Absence of autoantigen Ku in mature human neutrophils and human promyelocytic leukemia line (HL-60) cells and lymphocytes undergoing apoptosis.
Absence of autoantigen Ku in mature human neutrophils and human promyelocytic leukemia line (HL-60) cells and lymphocytes undergoing apoptosis.
复制标题
DOI：
10.1084/jem.181.6.2049
复制
发表时间：
1995-06-01
期刊：
影响因子：
--
通讯作者：
Reeves WH
中科院分区：
文献类型：
--
作者：
Ajmani AK;Satoh M;Reap E;Cohen PL;Reeves WH
Ku自身抗原是一种70- kd和80-kD蛋白的异源二聚体，可被系统性红斑狼疮及相关疾病患者的自身抗体识别，是一种dna依赖性蛋白激酶的dna结合成分。dna依赖性蛋白激酶的催化活性由一个350-kD亚基（p350）携带。鉴于最近描述的Ku在修复双链DNA断裂中的作用，我们研究了Ku和p350水平在中性粒细胞中的调节，中性粒细胞是一种终末分化的细胞类型，注定要经历凋亡。由于双链DNA断裂的出现是细胞凋亡的特征，我们对Ku可能反对程序性细胞死亡的可能性感兴趣。用抗ku和抗p350单克隆抗体流式细胞术分析外周血细胞，中性粒细胞未染色，静息（G0）淋巴细胞阳性。通过Western blotting和酶联免疫吸附法检测Ku抗原，证实成熟中性粒细胞中Ku不存在。相比之下，人早幼粒细胞白血病HL-60在二甲亚砜处理后向中性粒细胞分化，Ku和p350呈阳性。鉴于中性粒细胞的寿命较短，而Ku和p350的半衰期较长（bbb50 d），这些数据表明Ku在髓细胞分化过程中被主动降解。流式细胞术分析HL-60细胞Ku呈双峰染色。G1/G0、S和G2/M组细胞均呈阳性染色，而亚二倍体DNA含量为凋亡特征的细胞呈Ku阴性。植物血凝素刺激的人淋巴细胞也获得了类似的结果。这些数据表明，Ku抗原在注定要经历凋亡的髓细胞和凋亡的淋巴细胞中都被积极降解，这提高了Ku的降解可能有助于防止细胞凋亡过程中核DNA片段的不适当修复的可能性。
The Ku autoantigen is a heterodimer of 70- and 80-kD proteins recognized by autoantibodies from patients with systemic lupus erythematosus and related diseases that is the DNA-binding component of a DNA-dependent protein kinase. The catalytic activity of DNA-dependent protein kinase is carried by a 350-kD subunit (p350). In light of the recently described role of Ku in repairing double-strand DNA breaks, we investigated the regulation of Ku and p350 levels in neutrophils, a terminally differentiated cell type destined to undergo apoptosis. Since the appearance of double-strand DNA breaks is characteristic of apoptosis, we were interested in the possibility that Ku might oppose programmed cell death. Analysis of peripheral blood cells by flow cytometry using anti-Ku and anti-p350 monoclonal antibodies revealed that neutrophils were unstained, whereas resting (G0) lymphocytes were positive. The absence of Ku in mature neutrophils was confirmed by Western blotting and enzyme-linked immunosorbent assay for Ku antigen. In contrast, the human promyelocytic leukemia line, HL-60, which undergoes differentiation toward neutrophils after dimethylsulfoxide treatment, was positive for Ku and p350. In view of the short lifespan of neutrophils and the prolonged half-life of Ku and p350 (> 5 d), these data suggested that Ku was actively degraded during myeloid differentiation. Analysis of HL-60 cells by flow cytometry revealed that Ku staining was bimodal. Cells in G1/G0, S, or G2/M were all stained positively, whereas cells with a subdiploid DNA content characteristic of apoptosis were Ku negative. Similar results were obtained with phytohemagglutin-stimulated human lymphocytes. These data suggest that the Ku antigen is actively degraded in both myeloid cells destined to undergo apoptosis and apoptotic lymphocytes, raising the possibility that degradation of Ku may help to prevent the inappropriate repair of fragmented nuclear DNA during apoptosis.