Inhibition if cyclin A kinase activity in E2F-1 chemogene therapy of colon cancer
Inhibition if cyclin A kinase activity in E2F-1 chemogene therapy of colon cancer
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DOI：
10.1159/000069791
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发表时间：
2002-11-01
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影响因子：
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通讯作者：
McMasters, KM
中科院分区：
文献类型：
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作者：
Elliott, MJ;Baker, JD;McMasters, KM
腺病毒介导的凋亡基因e2 f-1的基因转移已显示在多种肿瘤细胞中诱导凋亡，并且与几种特异性化疗剂以相加或协同方式起作用以诱导肿瘤细胞死亡。E2 F-1的凋亡功能依赖于其结合DNA的能力;细胞周期蛋白A激酶活性已被证明负调节E2 F-1的DNA结合能力。在本研究中，我们试图确定细胞周期蛋白A激酶活性是否参与介导E2 F-1和结肠癌细胞化疗药物之间的相互作用。因此，用表达E2 F-1的腺病毒（Ad-E2 F-1，感染复数20）处理人结肠腺癌（SW 620）细胞。感染后立即以LD 25剂量给予一组具有不同细胞毒性作用模式的常规化疗剂。处理后3天，通过台盼蓝排除试验测定活力和生长抑制。细胞凋亡，确认使用细胞形态学，聚（ADP-核糖）聚合酶裂解，和流式细胞仪分析。免疫印迹法检测E2 F-1过表达和细胞周期蛋白A蛋白表达，激酶法测定细胞周期蛋白A激酶活性。发现曲马新碱（VIN）、喜树碱（CPT）和放线菌素D对E2 F-1介导的细胞凋亡具有协同作用（超过相加的单一治疗值的>38%）。依托泊苷、顺铂（CIS）和5-氟尿嘧啶（5-FU）与E2 F-1的协同作用最小（小于或等于11.5%，超过单药治疗值）。与Ad-LacZ对照相比，Ad-E2 F-1单独处理导致细胞周期蛋白A激酶活性增加3.4倍（p
Adenoviral-mediated gene transfer of the apoptotic gene e2f-1 has been shown to induce apoptosis in a variety of tumor cells and acts in an additive or cooperative fashion with several specific chemotherapeutic agents to induce tumor cell death. The apoptotic function of E2F-1 is dependent on its ability to bind DNA; cyclin A kinase activity has been shown to negatively regulate the DNA-binding capacity of E2F-1. In the present study, we sought to determine whether cyclin A kinase activity is involved in mediating the interaction between E2F-1 and chemotherapeutic agents in colon cancer cells. Therefore, human colon adenocarcinoma (SW620) cells were treated with an adenovirus expressing E2F-1 (Ad-E2F-1, multiplicity of infection 20). Immediately following infection, a panel of conventional chemotherapeutic agents with varying modes of cytotoxic action were administered at LD25 doses. Three days following treatment, viability and growth inhibition were determined by trypan blue exclusion assay. Apoptosis was confirmed using cellular morphology, poly (ADP-ribose) polymerase cleavage, and flow-cytometric analysis. E2F-1 overexpression and cyclin A protein expression were monitored by immunoblot, and cyclin A kinase activity was determined by kinase assay. Vincristine (VIN), camptothecin (CPT), and actinomycin D were found to have a cooperative (>38% over the additive single therapy values) effect on E2F-1-mediated apoptosis. Etoposide, cisplatin (CIS), and 5-fluorouracil (5-FU) showed the least cooperation (less than or equal to11.5% over the additive single therapy values) with E2F-1. Ad-E2F-1 treatment alone results in 3.4-fold increase of cyclin A kinase activity compared to Ad-LacZ control (p