CAJ | 학술논문

目的探讨经表皮生长因子受体介导进入肿瘤细胞的融合蛋白表皮生长因子-腺病毒早期转录区4第4编码蛋白(EGF-E4orf4)在细胞内的转运及对胞外信号调节激酶(ERK)磷酸化的影响.方法采用间接免疫荧光结合激光共聚焦显微镜观察融合蛋白在MDA-MB-231和BGC823细胞中的转运和代谢特点,Western blot检测融合蛋白引起的细胞内ERK磷酸化水平的改变.结果融合蛋白可在细胞内累积,向细胞核周围聚集,并与细胞早期内体和晚期溶酶体共定位;融合蛋白能快速触发细胞内ERK发生磷酸化,磷酸化水平在10min时最强,之后逐渐回落至刺激前水平,活化ERK的能力较表皮生长因子弱.结论融合蛋白EGF-E4orf4在细胞内经内体-溶酶体途径发生缓慢降解;在MDA-MB-231和BGC823细胞中,融合蛋白的作用特点存在明显差异,可能是导致融合蛋白对二者抑瘤率不同的原因之一.
목적 탐토경표피생장인자수체개도진입종류세포적융합단백표피생장인자-선병독조기전록구4제4편마단백(EGF-E4orf4)재세포내적전운급대포외신호조절격매(ERK)린산화적영향.방법 채용간접면역형광결합격광공취초현미경관찰융합단백재MDA-MB-231화BGC823세포중적전운화대사특점,Western blot검측융합단백인기적세포내ERK린산화수평적개변.결과 융합단백가재세포내루적,향세포핵주위취집,병여세포조기내체화만기용매체공정위;융합단백능쾌속촉발세포내ERK발생린산화,린산화수평재10min시최강,지후축점회락지자격전수평,활화ERK적능력교표피생장인자약.결론 융합단백EGF-E4orf4재세포내경내체-용매체도경발생완만강해;재MDA-MB-231화BGC823세포중,융합단백적작용특점존재명현차이,가능시도치융합단백대이자억류솔불동적원인지일.
Objective To investigate the intracellular trafficking pathway of fusion protein epidermal growth factor-adenovirus early region 4 open reading frame 4 protein (EGF-E4orf4) internalized via epidermal growth factor (EGF) receptor and its affectivity on extracellular signal-regulated kinase(ERK) phosphorylation. MethodsMDA-MB-231 and BGC823 cells were incubated with fluorescein isothiocyanate-EGF-E4orf4 or EGF at different time points. The specific molecular mark of early endosome or late lysosome was labeled by indirect immunofluorescence,and then colocalization staining was observed using confocal laser microscopy. The levels of ERK phosphorylation were detected by Western blot. ResultsThe fluorescent signal of fusionprotein EGF-E4orf4 accumulated within the cells and congregated to the perinuclear region. A nucleus localization of the fusion protein was only at MDA-MB-231 cell. Colocalization of EGF-E4orf4 with early endosome/late lysosome was observed. EGF-E4orf4 stimulated ERK phosphorylation, which was most obvious 10 minutes after stimulation, and then gradually attenuated, which was similar to EGF stimulation but with a less decrease. ConclusionsInternalized EGF-E4orf4 can be slowly degraded via endosome-lysosome pathway. The action features of EGF-E4orf4 are remarkably different between MDA-MB-231 and BGC823 cells, which may help explain the differences in its anti-tumor potency and in the special selectivity toward different tumor cells.