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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EBV在体外感染人NK细胞尚无直接证据。在这项研究中，我们利用含有增强绿色荧光（EGFP）的重组EBV证明了EBV进入NK细胞。48小时后，约30%的NK细胞系和40%以上的正常外周血NK细胞EGFP信号阳性。ebna和BHLFs的原位杂交表明，EBV感染早期潜伏性感染和裂解性感染并存。潜伏性而非溶性EBV感染的NK细胞呈奇异而巨大的形状。Cdc2、细胞周期蛋白B和Cdc25C在G2/M转变中起重要作用，虽然它们的激活被抑制，但它们的蛋白水平增加并部分迁移到细胞核。流式细胞术分析显示ebv感染的NK细胞主要出现G2阻滞。尽管PML、SC35和HP1的核分布不变，但由肌动蛋白、微管蛋白和纤层蛋白组成的细胞骨架和核骨架明显重排，中心体数量异常。我们建立了8个携带ebv的克隆，潜伏期均为I型和II型。它们都在ebv相关的nk细胞肿瘤中被识别。在携带ebv的NKL克隆中，细胞大小变得更大，微核形成的频率（基因组不稳定性的标志）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。