Zheng Li-Duan, Xiong Zhou-Fang, Zhu Jian-Wen, Wang Ze-Hua
Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Chin Med J (Engl). 2005 Feb 5;118(3):226-30.
The second mitochondria-derived activator of caspases (Smac) is a novel proapoptotic gene, which plays an important role in the apoptosis-inducing effects of irradiation on tumor cells. The purpose of this study was to investigate the effects of extrinsic Smac gene transfer and its over-expression in radiotherapeutic sensitivities of cervical cancer cells.
After the Smac gene was transferred into the cervical cancer cell line HeLa, subcloned cells were obtained by persistent G418 selection. Cellular Smac gene expression was detected by RT-PCR and Western blot, while in vitro cell viabilities were detected by trypan blue staining assay. After treatment with X-ray irradiation, cellular radiotherapeutic sensitivities were investigated by tetrazolium bromide colorimetry. Cellular apoptosis and its rate were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. The expression and activities of cellular caspase-3 were assayed by Western blot and colorimetry.
Smac mRNA and protein levels in HeLa/Smac cells and the selected subclone cell line of cervical cancer were significantly higher than those of HeLa (P < 0.01). There was no significant difference in cellular viabilities between them (P > 0.05). However, after irradiation with 8 Gy X-ray, growth activities of HeLa/Smac were reduced by 22.42% (P < 0.01). When compared with those of HeLa, partial HeLa/Smac cells presented characteristic morphological changes of apoptosis under electronic microscope, with higher apoptosis rates (16.4% vs. 6.2%, P < 0.01); the caspase-3 expression levels in HeLa/Smac cells were improved significantly (P < 0.01), while its activities were increased by 3.42 times (P < 0.01).
Stable transfer of the extrinsic Smac gene and its over-expression in cervical cancer cell line could significantly enhance the expression and activities of cellular caspase-3 and ameliorate apoptosis-inducing effects of irradiation on cancer cells, which was a novel strategy to improve radiotherapeutic effects on cervical cancer.
第二线粒体衍生半胱天冬酶激活剂（Smac）是一种新型促凋亡基因，在辐射诱导肿瘤细胞凋亡效应中起重要作用。本研究旨在探讨外源性Smac基因转染及其过表达对宫颈癌细胞放射治疗敏感性的影响。
将Smac基因转染至宫颈癌细胞系HeLa后，通过持续G418筛选获得亚克隆细胞。采用RT-PCR和蛋白质免疫印迹法检测细胞Smac基因表达，用台盼蓝染色法检测体外细胞活力。经X射线照射处理后，采用四氮唑蓝比色法研究细胞放射治疗敏感性。通过电子显微镜、膜联蛋白V-FITC和碘化丙啶染色流式细胞术测定细胞凋亡及其发生率。采用蛋白质免疫印迹法和比色法检测细胞半胱天冬酶-3的表达和活性。
HeLa/Smac细胞及所选宫颈癌亚克隆细胞系中Smac mRNA和蛋白水平显著高于HeLa细胞（P < 0.01）。两者细胞活力无显著差异（P > 0.05）。然而，经8 Gy X射线照射后，HeLa/Smac细胞的生长活性降低了22.42%（P < 0.01）。与HeLa细胞相比，部分HeLa/Smac细胞在电子显微镜下呈现典型的凋亡形态学变化，凋亡率更高（16.4%对6.2%，P < 0.01）；HeLa/Smac细胞中半胱天冬酶-3表达水平显著提高（P < 0.01），其活性增加了3.42倍（P < 0.01）。
外源性Smac基因在宫颈癌细胞系中的稳定转染及其过表达可显著增强细胞半胱天冬酶-3的表达和活性，改善辐射诱导癌细胞凋亡的效应，这是提高宫颈癌放射治疗效果的一种新策略。
