Eriksson David, Löfroth Per-Olov, Johansson Lennart, Riklund Katrine Ahlström, Stigbrand Torgny
Department of Immunology, Umeå university, Umeå, Sweden.
Clin Cancer Res. 2007 Sep 15;13(18 Pt 2):5501s-5508s. doi: 10.1158/1078-0432.CCR-07-0980.
Experimental radioimmunotherapy delivering absorbed doses of 2.5 to 10 Gy has been shown to cause growth retardation of tumors. The purpose of this study was to elucidate the sequential molecular and cellular events occurring in HeLa Hep2 cells exposed to such doses.
Dose-response curves, activation of cell cycle checkpoints, and mitotic behavior were investigated in HeLa Hep2 cells following 2.5- to 10-Gy irradiation by carrying out 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, Western blots, fluorescence-activated cell sorting analysis, and immunofluorescence stainings. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining was used to detect apoptosis.
A G2-M arrest was shown by fluorescence-activated cell sorting analysis. p53 and p21 were found to be up-regulated but were not immediately related to the arrest. The G2-M arrest was transient and the cells reentered the cell cycle still containing unrepaired cellular damage. This premature entry caused an increase of anaphase bridges, lagging chromosomal material, and multipolar mitotic spindles as visualized by propidium iodide staining and immunofluorescence staining with alpha-tubulin and gamma-tubulin antibodies. Furthermore, a dose-dependent significant increase in centrosome numbers from 12.6+/-6.6% to 67+/-5.3% was identified as well as a dose-dependent increase of polyploid cells from 2.8+/-1.3% to 17.6+/-2.1% with the highest absorbed dose of 10 Gy. These disturbances caused the cells to progress into mitotic catastrophe and a fraction of these dying cells showed apoptotic features as displayed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining 5 to 7 days after irradiation.
An absorbed dose of 2.5 to 10 Gy was shown to force HeLa Hep2 cells into mitotic catastrophe and delayed apoptosis. These might be important cell death mechanisms involved in tumor growth retardation following radioimmunotherapy of solid tumors.
实验性放射免疫疗法给予2.5至10 Gy的吸收剂量已被证明可导致肿瘤生长迟缓。本研究的目的是阐明暴露于此类剂量的HeLa Hep2细胞中发生的一系列分子和细胞事件。
通过进行3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐检测、蛋白质免疫印迹、荧光激活细胞分选分析和免疫荧光染色，研究HeLa Hep2细胞在2.5至10 Gy照射后的剂量反应曲线、细胞周期检查点激活和有丝分裂行为。采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记染色检测细胞凋亡。
荧光激活细胞分选分析显示出现G2-M期阻滞。发现p53和p21上调，但与阻滞无直接关系。G2-M期阻滞是短暂的，细胞重新进入细胞周期时仍含有未修复的细胞损伤。这种过早进入导致后期桥、落后染色体物质和多极有丝分裂纺锤体增加，这通过碘化丙啶染色以及用α-微管蛋白和γ-微管蛋白抗体进行的免疫荧光染色得以观察到。此外，还发现中心体数量从12.6±6.6%呈剂量依赖性显著增加至67±5.3%，并且多倍体细胞从2.8±1.3%呈剂量依赖性增加至17.6±2.1%，最高吸收剂量为10 Gy。这些紊乱导致细胞进入有丝分裂灾难，并且在照射后5至7天，一部分死亡细胞表现出末端脱氧核苷酸转移酶介导的dUTP缺口末端标记染色所显示的凋亡特征。
2.5至10 Gy的吸收剂量被证明可迫使HeLa Hep2细胞进入有丝分裂灾难并延迟细胞凋亡。这些可能是实体瘤放射免疫治疗后肿瘤生长迟缓所涉及的重要细胞死亡机制。
