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 fo r granulocyte-macrophage colony stimulating factor (GM CSF) and interl eukin-5 (IL-5), but not IL-3, We have found that this cell line produc es Gm-CSF in amounts up to 113 pg/ml in culture supernatants, Deprivat ion of endogenous GM-CSF by addition of neutralizing anti-GMCSF antibo dy strongly inhibits proliferation of the cells, suggesting a GM-CSF a utocrine growth mechanism. To examine whether endogenously produced GM -CSF activates intracellular GM-CSF/IL-3/IL-5-related signal transduct ion 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 n ot 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 act ivate signal transduction pathways, interfering with activation by exo genous cytokine until the endogenous stimulation is removed. We then a ssessed the activation of the p-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 phosph orylation of pc and JAK2 in response to GM-CSF and IL-5 could be marke dly enhanced by depriving cells of endogenous GM-CSF. Constitutive hyp erphosphorylation 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 demon stration of autocrine activation of intracellular cytokine signaling p athways by malignant hematopoietic cells, Because the addition of anti -GM-CSF to cell cultures improved responsiveness of intracellular sign al transducing molecules to exogenous GMCSF and IL-5, it can be inferr ed that endogenously produced GM-CSF exerts its effects by secretion a nd binding to surface GM-CSF receptors, although an intracellular comp onent to signaling cannot be excluded. These observations provide furt her information regarding an autocrine contribution to leukemic cell g rowth, and establish a new model for study of these events. (C) 1997 W iley-Liss, Inc.