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Changes of cell cycle distribution and nuclear structure in NK cells by EBV infection

EBV感染后NK细胞细胞周期分布和核结构的变化

基本信息

批准号：
14570997
负责人：
SUGIMOTO Koichi
金额：
$ 1.73万
依托单位：
Juntendo University
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-14570997/
关键词：
EGFP-EBV NK cell Latent infection G2 arrest Latency type I Genomic instability Resistance to chemotherapy 抗癌剤への耐性 EBウイルス GFP蛋白質多核細胞 cdc2 cdc25C actin α-tubulin

项目摘要

Although considerable part of natural killer (NK) cell neoplasms possess EBV genome, there has been no direct evidence that EBV infects human NK cells in vitro. In this study, we demonstrated EBV entry into the NK cells using enhanced green fluorescence (EGFP)-containing recombinant EBV. After 48 hr, about 30% of NK cell lines and more than 40% of normal peripheral blood NK cells were positive for EGFP signal. In situ hybridization for EBNAs and BHLFs showed that latent and lytic infections coexisted at the early phase of EBV infection. NK cells in latent but not lytic EBV infection tended to show a bizzare and giant shape. Cdc2, cyclin B, and Cdc25C, which are important for G2/M transition, increased in protein levels and partially migrated to the nucleus though their activation was inhibited. Flow cytometric analysis showed mainly G2 arrest in EBV-infected NK cells. Although nuclear distribution of PML, SC35, and HP1 was unchanged, cyto- and nuclear-skeletons made of actin, tubulin and lamin has apparently rearranged and abnormalities of centrosome numbers were detected. We established eight EBV-carrying clones, which showed both latency types I and II. Both of them are recognized in EBV-associated NK-cell neoplasms. The cell size became larger and the frequency of micronuclei formation, a hallmark of genomic instability, increased in EBV-carrying NKL clones. Furthermore, these clones were resistant to various chemotherapeutic agents and cell dilution compared to the parent NK cell lines.

尽管相当一部分自然杀伤（NK）细胞肿瘤具有EBV基因组，但目前还没有直接证据表明EBV在体外感染人类NK细胞。在这项研究中，我们使用含有增强型绿色荧光 (EGFP) 的重组 EBV 证明了 EBV 能够进入 NK 细胞。 48小时后，约30%的NK细胞系和超过40%的正常外周血NK细胞呈EGFP信号阳性。 EBNA和BHLF的原位杂交表明，EBV感染早期潜伏感染和裂解感染并存。潜伏但非裂解性 EBV 感染中的 NK 细胞往往表现出奇异且巨大的形状。对于 G2/M 转变很重要的 Cdc2、细胞周期蛋白 B 和 Cdc25C 的蛋白质水平增加，尽管其激活受到抑制，但部分迁移到细胞核。流式细胞术分析显示，EBV 感染的 NK 细胞主要发生 G2 期停滞。尽管 PML、SC35 和 HP1 的核分布没有变化，但由肌动蛋白、微管蛋白和核纤层蛋白组成的细胞骨架和核骨架明显重新排列，并且检测到中心体数量异常。我们建立了 8 个携带 EBV 的克隆，它们均表现出 I 型和 II 型潜伏期。两者均在 EBV 相关 NK 细胞肿瘤中被识别。在携带 EBV 的 NKL 克隆中，细胞尺寸变大，微核形成频率（基因组不稳定的标志）增加。此外，与亲本 NK 细胞系相比，这些克隆对各种化疗药物和细胞稀释具有抗性。