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

慢性肉芽肿性疾病的基因治疗结合转导造血细胞的体内扩增。

• 批准号: 14570993
• 负责人: MORI Masaki
• 金额: $ 2.24万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570993/
• 关键词: erythropoietin; apoptosis; MAP kinase; caspase; Bcl-xL; MAPキナゼ

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.

促红细胞生成素（EPO）可以在红系发育过程中拯救红系细胞免于凋亡，导致红细胞产生。然而，EPO如何保护红系细胞免于凋亡的详细机制仍然是一个问题。为了解决这个问题，我们使用了人EPO依赖性白血病细胞系UT-7/EPO和正常红系祖细胞。EPO剥夺72 h后，32.5%的UT-7/EPO细胞发生凋亡，Bcl-xL蛋白表达下调。此外，在这些细胞中检测到caspase-3，p11和p21的裂解产物，以及caspase激活的DNA酶抑制剂（ICAD）的一些裂解形式。经pancasepase抑制剂Z-VAD-FMK预处理后，UT-7/EPO细胞的凋亡率明显降低（32.1%vs12.6%），提示EPO通过抑制caspase活性来保护UT-7/EPO细胞免于凋亡。当MEK 1/2抑制剂U 0 126抑制细胞外信号调节激酶（ERK 1和ERK 2）的活性时，Bcl-xL蛋白的表达下调，并诱导细胞凋亡。有趣的是，Z-VAD-FMK阻断了U 0 126诱导的Bcl-xL蛋白的下调和凋亡，强烈地表明Bcl-xL表达受到位于EPO信号转导中ERK活化途径下游的半胱天冬酶的调节。重要的是，在正常红系祖细胞中也观察到这些发现。VAD预处理抑制U 0 12 6诱导的UT 7 EPO细胞凋亡，G 0/G1期细胞比例增加（35%~ 6 7%），p2 7/Kip 1蛋白表达上调。总之，EPO激活ERK通过抑制caspase活性上调Bcl-xL表达，从而保护红系细胞免于凋亡。

项目成果

Muri, M: "Activation of extracellular signal-regulated kinases ERK1 and ERK2 induces Bcl-xL up-regulation via inhibition of caspase activities in erythropoietin signaling"J Cell Physiol. 195. 290-297 (2003)

Muri, M：“细胞外信号调节激酶 ERK1 和 ERK2 的激活通过抑制促红细胞生成素信号传导中的 caspase 活性诱导 Bcl-xL 上调”J Cell Physiol。

Komatsu, N: "A member of Forkhead transcription factor FKHRL1 is a downstream effector of ST1571 induced cell cycle arrest in BCR-ABL expressing cells"J Biol Chem. 278. 6411-6419 (2003)

Komatsu, N：“Forkhead 转录因子 FKHRL1 的成员是 BCR-ABL 表达细胞中 ST1571 诱导的细胞周期停滞的下游效应子”J Biol Chem。

Mishima Y, Matsumoto-Mishima Y, Terui Y, Katsuyama M, Yamada M, Mori M, Ishizaka Y Ikeda K, Watanabe J, Mizunuma N, Hayasawa H, Hatake K.: "Leukemic cell-surface CD13/aminopeptidase N and resistance to apoptosis mediated by endothelial cells."J Natl Cance

Mishima Y、Matsumoto-Mishima Y、Terui Y、Katsuyama M、Yamada M、Mori M、Ishizaka Y Ikeda K、Watanabe J、Mizunuma N、Hayasawa H、Hatake K.：“白血病细胞表面 CD13/氨肽酶 N 和耐药性

Komatsu N, Watanabe T, Uchida M, Mori M, Kirito T, Kikuchi S, Liu Q, Tauchi T, Miyazawa K, Endo H, Nagai T, Ozawa K.: "A member of forkhead transcription factor FKHRLI is a downstream effector of ST1571-induced cell cycle arrest in BCR-ABL expressing cell

Komatsu N, Watanabe T, Uchida M, Mori M, Kirito T, Kikuchi S, Liu Q, Tauchi T, Miyazawa K, Endo H, Nagai T, Ozawa K.：“叉头转录因子 FKHRLI 的成员是下游效应子

Mishima Y, Terui Y, Mishima Y, Katsuyama M, Mori M, Tomizuka H, Takizawa T, Miyazato A, Ueda M, Yamada M, Hayasawa H, Mizunuma N, Ishizaka Y, Ikeda K, Kato T, Ozawa K, Hatake K.: "New human myelodysplastic cell line, TER-3 : G-CSF specific downregulation

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