Hematopoietic cytokines enhance Chk1-dependent G2/M checkpoint activation by etoposide through the Akt/GSK3 pathway to inhibit apoptosis

Hematopoietic cytokines play crucial roles in regulation of cell cycle progression and apoptosis of hematopoietic cells. However, the effects of cytokines on cellular responses to chemotherapeutic agents and the mechanisms involved have remained elusive. Here we report that erythropoietin or IL-3 promotes G2/M arrest and prevents apoptosis induced by the topoisomerase II inhibitor etoposide in murine hematopoietic 32D cells and human leukemic UT7 cells. Erythropoietin or IL-3 significantly enhanced etoposide-induced activation-specific phosphorylation of Chk1, a checkpoint kinase that inhibits Cdc2 activation by Cdc25 phosphatases, and led to the inhibition of Cdc2 kinase activity with the persistent inhibitory phosphorylation on Tyr15. The inhibitory Cdc2 phosphorylation and G2/M block by etoposide were enhanced or inhibited by overexpression of Chk1 or by the specific Chk1 inhibitor SB218078, respectively. The G2/M arrest induced by etoposide was also enhanced or inhibited by expression of a constitutively activated or dominant-negative Akt mutant, respectively. Furthermore, SB216763 or LiCl, a specific inhibitor for the GSK3 kinase inhibited by Akt, enhanced the Chk1 phosphorylation and G2/M arrest by etoposide. These results indicate that hematopoietic cytokines protect etoposide-treated cells from DNA damage-induced apoptosis by promoting, through the PI3K/Akt/GSK3 signaling pathway, G2/M checkpoint that is dependent on Chk1-mediated inhibition of Cdc2.