Gene therapy for chronic granulomatous disease combined with in vivo expansion of transduced hematopoietic cells.

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Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO-dependent leukemia cell line UT-7/EPO and normal erythroid progenitor cells. After deprivation of EPO for 72 hours, 32.5 % of UT-7/EPO cells underwent apoptosis, accompanied by, down-regulation of Bcl-xL protein. In addition, the cleaved products of caspase-3, p11 and p21, and a few cleaved forms of inhibitor of caspase-activated DNase (ICAD) were detected in these cells. When the cells were pre-treated with pancaspase inhibitor Z-VAD-FMK, the ratio of apoptotic cells was significantly reduced in these cells (32.1% vs 12.6%), suggesting that EPO protects the UT-7/EPO cells from apoptosis via inhibition of caspase activities. When a MEK 1/2 inhibitor U0 126 inhibited activities of extracellular signal-regulated kinases (ERK1 and ERK2), the expression of Bcl-xL protein was down-regulated and subsequently apoptosis was induced. Interestingly, Z-VAD-FMK blocked U0 126-induced down-regulation of Bcl-xL protei and apoptosis, strongly suggesting that Bcl-xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. Moreover, the ratio of G0/G1 phase cells increased (35% to 67%) and the expression of p27/Kip 1 protein was up-regulated when the pretreatment of VAD inhibited U0 126-induced apoptosis in UT-7/EPO cells. In conclusion, the activation of ERKs by EPO up-regulates Bcl-xL expression via inhibition of caspase activities, resulting in the protection of erythroid cells from apoptosis.