Department of Virology and Preventive Medicine, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Int J Radiat Oncol Biol Phys. 2010 Jan 1;76(1):229-41. doi: 10.1016/j.ijrobp.2009.08.054.
We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions).
A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated beta-galactosidase (SA-beta-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependent kinase inhibitor p21(WAF1/CIP1) in the irradiated cells was analyzed by Western blotting.
When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells (<1% of initially irradiated cells) could not form a colony: however, they showed a morphological phenotype consistent with cellular senescence, that is, enlarged and flattened appearance. The senescent nature of these attached cells was further indicated by staining for SA-beta-gal. The mean telomere length was not changed after irradiation with C-ions. Phosphorylation of p53 at serine 15 as well as the expression of p21(WAF1/CIP1) was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53.
Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.
我们研究了碳离子束（C-ions）照射对人神经胶质瘤细胞的生物学反应。
用 C-ions 照射人神经胶质瘤来源的细胞系 NP-2。用 Hoechst 33342 染色凋亡的细胞核。通过用单丹磺酰尸胺（MDC）染色或通过 Western blot 检测微管相关蛋白轻链 3（MAP-LC3）（LC3-I）转化为膜结合形式（LC3-II）来检测自噬的诱导。通过检测衰老相关β-半乳糖苷酶（SA-β-gal）的诱导来检测细胞衰老的标志物。通过 Southern blot 杂交测定照射细胞的平均端粒长度。通过 Western blot 分析照射细胞中肿瘤抑制因子 p53 和细胞周期/细胞周期依赖性激酶抑制剂 p21（WAF1/CIP1）的表达。
当 NP-2 细胞用 C-ions 照射 6 Gy 时，大多数细胞死于凋亡和自噬。附着细胞的剩余部分（初始照射细胞的<1%）不能形成菌落：然而，它们表现出与细胞衰老一致的形态表型，即增大和平坦的外观。这些附着细胞的衰老性质进一步通过 SA-β-gal 染色得到证实。用 C-ions 照射后，端粒长度没有改变。NP-2 细胞照射后，p53 丝氨酸 15 磷酸化以及 p21（WAF1/CIP1）的表达被诱导。此外，我们发现 C-ions 照射诱导缺乏功能性 p53 的人神经胶质瘤细胞的细胞衰老。
C-ions 照射诱导人神经胶质瘤细胞凋亡、自噬和细胞衰老。
