中文摘要：

为探索八氯腺苷的抗肿瘤作用机制,以神经母细胞瘤SH-SY5Y和SK-N-SH细胞为对象,采用四唑盐比色实验（MTT法）证明,八氯腺苷具有明显的抑制肿瘤细胞增殖的作用,这种抑制作用呈剂量-时间依赖性.流式细胞分析显示,10 μmol/L八氯腺苷作用48 h后可导致靶细胞生长停滞于G2/M期;SH-SY5Y细胞发生明显细胞凋亡,但SK-N-SH细胞却未见凋亡.Hoechst 33342染色显示,SK-N-SH细胞发生了核分裂异常.蛋白质免疫印迹分析证明,10μmol/L八氯腺苷处理SH-SY5Y 48～72 h后,G2检验点调节蛋白ATM、Chk1、Cdc25C和Cdc2磷酸化形式明显上调,同时伴有caspase-3的激活,提示SH-SY5Y细胞发生了G3检验点通路和细胞凋亡途径的激活.与SH-SY5Y细胞不同,在SK-N-SH细胞中,八氯腺苷处理24～96 h时,磷酸化ATM、磷酸化Chk1/Chk2、磷酸化Cdc25C以及磷酸化Cdc2的水平呈现逐渐降低的趋势.结果提示,SK-N-SH细胞在八氯腺苷处理后发生了G2检验点失败.蛋白质免疫印迹分析还显示,八氯腺苷可诱导p53在SH-SY5Y细胞的表达,但却不能影响SK-N-SH细胞的p53组成性表达水平.p21在SK-N-SH的组成性表达随八氯腺苷处理时间延长而逐渐减少,但在处理前后的SH-SY5Y细胞均未检测到p21蛋白的表达.上述实验结果提示,八氯腺苷抑制两种细胞增殖的机制不同：在SH-SY5Y细胞,八氯腺苷可激活ATM-Chk-Cdc25C-Cdc2/cyclin途径和凋亡通路,使细胞发生G2/M期阻滞和细胞凋亡;在SK-N-SH细胞,八氯腺苷诱导G2检验点失败,导致细胞阻滞在有丝分裂期,并发生有丝分裂异常.2种不同的细胞命运可能还与p53和p21表达不同有关.

英文摘要：

To explore the mechanisms of 8-chloro-adenosine （8-Cl-Ado）-antitumor effect, the inhibitory proliferation of human neuroblastoma cell lines SH-SY5Y and SK-N-SH induced by 8-Cl-Ado was evaluated with MTr methods which showed a time- and dose-dependent manner. Flow cytometry analysis and Hoechst 33342 staining showed that long exposure （48 hours） of SH-SY5Y cells to 8-Cl-Ado （10μmol/L） could induce G2/M arrest followed by apoptosis, while SK-N-SH cells arrested in G2/M phase accompanied by aberrant mitotic division. Westem blotting showed that the levels of phospho-ATM, phospho-Chk1, phospho-Cdc25C and phospho-Cdc2 were upregulated in SH-SY5Y cell lines, indicating that the ATM/Chk1 pathway was activated that arrests the cells in the G2 phase. However, the loss of phosphorylated forms of Chk2, Cdc25C and Cdc2 occurred in SK-N-SH cells, indicating induction of G2 checkpoint failure that allowed the cell entry into mitosis. Westem blotting also showed the activation of caspase-3 in SH-SY5Y cells but not SK-N-SH cell. Meanwhile, the expression of p53 was upregulated on the basic level in SH-SY5Y cells after 8-Cl-Ado exposure, but the constitutive expression of p53 as well as p21 in SK-N-SH cells was downregulated. These results suggest involvement of different mechanisms in response to 8-Cl-Ado-induced G2/M arrest in SH-SY5Y and SK-N-SH cells. 8-Cl-Ado appears to exert its cytotoxicity toward the SH-SY5Y cells by triggering ATM- Chk-Cdc25C-Cdc2 pathway followed by apoptosis. In SK-N-SH cell lines, however, 8-Cl-Ado induces G2 checkpoint failure that allows the cells entry into mitosis accompanied by aberrant mitotic division. Two different fates may also be related to the expression of p53 as well as p21.