Monroe ◽  
E Haff ◽  
RC Holman ◽  
...  

Abstract Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) following interleukin-3 (IL-3) priming has been shown to increase thrombopoiesis. To elucidate the comparative abilities of IL-3 and GM- CSF in influencing megakaryocyte development in vivo, serial bone marrow analyses were performed on rhesus monkeys treated with 5 micrograms/kg/d of IL-3 and 5 micrograms/kg/d of GM-CSF sequentially for 4 days each, simultaneously for 8 days, and as single agents for 8 days. Platelet counts maximally increased to a mean of 7.5 x 10(5)/microL (n = 3) on days 11 through 12 in monkeys treated with sequential IL-3/GM-CSF. In contrast, neither IL-3 alone nor simultaneously administered IL-3/GM-CSF elicited increases in thrombopoiesis between days 3 and 15. GM-CSF elicited a variable platelet response. Megakaryocyte ploidy distributions were significantly (P < .001) shifted between days 7 and 10 in monkeys treated sequentially and between days 3 and 15 in monkeys treated with combined IL-3/GM-CSF and with GM-CSF alone but not in monkeys treated with IL-3 alone. The changes in mean DNA content and megakaryocyte size, as determined by digital image analysis, were larger in monkeys treated with sequential IL-3/GM-CSF and with GM-CSF alone than in simultaneously treated monkeys. In addition, sequentially but not simultaneously treated monkeys showed increased numbers of megakaryocytes on bone marrow biopsy. We conclude that administration of IL-3 followed by GM-CSF treatment increases thrombopoiesis by sequentially increasing megakaryocyte numbers and maturation and that these effects are diminished by simultaneous administration of the two cytokines.


Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1673-1678 ◽  
Author(s):  
E Naparstek ◽  
Y Hardan ◽  
M Ben-Shahar ◽  
A Nagler ◽  
R Or ◽  
...  

We studied an alternative method of using hematopoietic growth factors (HGFs) to enhance hematopoietic recovery in patients undergoing bone marrow transplantation (BMT), by short in vitro preincubation. Twenty consecutive patients with leukemia received T-cell-depleted allografts using Campath-1G. Two thirds of the marrow was infused on the scheduled day of transplant and one third of the marrow following preincubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) on day 4. Engraftment parameters and duration of hospitalization were compared by actuarial analysis to those of 40 historical controls. Patients receiving the incubated boost had significantly faster platelet recovery (P = .017) and shorter hospitalization period (P = .001) when compared with the control subjects. Platelet count reached greater than 25 x 10(9)/L on day 17 (median) in the study group and on day 23 in the controls. The median duration of hospitalization was 20 and 36 days, respectively. In the early posttransplantation follow-up, two of four patients in the study group died as a result of graft rejection, while all 13 deaths in the control group resulted from complications associated with marrow suppression. We suggest that pretransplant in vitro activation of bone marrow cells with IL-3 and GM-CSF may prove to be an efficient method for enhancing marrow recovery after BMT.


Blood ◽  
1989 ◽  
Vol 74 (8) ◽  
pp. 2652-2656 ◽  
Author(s):  
T Gesner ◽  
RA Mufson ◽  
KJ Turner ◽  
SC Clark

Granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) each bind specifically to a small number of high- affinity receptors present on the surface of the cells of the acute myelogenous leukemia line, KG-1. Through chemical cross-linking of IL-3 and GM-CSF to KG-1 cells, we identified distinct binding proteins for each of these cytokines with approximate molecular masses of 69 and 93 Kd, respectively. Although these two binding proteins are distinct, GM- CSF and IL-3 compete with each other for binding to KG-1 cells. Other cell lines, which express receptors for either factor but not for both do not display this cross-competition for binding with IL-3 and GM-CSF. These findings imply that distinct IL-3 and GM-CSF binding proteins are expressed on the cell surface and that an association exists between these proteins on KG-1 cells.


Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1193-1199 ◽  
Author(s):  
CC Paul ◽  
M Tolbert ◽  
S Mahrer ◽  
A Singh ◽  
MJ Grace ◽  
...  

Abstract The cytokines interleukin-3 (IL-3); IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to contribute to the proliferation and differentiation of eosinophil progenitors. Recently, it was determined that the cellular receptors for these three cytokines share a common beta-chain while having unique alpha-chains. Thus, there is considerable interest in how these cytokines and their receptors interact in promoting production of eosinophils. We have established a cell line (AML14) from a patient with acute myelogenous leukemia that will consistently exhibit eosinophilic differentiation in suspension in response to IL-3, IL-5, and GM-CSF. Proliferation with only modest differentiative effects was observed in response to a single cytokine. Combinations of two cytokines gave variable results, with GM-CSF + IL-3 and IL-3 + IL-5 causing more proliferation than a single cytokine but little more differentiation. The combination of GM-CSF + IL-5 caused marked enhancement of eosinophilic differentiation with only modest augmentation of proliferation. The combination of all three cytokines was most effective in stimulating both proliferation and eosinophilic differentiation (up to 70% of cells) of AML14 cells. Specific binding of GM-CSF and IL-5 to AML14 cells can be conveniently studied by flow cytometric methods, and cross-competition of these two cytokines for their respective receptors was demonstrated. IL-3 was shown to partially compete for IL-5 binding on AML14 cells. Although specific IL- 3 binding could not be demonstrated by flow cytometry, mRNA for the alpha-chains of the IL-3, IL-5, and GM-CSF receptors and the beta-chain common to all three receptors was detected in AML14 cells. The AML14 cell line may be a useful model for the study of cooperative interactions of IL-3, IL-5, GM-CSF, and their respective receptors in the promotion of eosinophil progenitor growth and differentiation.


Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2479-2485
Author(s):  
CP Stahl ◽  
EF Winton ◽  
MC Monroe ◽  
E Haff ◽  
RC Holman ◽  
...  

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) following interleukin-3 (IL-3) priming has been shown to increase thrombopoiesis. To elucidate the comparative abilities of IL-3 and GM- CSF in influencing megakaryocyte development in vivo, serial bone marrow analyses were performed on rhesus monkeys treated with 5 micrograms/kg/d of IL-3 and 5 micrograms/kg/d of GM-CSF sequentially for 4 days each, simultaneously for 8 days, and as single agents for 8 days. Platelet counts maximally increased to a mean of 7.5 x 10(5)/microL (n = 3) on days 11 through 12 in monkeys treated with sequential IL-3/GM-CSF. In contrast, neither IL-3 alone nor simultaneously administered IL-3/GM-CSF elicited increases in thrombopoiesis between days 3 and 15. GM-CSF elicited a variable platelet response. Megakaryocyte ploidy distributions were significantly (P < .001) shifted between days 7 and 10 in monkeys treated sequentially and between days 3 and 15 in monkeys treated with combined IL-3/GM-CSF and with GM-CSF alone but not in monkeys treated with IL-3 alone. The changes in mean DNA content and megakaryocyte size, as determined by digital image analysis, were larger in monkeys treated with sequential IL-3/GM-CSF and with GM-CSF alone than in simultaneously treated monkeys. In addition, sequentially but not simultaneously treated monkeys showed increased numbers of megakaryocytes on bone marrow biopsy. We conclude that administration of IL-3 followed by GM-CSF treatment increases thrombopoiesis by sequentially increasing megakaryocyte numbers and maturation and that these effects are diminished by simultaneous administration of the two cytokines.


Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1673-1678 ◽  
Author(s):  
E Naparstek ◽  
Y Hardan ◽  
M Ben-Shahar ◽  
A Nagler ◽  
R Or ◽  
...  

Abstract We studied an alternative method of using hematopoietic growth factors (HGFs) to enhance hematopoietic recovery in patients undergoing bone marrow transplantation (BMT), by short in vitro preincubation. Twenty consecutive patients with leukemia received T-cell-depleted allografts using Campath-1G. Two thirds of the marrow was infused on the scheduled day of transplant and one third of the marrow following preincubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) on day 4. Engraftment parameters and duration of hospitalization were compared by actuarial analysis to those of 40 historical controls. Patients receiving the incubated boost had significantly faster platelet recovery (P = .017) and shorter hospitalization period (P = .001) when compared with the control subjects. Platelet count reached greater than 25 x 10(9)/L on day 17 (median) in the study group and on day 23 in the controls. The median duration of hospitalization was 20 and 36 days, respectively. In the early posttransplantation follow-up, two of four patients in the study group died as a result of graft rejection, while all 13 deaths in the control group resulted from complications associated with marrow suppression. We suggest that pretransplant in vitro activation of bone marrow cells with IL-3 and GM-CSF may prove to be an efficient method for enhancing marrow recovery after BMT.


1992 ◽  
Vol 10 (3) ◽  
pp. 398-405 ◽  
Author(s):  
H M Kantarjian ◽  
M Talpaz ◽  
D Kontoyiannis ◽  
J Gutterman ◽  
M J Keating ◽  
...  

PURPOSE The study was undertaken to improve the results of intensive chemotherapy in chronic myelogenous leukemia (CML) in accelerated (CML-AP) and blastic phases (CML-BP) by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) as supportive therapy. PATIENTS AND METHODS Forty-eight patients were treated with daunorubicin 120 mg/m2 intravenously on day 1, cytarabine (ara-C) 1.5 g/m2/d by continuous infusion over 24 hours for 4 days, and Solu-Medrol (methylprednisolone; The Upjohn Co, Kalamazoo, MI) 100 mg/d for 5 days, followed on day 5 by GM-CSF 125 micrograms/m2/d over 6 hours until recovery of granulocyte count above 2.0 x 10(3)/microliters. Twenty-four patients had CML-BP, and 24 had CML-AP. RESULTS During remission induction, 45 patients (94%) developed febrile episodes (fever of unknown origin, 23 patients [48%]; documented infections, 22 patients [46%]). The median time to recovery of granulocyte count above 0.5 x 10(3)/microliters was 29 days and to platelet count above 30 x 10(3)/microliters, 28 days. Overall, 14 of 48 patients (29%) achieved a complete hematologic remission (CHR), and seven (15%) reverted to a second chronic phase. CHR was noted in eight of 24 patients with CML-BP (33%), and in six of 24 patients with CML-AP (25%). Cytogenetic responses were observed in 11 patients (23%), but were transient. Sixteen patients developed either fluid retention, hypotension, pleuropericardial effusions, or pericarditis, or a combination of these side effects. These side effects were severe in four patients and are likely to be disease-associated, as a similar regimen of intensive chemotherapy and GM-CSF at the same dose and schedule in acute lymphocytic leukemia was not associated with these side effects. CONCLUSIONS The results pertinent to remission rates, induction mortality, myelosuppression profile and related complications, and overall survival were not significantly improved compared with previous experience. In summary, the results of intensive chemotherapy in CML-transformed phases remain poor, despite the addition of GM-CSF as a supportive measure.


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