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1.
Exp Hematol ; 28(6): 716-25, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10880758

ABSTRACT

By virtue of its high expression in both developing hematopoietic tissues and many myeloid leukemia cells lines, the embryonic tyrosine kinase receptor ETK2 (also known as Tyro3, Sky, and Rse) has been postulated to play a role in early hematopoiesis. To investigate this role, we expressed murine ETK2 in the interleukin 3 (IL-3) dependent myeloid progenitor cell line FDC-P1 and examined its effect on growth factor dependence.ETK2 cDNAs encoding full-length or kinase domain-deleted receptor were retrovirally transduced into murine FDC-P1 cells. Survival, cell cycle status, and proliferative responses of ETK2 expressing clones were studied at normal and reduced growth factor concentrations. ETK2 was expressed as a functional tyrosine kinase of 110 and 150 kDa. This proto-oncogene altered the growth of FDC-P1 cells, allowing survival at reduced growth factor concentrations and delaying apoptosis after IL-3 withdrawal. ETK2-expressing clones contained a higher fraction of cells in the S/G2/M phases of the cell cycle, both after cytokine withdrawal and in the presence of IL-3. Furthermore, these cells had a modestly enhanced proliferative response to IL-3 and granulocyte-macrophage colony-stimulating factor, suggesting that ETK2 intracellular signaling may converge with that of hematopoietic growth factors. The effects of ETK2 expression on viability and proliferation were largely dependent on a functional intracellular tyrosine kinase domain. These results support a role for ETK2 in the survival and/or expansion of primitive hematopoietic cells and suggest that this tyrosine kinase may be implicated in myeloid leukemogenesis as well.


Subject(s)
Hematopoietic Stem Cells/cytology , Receptor Protein-Tyrosine Kinases/physiology , Animals , Apoptosis/drug effects , Cell Cycle , Cell Survival , Cell Transformation, Neoplastic , Cells, Cultured/drug effects , Culture Media/pharmacology , Culture Media, Serum-Free/pharmacology , Genes, Synthetic , Genetic Vectors/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cells/drug effects , Interleukin-3/pharmacology , Leukemia/etiology , Mice , Phosphorylation , Protein Processing, Post-Translational , Protein Structure, Tertiary , Proto-Oncogene Mas , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/genetics , Recombinant Fusion Proteins/physiology , Retroviridae/genetics , Sequence Deletion , Signal Transduction/drug effects , Transfection
2.
Science ; 285(5426): 412-5, 1999 Jul 16.
Article in English | MEDLINE | ID: mdl-10411505

ABSTRACT

Graft versus host disease, an alloimmune attack on host tissues mounted by donor T cells, is the most important toxicity of allogeneic bone marrow transplantation. The mechanism by which allogeneic T cells are initially stimulated is unknown. In a murine allogeneic bone marrow transplantation model it was found that, despite the presence of numerous donor antigen-presenting cells, only host-derived antigen-presenting cells initiated graft versus host disease. Thus, strategies for preventing graft versus host disease could be developed that are based on inactivating host antigen-presenting cells. Such strategies could expand the safety and application of allogeneic bone marrow transplantation in treatment of common genetic and neoplastic diseases.


Subject(s)
Antigen-Presenting Cells/immunology , Bone Marrow Transplantation/immunology , CD8-Positive T-Lymphocytes/immunology , Graft vs Host Disease/prevention & control , Animals , Bone Marrow Transplantation/adverse effects , Dendritic Cells/immunology , Graft vs Host Disease/immunology , H-2 Antigens/immunology , Lymph Nodes/immunology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Knockout , Minor Histocompatibility Antigens/immunology , Spleen/immunology , Transplantation Chimera
3.
Blood ; 91(8): 2935-47, 1998 Apr 15.
Article in English | MEDLINE | ID: mdl-9531604

ABSTRACT

Mice lacking interleukin-2 (IL-2) developed a severe hematopoietic disorder characterized by the abnormal development of myeloid cells and neutropenia. Analysis of the bone marrow of IL-2-deficient (IL-2(-/-)) mice showed that the number of mature polymorphonuclear cells was decreased by 65% to 75%, and granulocyte/macrophage precursor cells were reduced by 50%. Bone marrow cells from IL-2(-/-) mice were unable to sustain myelopoiesis in lethally irradiated mice and in long-term bone marrow cultures (LTBMC). The addition of exogenous IL-2 to LTBMC of IL-2(-/-) cells partially restored hematopoietic progenitor activity. In the bone marrow of wild-type mice, immature (Mac-1(lo)) myeloid cells, including myeloblasts and promyelocytes, constitutively expressed the beta-chain of the IL-2R, and the number of Mac-1(lo)IL-2Rbeta+ cells was increased by twofold to threefold in IL-2(-/-) mice. During culture in the presence of IL-2 and the absence of stromal cells, Mac-1(lo)IL-2Rbeta+ immature myeloid cells proliferated and gave rise to mature granulocytes and macrophages. Collectively, these observations indicate that defective myelopoiesis in IL-2(-/-) mice is at least in part a consequence of their direct dependency on IL-2, and by regulating the growth of immature myeloid cells, IL-2 plays an important role in the homeostatic regulation of myelocytic cell generation.


Subject(s)
Interleukin-2/deficiency , Leukopoiesis , Animals , Cell Differentiation/drug effects , Cells, Cultured , Granulocytes/cytology , Granulocytes/physiology , Interleukin-2/genetics , Interleukin-2/pharmacology , Leukopoiesis/drug effects , Mice , Mice, Mutant Strains , Neutrophils/cytology , Neutrophils/physiology
4.
J Cell Physiol ; 164(1): 99-107, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7790403

ABSTRACT

In order to study a previously described genetic difference manifested in stem cell kinetics of specific mouse strains, effects of this putative gene, stk, were measured on growth and expansion of stem and progenitor cell populations ex vivo. Bone marrow cells from each of two inbred mouse strains, C57BL/6J and DBA/2J, were placed into separate bioreactor cultures perfused continuously with growth medium containing erythropoietin (Epo), interleukin-3 (IL-3), granulocyte-macrphage colony stimulating factor (GM-CSF), and Kit ligand as well as 5% CO2. Expansion of cell numbers reached 20-fold for DBA/2J and 10-fold for C57BL/6J marrow within about 1 week of culture. Significant production was also seen of colony-forming unit (CFU)-GM (up nine-fold from input levels) just prior to the cell production peak, and, importantly, moderate expansion of day 12 colony-forming unit-spleen (CFU-S; two- to threefold) occurred as well, although CFU-S production peaked at a relatively short 4 days. CFU-S and CFU-GM levels declined rapidly in culture, either because of unfavorable growth conditions or terminal differentiation. Attempts to remove toxic metabolites by increasing the media perfusion rate resulted in a boost in cell expansion capability by DBA/2J marrow. In bioreactors in which stromal cells were established before marrow inoculation, there was greater expansion of CFU-S (especially by DBA/2J) and CFU-GM, although total cell yield appeared to be unaffected, perhaps because the maximum cell density had already been reached. The relative high potential for CFU-S expansion measured in DBA/2J marrow over that of C57BL/6J will be useful in following genetic contributions to bone marrow production capacity.


Subject(s)
Genes , Hematopoiesis , Hematopoietic Stem Cells/cytology , Mice/physiology , Animals , Bone Marrow Cells , Cell Division , Cells, Cultured , Colony-Forming Units Assay , Female , Kinetics , Lactates/metabolism , Lactic Acid , Spleen/cytology
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