摘要
目的 阐明EB病毒潜伏膜蛋白 1(LMP1)在鼻咽癌 (NPC)中调节表皮生长因子受体(EGFR)磷酸化水平。方法 采用已建立的、受四环素调控的、LMP1表达的NPC细胞系 (pTet on LMP1HNE2 ) ,定量诱导pTet on LMP1HNE2细胞LMP1动态表达水平 ,并观察EGFR表达和磷酸化水平。采用瞬间转染方法 ,将EGFR显性负性突变体和LMP1antisense表达质粒分别导入pTet on LMP1HNE2细胞中 ,采用免疫共沉淀和Westernblot检测EGFR磷酸化 ;同时观察EGF刺激后对EGFR磷酸化前后的影响。结果 在pTet on LMP1HNE2细胞中 ,LMP1能诱导EGFR的表达和磷酸化 ,并随LMP1表达增加而增加。pTet on LMP1HNE2细胞在导入EGFR显性负性突变体后 ,不但完全阻断EGFR磷酸化 ,而且完全抑制EGF所引起的EGFR磷酸化 ;而导入LMP1antisense后 ,可显著地抑制EGFR磷酸化水平。结论 LMP1能同时诱导EGFR表达和上调EGFR磷酸化 ,并呈剂量相关效应 ,提示EGFR磷酸化可能在NPC发生发展过程中起着重要作用。
Objective To elucidate the regulation of the phosphorylation of epidermal growth factor receptor (EGFR) by the EB virus encoded latent membrane protein 1(LMP1) in nasopharyngeal carcinoma cell line. Methods The levels of EGFR expression and phosphorylation in pTet-on LMP1 HNE2 cell, a nasopharyngeal carcinoma (NPC) cell line, in the dynamic expression of LMP1 induced by different concentrations of doxycycline (Dox) were observed. The EGFR dominant negative mutant and LMP1 antisense expression plasmid were transiently transfected into pTet-on LMP1 HNE2 cells by lipofectamine, and the changes in EGFR phosphorylation were observed by immunocoprecitation and Western blot. The changes in EGFR phosphorylation were observed after EGF treatment. Results In pTet-on LMP1 HNE2 cells, Dox-induced LMP1 upregulated EGFR expression and phosphorylation in a dose-dependent manner. After EGFR dominant negative mutant was transfected into pTet-on LMP1 HNE2 cells, the increase of EGFR phosphorylation was inhibited completely. When LMP1 antisense expression plasmid was transfected into pTet-on LMP1 HNE2 cells, the levels of EGFR phosphorylation were also inhibited significantly. Meanwhile, after EGF had been added into pTet-on LMP1 HNE2 cells, increase of EGFR phosphorylation was induced, but it was completely blocked by EGFR dominant negative mutant and the introduction of LMP1 antisense. Conclusion EB virus encoded LMP1 not only induces the dose-dependent expression of EGFR, but also the dose-dependent phosphorylation of EGFR. The phosporylation of EGFR may play a vital role in the development of nasopharyngeal carcinoma.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2002年第3期226-229,共4页
Chinese Journal of Oncology
基金
国家重点基础研究发展规划 ( 973)资助项目(G19980 5 12 0 1)
国家自然科学基金杰出青年基金资助项目( 95 2 5 0 2 2 )
国家自然科学基金资助项目 ( 30 0 0 0 0 87)
关键词
EB病毒
潜伏膜蛋白1
鼻咽癌
表皮生长因子受体
磷酸化
研究
Epstein-Barr virus
Nasopharyngeal neoplasms
Latent membrane protein 1
Epidermal growth factor receptor
