Gao Xuejuan, Chen Tongsheng, Xing Da, Wang Fang, Pei Yihui, Wei Xunbin
Institute of Laser Life Science, South China Normal University, Guangzhou, China, and Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, USA.
J Cell Physiol. 2006 Feb;206(2):441-8. doi: 10.1002/jcp.20484.
Laser irradiation has been shown to trigger cellular proliferation and apoptosis in various cell types. Studying the signaling pathways involved in the laser irradiation is important for understanding these processes. In present study, to monitor the protein kinase Cs (PKCs) activity in living cells in real time, we transfected and screened human lung adenocarcinoma cells (ASTC-a-1) stably expressing C kinase activity reporter (CKAR) constructed based on fluorescence resonance energy transfer (FRET) technique. The CKAR is a specific, reversible reporter of phosphorylation by PKCs and it can monitor the ongoing balance between PKCs and phosphatases. The increasing dynamics of PKCs activity is monitored during cell proliferation induced by low-power laser irradiation (LPLI) (0.8 J/cm2) in serum-starved ASTC-a-1 cells stably expressing CKAR reporter using FRET imaging on laser scanning confocal microscope and using spectrofluorometric analysis on a luminescence spectrometer, respectively. However, the decreasing dynamics of PKCs activity has been monitored in real time using FRET imaging for the cells treated with high fluence LPLI (60 J/cm2), which was previously found to induce cell apoptosis. Taken together, LPLI induces the ASTC-a-1 cell proliferation by specifically activating PKCs. However, PKCs activity decreases during cell apoptosis induced by high fluence LPLI. Our results indicate that PKCs play an important role in the laser irradiation-induced biological effects.
激光照射已被证明能触发多种细胞类型的细胞增殖和凋亡。研究激光照射所涉及的信号通路对于理解这些过程很重要。在本研究中,为实时监测活细胞中的蛋白激酶C(PKCs)活性,我们对稳定表达基于荧光共振能量转移(FRET)技术构建的C激酶活性报告基因(CKAR)的人肺腺癌细胞(ASTC-a-1)进行了转染和筛选。CKAR是PKCs磷酸化的特异性、可逆报告基因,它可以监测PKCs和磷酸酶之间的动态平衡。分别使用激光扫描共聚焦显微镜上的FRET成像和发光光谱仪上的荧光分光光度分析,监测稳定表达CKAR报告基因的血清饥饿ASTC-a-1细胞在低功率激光照射(LPLI)(0.8 J/cm2)诱导细胞增殖过程中PKCs活性的增加动态。然而,对于先前发现可诱导细胞凋亡的高剂量LPLI(60 J/cm2)处理的细胞,已使用FRET成像实时监测到PKCs活性的降低动态。综上所述,LPLI通过特异性激活PKCs诱导ASTC-a-1细胞增殖。然而,在高剂量LPLI诱导的细胞凋亡过程中PKCs活性降低。我们的结果表明PKCs在激光照射诱导的生物学效应中起重要作用。