Effect of human granulocyte macrophage-colony stimulating factor on differentiation and apoptosis of the human...

We investigated the effects of human granulocyte macrophage-colony stimulating factor (GM-CSF) on the relation between differentiation and apoptosis in SaOS-2 cells, an osteoblast-like cell line. To determine the relationship between these cellular processes, SaOS-2 cells were treated in vitro for 1, 7 and 14 days with 200 ng/mL GM-CSF and compared with untreated cells. Five nM insulin-like growth factor (IGF-I) and 30 nM okadaic acid were used as negative and positive controls of apoptosis, respectively. Effects on cell differentiation were determined by ECM (extracellular matrix) mineralization, morphology of some typical mature osteoblast differentiation markers, such as osteopontin and sialoprotein II (BSP-II), and production of bone ECM components such as collagen I. The results showed that treatment with GM-CSF caused cell differentiation accompanied by increased production of osteopontin and BSP-II, together with increased ECM deposition and mineralization. Flow cytometric analysis of annexin V and propidium iodide incorporation showed that GM-CSF up-regulated apoptotic cell death of SaOS-2 cells after 14 days of culture in contrast to okadaic acid, which stimulated SaOS-2 apoptosis only during the early period of culture. Endonucleolytic cleavage of genomic DNA, detected by "Aúladdering analysis"Aù, confirmed these data. The results suggest that GM-CSF induces osteoblastic differentiation and long-term apoptotic cell death of the SaOS-2 human osteosarcoma cell line, which in turn suggests a possible in vivo physiological role for GM-CSF on human osteoblast cells.