Specific inhibition of Stat5a/b promotes apoptosis of IL-2-responsive primary and tumor-derived lymphoid cells
Specific inhibition of Stat5a/b promotes apoptosis of IL-2-responsive primary and tumor-derived lymphoid cells
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DOI：
10.4049/jimmunol.171.8.3919
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发表时间：
2003-10-15
影响因子：
4.4
通讯作者：
Kirken, RA
中科院分区：
文献类型：
--
作者：
Behbod, F;Nagy, ZS;Kirken, RA
Stat5a/b 表现出 96% 的同源性，是正常免疫功能所必需的。目前的研究通过使用新型硫代磷酸酯-2'-O-甲氧基乙基反义寡脱氧核苷酸 (asODN) 特异性且同时破坏两种蛋白质来检查淋巴细胞中 Stat5a/b 的功能。通过在人原代细胞系和肿瘤细胞系中分别存在大于或等于 55% 和 95% 的荧光 TAMRA 标记 ODN，证实了有效递送。急性asODN给药在6小时内降低了Stat5a的水平（90%），而Stat5b需要近48小时才能达到相同的抑制作用，这表明Stat5a的表观周转率比Stat5b高8倍。然而，密切相关的 Stat3 蛋白的表达在 asODN 处理后没有变化。 Stat5a/b 的分子消融在 24 小时内促进大量原代 PHA 激活的 T 细胞 (72%) 和淋巴肿瘤细胞系 (例如 YT；74%) 的凋亡细胞死亡，评估方法为 1) 核溶解核变性和其他普遍细胞结构改变的可视化，2) TdT 阳性 DNA 降解的酶检测，以及 3) 自动细胞计数检测膜联蛋白V易位。与 Stat5a/b 缺失小鼠的研究结果相反，细胞周期进程似乎并未受到显着影响。有趣的是，IL-2 不敏感且未引发的 T 细胞和 Jurkat 细胞几乎不受影响。最后，有证据表明与 Stat5a/b 消融相关的细胞毒性可能源自 caspase-8 的激活，caspase-8 是一种有助于凋亡细胞定型的起始蛋白酶。我们认为，在能够激活 Stat5a 和 Stat5b 的淋巴细胞中，这两种蛋白优先介导抗凋亡存活影响。
Stat5a/b exhibits 96% homology and are required for normal immune function. The present studies examined Stat5a/b function in lymphoid cells by specific and simultaneous disruption of both proteins using novel phosphorothioate-2'-O-methoxyethyl antisense oligodeoxynucleotides (asODN). Efficient delivery was confirmed by the presence of fluorescent TAMRA-labeled ODN in greater than or equal to 55 and 95% in human primary and tumor cell lines, respectively. Acute asODN administration reduced levels of Stat5a (90%) in 6 h, whereas Stat5b required nearly 48 h to attain the same inhibition, suggesting that the apparent turnover rate for Stat5a was 8-fold higher than that for Stat5b. Expression of the closely related Stat3 protein was unchanged after asODN treatment, however. Molecular ablation of Stat5a/b promoted apoptotic cell death in a significant population of primary PHA-activated T cells (72%) and lymphoid tumor cell line (e.g., YT; 74%) within 24 h, as assessed by 1) visualization of karyolytic nuclear degeneration and other generalized cytoarchitectural alterations, 2) enzymatic detection of TdT-positive DNA degradation, and 3) automated cytometric detection of annexin V translocation. Contrary to findings from Stat5a/b-null mice, cell cycle progression did not appear to be significantly affected. Interestingly, IL-2-insensitive and unprimed T cells and Jurkat cells remained mostly unaffected. Finally, evidence is provided that the cytotoxicity associated with Stat5a/b ablation may derive from activation of caspase-8, an initiator protease that contributes to apoptotic cell commitment. We propose that in lymphoid cells competent to activate Stat5a and Stat5b, both proteins preferentially mediate an antiapoptotic survival influence.