Cheng Zhi Y, Guo Xiao L, Yang Xiao Y, Niu Zhi Y, Li Shi H, Wang Su Y, Chen Hao, Pan Ling
Department of Hematology and Heibei Institute of Hematology, The Second Hospital of Hebei Medical University, Shijiazhuang, PR China.
J Exp Clin Cancer Res. 2008 Dec 30;27(1):87. doi: 10.1186/1756-9966-27-87.
To investigate, in vitro, the regulatory effects of tumor-suppressing gene PTEN on mTOR (mammalian target of rapamycin) signaling pathway, the effects of transfected PTEN and rapamycin on the growth inhibition, and apoptosis induction for human leukemia cell line K562 cells.
K562 cells were transfected with recombined adenovirus-PTEN vector containing green fluorescent protein (Ad-PTEN-GFP), followed by the treatment of the cells with or without rapamycin. The proliferation inhibition rate and apoptotic rate of these transfected and/or rapamycin treated K562 cells were measured by MTT assay and flow cytometry (FCM), the expression levels of PTEN-, mTOR-, cyclinD1- and P27kip1- mRNA were measured by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR), the protein expression levels of PTEN, Akt, p-Akt were detected by western blotting.
The proliferation of K562 cells was inhibited by PTEN gene transfection with/without the treatment of rapamycin. The expression levels of PTEN- and P27kip1- mRNA were up-regulated, and the mTOR- and cyclinD1- mRNA were down-regulated in K562 cells after the cells transfected with wild type PTEN gene and treated with rapamycin.
PTEN and rapamycin inhibited mTOR expression by acting as an upstream regulator of mTOR. Low dose rapamycin in combination with over-expressed PTEN might have synergistic effects on inhibiting the proliferation and promoting apoptosis of K562 cells.
在体外研究抑癌基因PTEN对哺乳动物雷帕霉素靶蛋白（mTOR）信号通路的调控作用，以及转染PTEN和雷帕霉素对人白血病细胞株K562细胞生长抑制和凋亡诱导的影响。
用含绿色荧光蛋白的重组腺病毒-PTEN载体（Ad-PTEN-GFP）转染K562细胞，然后用或不用雷帕霉素处理细胞。通过MTT法和流式细胞术（FCM）检测这些转染和/或经雷帕霉素处理的K562细胞的增殖抑制率和凋亡率，通过实时荧光相对定量逆转录聚合酶链反应（FQ-PCR）检测PTEN、mTOR、细胞周期蛋白D1和P27kip1 mRNA的表达水平，通过蛋白质印迹法检测PTEN、Akt、p-Akt的蛋白表达水平。
无论是否用雷帕霉素处理，PTEN基因转染均抑制K562细胞的增殖。野生型PTEN基因转染并用雷帕霉素处理后的K562细胞中，PTEN和P27kip1 mRNA的表达水平上调，mTOR和细胞周期蛋白D1 mRNA的表达水平下调。
PTEN和雷帕霉素作为mTOR的上游调节因子抑制mTOR表达。低剂量雷帕霉素与过表达的PTEN联合使用可能对抑制K562细胞的增殖和促进其凋亡具有协同作用。
