Radioprotective synergistic action of cytokines on the clonal growth of x-irradiated human megakaryocytic progenitor cells

Full text: In radiation-induced myelosuppression in animals, treatment with recombinant human thrombopoietin (TPO) or its derivatives has a profound effect on thrombocytopenia by effectively decreasing the platelet reduction and accelerating platelet recovery. However, previous in vivo or in vitro studies have shown that cytokine cocktail is more effective to promote megakaryopoiesis and thrombopoiesis than TPO alone. Therefore, clarifying the optimal cytokine combinations is very important for the recovery of hematopoietic systems following radiation exposure. Our previous studies characterized the radiosensitivity of CD34+ CFU-Meg (megakaryocytic progenitors) from human placental and umbilical cord blood and the effects of various early-acting cytokines. The results demonstrated that a combination of TPO and stem cell factor (SCF) support the maximum clonal growth in vitro and protect against radiation damage of CFU-Meg by X-rays. As the mechanisms of the synergistic effect by these cytokines are not clear, we extended these studies to investigate the radioprotective synergistic action of TPO and SCF. This combination activated mitogen-activated protein kinase (MAPK) and extracellular-signal-regulated protein kinase (ERK) and suppressed caspase-3 in 5Gy-irradiated CD34+ cells. specific inhibitors against each protein, PD98059 and various synthetic substrates, showed a partial activity of this combination in the clonal growth of X-irradiated CD34+ CFU-Meg. Propidium iodide experiments showed nuclear condensation and apoptotic bodies in X-irradiated cells, 52% of which were scored as apoptotic cells. A combination of TPO plus SCF or Z-VAD-FMK (general caspase inhibitor) resulted in the strongest inhibition of apoptosis at about 21% and 18%, respectively. However, wartomanin, a specific inhibitor of the phosphatidylinositol-3-kinase pathway, showed no effect on the synergistic action of TPO plus SCF. We suggest that part of the synergistic effect of TPO plus SCF can be explained by the activation of MAPK/ERK and the suppression of caspase cascade, but may not be the only mechanism taking part in the synergism