Peng Li-ping, Huang Jian-ming, Zhang Guo-nan, Zha Xiao, Ren Yuan, Fan Ying, Deng Bi-fang
Department of Gynecological Oncology, Sichuan Cancer Hospital, Chengdu 610041, China.
Zhonghua Fu Chan Ke Za Zhi. 2010 Nov;45(11):860-4.
To study the influence of survivin mutant-T34A (survivin(T34A)) and survivin deletant-N-terminal 8 amino acids residues (survivin(N-8AA)) on the cell cycle distribution and chemosensitivity in human ovarian cancer HO-8910 cells for explorating the roles of modified survivin-mediated apoptosis induced by chemotherapeutic agents and possible signaling pathways involved.
pcDNA3.1 plasmid contained wild-type, survivin(T34A) and survivin(N-8AA) genes were transfected into HO-8910 cells, respectively, the control groups were HO-8910 cells transfected with pcDNA3.1 plasmids. The expression of mRNA was examined by reverse transcription(RT) PCR and identified by DNA sequencing; the cell cycles were determined by flow cytometer analysis (FCM); the growth inhibitions rate of cisplatin (DDP), paclitaxel (PTX) and LY294002 on the transfected cells were determined using methyl thiazolyl tetrazolium (MTT) assay.
(1) The RT-PCR procedures and genome sequences showed that the survivin mRNA were expressed stable in the transfected HO-8910 cells. (2) There was lower percent of G(0)/G(1) phase cells in SN-HO-8910 cells than that in PC-HO-8910 cells (44.72% vs. 49.64%, P < 0.05); while higher percentage of G(2)/M phase and S phase cells (1.06% and 54.22% vs. 0.56% and 49.80%, P < 0.05). There was lower the G(2)/M phase and S phase cells in M-HO-8910 cells 0.16% and 36.33%, than that in PC-HO-8910 cells (P < 0.05); while higher percentage of G(0)/G(1) phase cells (63.51%, P < 0.05). G(0)/G(1), G(2)/M and S phase cells in Sur-HO-8910 cells were 54.46%, 0.62% and 44.92%, and there were not significantly difference (P > 0.05), compared to those in PC-HO-8910 cells. (3) The inhibitory concentration (IC(50)) of DDP and PTX were higher in Sur-HO-8910 cells than those in control cells [(20.4 ± 6.1) vs. (14.4 ± 3.9) µmol/L, (36.7 ± 4.0) vs. (28.6 ± 3.6) µmol/L; all P < 0.05]. The IC(50) of DDP and LY294002 in SN-HO-8910 cells were lower than those in control cells [(7.6 ± 1.0) vs. (14.4 ± 3.9) µmol/L, (13.2 ± 4.0) vs. (41.0 ± 7.9) µmol/L; all P < 0.01]. The IC(50) of PTX [(37.9 ± 4.8) µmol/L] in SN-HO-8910 cells were higher than that in control cells (P < 0.05). The IC(50) of DDP in M-HO-8910 cells [(9.9 ± 1.2) µmol/L] were lower than that in control cells (P < 0.05), and the IC(50) of LY294002 in M-HO-8910 cells [(66.9 ± 4.8) µmol/L] higher than that in control cells (P < 0.01).
The changes of cells cycle distribution caused by survivin(T34A) or survivin(N-8AA) enhanced the G(2)/M cell cycle-dependent chemosensitivity of PTX. Compared to survivin(T34A), survivin(N-8AA) preferentially to mediate the cytotoxicity of DDP and LY294002, suggesting that it may be related to the cell cycle-dependence of survivin function and to blockage of the formation of its active dimer.
研究生存素突变体-T34A（survivin(T34A)）和生存素N端缺失8个氨基酸残基（survivin(N-8AA)）对人卵巢癌HO-8910细胞周期分布及化疗敏感性的影响，以探讨修饰的生存素介导化疗药物诱导凋亡的作用及可能涉及的信号通路。
将分别含有野生型、survivin(T34A)和survivin(N-8AA)基因的pcDNA3.1质粒转染入HO-8910细胞，对照组为转染pcDNA3.1质粒的HO-8910细胞。采用逆转录（RT）PCR检测mRNA表达并经DNA测序鉴定；用流式细胞仪分析（FCM）检测细胞周期；采用甲基噻唑基四氮唑（MTT）法测定顺铂（DDP）、紫杉醇（PTX）和LY294002对转染细胞的生长抑制率。
（1）RT-PCR及基因组序列结果显示，生存素mRNA在转染的HO-8910细胞中稳定表达。（2）SN-HO-8910细胞中G(0)/G(1)期细胞百分比低于PC-HO-8910细胞（44.72%对49.64%，P<0.05）；而G(2)/M期和S期细胞百分比高于PC-HO-8910细胞（1.06%和54.22%对0.56%和49.80%，P<0.05）。M-HO-8910细胞中G(2)/M期和S期细胞百分比低于PC-HO-8910细胞（0.16%和36.33%，P<0.05）；而G(0)/G(1)期细胞百分比高于PC-HO-8910细胞（63.51%，P<0.05）。Sur-HO-8910细胞中G(0)/G(1)、G(2)/M和S期细胞分别为54.46%、0.62%和44.92%，与PC-HO-8910细胞相比无显著差异（P>0.05）。（3）Sur-HO-8910细胞中DDP和PTX的半数抑制浓度（IC(50)）高于对照细胞[（20.4±6.1）对（14.4±3.9）μmol/L，（36.7±4.0）对（28.6±3.6）μmol/L；均P<0.05]。SN-HO-8910细胞中DDP和LY294002的IC(50)低于对照细胞[（7.6±1.0）对（14.4±3.9）μmol/L，（13.2±4.0）对（41.0±7.9）μmol/L；均P<0.01]。SN-HO-8910细胞中PTX的IC(50)[（37.9±4.8）μmol/L]高于对照细胞（P<0.05）。M-HO-8910细胞中DDP的IC(50)[（9.9±1.2）μmol/L]低于对照细胞（P<0.05），而M-HO-8910细胞中LY294002的IC(50)[（66.9±4.8）μmol/L]高于对照细胞（P<0.01）。
survivin(T34A)或survivin(N-8AA)引起的细胞周期分布变化增强了PTX对G(2)/M期细胞周期依赖性的化疗敏感性。与survivin(T34A)相比，survivin(N-8AA)优先介导DDP和LY294002的细胞毒性，提示这可能与生存素功能的细胞周期依赖性及活性二聚体形成受阻有关。
