Xiao Jie, Yin Songmei, Li Yiqing, Xie Shuangfeng, Nie Danian, Ma Liping, Wang Xiuju, Wu Yudan, Feng Jianhong
Department of Hematology, Second Afflicted Hospital of Sun Yat-Sen University, Guangzhou, China.
Acta Biochim Biophys Sin (Shanghai). 2009 Aug;41(8):699-708. doi: 10.1093/abbs/gmp058.
S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.
S期激酶相关蛋白2（SKP2）基因是一种肿瘤抑制基因，参与泛素介导的P27kip1降解过程。SKP2和P27kip1通过调节白血病细胞的增殖、凋亡和分化，影响白血病的进程和预后。在本研究中，我们探讨了通过下调SKP2来逆转HL-60/A耐药的机制。采用Amaxa Nucleofector系统将SKP2小干扰RNA（siRNA）转染至HL-60/A细胞。分别通过逆转录-聚合酶链反应和蛋白质印迹分析检测Skp2、P27kip1和多药耐药相关蛋白（MRP）的基因和蛋白表达水平。通过流式细胞术分析细胞周期。根据不同浓度化疗药物作用下这两种细胞的死亡率，采用细胞毒性分析计算50%抑制浓度值，以比较细胞的敏感性。由于MRP的表达，HL-60/A细胞表现出对阿霉素、柔红霉素和阿糖胞苷交叉耐药的多药耐药表型特征。我们发现HL-60/A细胞中SKP2的表达高于HL-60细胞，但P27kip1的表达较低。用SKP2 siRNA转染的HL-60/A细胞中SKP2的表达下调，而P27kip1的蛋白水平上调。与对照组（用对照siRNA转染）的MRP表达水平相比，用SKP2 siRNA转染的HL-60/A细胞中MRP的mRNA和蛋白表达水平较低，且后者细胞对阿霉素、柔红霉素和阿糖胞苷更敏感。下调SKP2表达并使细胞停滞于G0/G1期可提高MRP表达下调的白血病细胞的药物敏感性。
