Autocrine activation of the IL-3/GM-CSF/IL-5 signaling pathway in leukemic cells.
The AML14.3D10 human myeloid leukemic cell line expresses receptors for granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-5 (IL-5), but not IL-3. We have found that this cell line produces GM-CSF in amounts up to 113 pg/ml in culture supernatants. Deprivation of endogenous GM-CSF by addition of neutralizing anti-GM-CSF antibody strongly inhibits proliferation of the cells, suggesting a GM-CSF autocrine growth mechanism. To examine whether endogenously produced GM-CSF activates intracellular GM-CSF/IL-3/IL-5-related signal transduction pathways, we performed antiphosphotyrosine immunoblotting of cell lysates of AML14.3D10 cells before and after deprivation of endogenous GM-CSF. We found constitutive tyrosine-phosphorylation of a number of proteins in AML14.3D10 that could not be detectably increased by the addition of exogenous GM-CSF, IL-3, or IL-5. However, GM-CSF-deprived cells demonstrated a marked increase in phosphorylation of proteins of identical molecular mass following addition of GM-CSF and IL-5, but not IL-3, consistent with the receptor expression of the cells and the known use of the same signaling pathways by the three cytokines. This suggests that AML14.3D10 cells use endogenously produced GM-CSF to activate signal transduction pathways, interfering with activation by exogenous cytokine until the endogenous stimulation is removed. We then assessed the activation of the beta-subunit common to the GM-CSF/IL-3/IL-5 receptors (beta c), JAK2 and p53/56 lyn, known to be involved in the common signaling pathways of the three cytokines. We found that phosphorylation of beta c and JAK2 in response to GM-CSF and IL-5 could be markedly enhanced by depriving cells of endogenous GM-CSF. Constitutive hyperphosphorylation of lyn was found in AML14.3D10 cells, and no further activation of lyn in response to cytokine was demonstrable in GM-CSF-deprived cells, suggesting that lyn is activated in this cell line by a mechanism other than GM-CSF. These studies represent the first demonstration of autocrine activation of intracellular cytokine signaling pathways by malignant hematopoietic cells. Because the addition of anti-GM-CSF to cell cultures improved responsiveness of intracellular signal transducing molecules to exogenous GM-CSF and IL-5, it can be inferred that endogenously produced GM-CSF exerts its effects by secretion and binding to surface GM-CSF receptors, although an intracellular component to signaling cannot be excluded. These observations provide further information regarding an autocrine contribution to leukemic cell growth, and establish a new model for study of these events.
Paul
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CC
Research Service, VA Medical Center, Wright State University, Dayton, Ohio 45428, USA.
Mahrer
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S
McMannama
K
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Baumann
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MA
D000906:Antibodies / Q000276:immunology; D002455:Cell Division / Q000502:physiology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000276:immunology / Q000378:metabolism*; D006728:Hormones / Q000502:physiology*; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism*; D015848:Interleukin-5 / Q000378:metabolism*; D007951:Leukemia, Myeloid / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D000906:Antibodies; D006728:Hormones; D007377:Interleukin-3; D015848:Interleukin-5; D016178:Granulocyte-Macrophage Colony-Stimulating Factor