Takahashi A, Ohnishi K, Ota I, Asakawa I, Tamamoto T, Furusawa Y, Matsumoto H, Ohnishi T
Department of Biology, Nara Medical University, Kashihara, Nara 634-8521, Japan.
Int J Radiat Biol. 2001 Oct;77(10):1043-51. doi: 10.1080/09553000110066095.
To investigate the dependence on p53 gene status of the thermal enhancement of cellular sensitivity against different levels of linear energy transfer (LET) from X-rays or carbon-ion (C-) beams.
Two kinds of human squamous cell carcinoma cell lines were used with an identical genotype except for the p53 gene. SAS/mp53 cells were established by transfection with mutated p53 (mp53) gene to SAS cells having functional wild-type p53 (wtp53). As the control, a neo vector was transfected to the SAS cells (SAS/neo cells). Both cells were exposed to X-rays or accelerated C-beams (30-150 KeV microm(-1)) followed by heating at 44 degrees C. Cellular sensitivity was determined by colony-forming activity. Induction of apoptosis was analysed by Hoechst 33342 staining of apoptotic bodies and agarose-gel electrophoresis for the formation of DNA ladders.
It was found that (1) there was no significant difference in cellular sensitivity between SAS/neo and SAS/mp53 cells to LET radiation of >30 KeV microm(-1), although the radiosensitivity of SAS/neo cells to X-rays was higher (1.2-fold) than that of SAS/mp53 cells; (2) there was an interactive thermal enhancement of radiosensitivity below an LET of 70 KeV microm(-1) in SAS/neo cells, although only additive thermal enhancement was observed in SAS/mp53 cells through all LET levels examined; (3) low-LET radiation induced apoptosis only in SAS/neo cells; (4) high-LET radiation at an isosurvival dose-induced apoptosis of SAS/neo cells at a higher frequency compared with that with low-LET radiation; (5) high-LET radiation-induced p53-independent apoptosis in SAS/mp53 cells; and (6) thermal enhancement of cellular sensitivity to X-rays was due to induction of p53-dependent apoptosis.
The findings suggest that thermal enhancement of radiosensitivity may result from p53-dependent apoptosis induced by inhibition of p53-dependent cell survival system(s) through either regulation of the cell cycle or induction of DNA repair. It is also suggested that the analysis of p53 gene status of cancer cells may predict response to combined therapies with low-LET radiation and hyperthermia.
研究细胞对X射线或碳离子(C-)束不同线性能量转移(LET)水平的辐射热增强敏感性对p53基因状态的依赖性。
使用两种人类鳞状细胞癌细胞系,除p53基因外,其基因型相同。通过将突变型p53(mp53)基因转染到具有功能性野生型p53(wtp53)的SAS细胞中,建立了SAS/mp53细胞。作为对照,将neo载体转染到SAS细胞中(SAS/neo细胞)。两种细胞均接受X射线或加速C束(30-150 KeV·μm⁻¹)照射,随后在44℃加热。通过集落形成活性测定细胞敏感性。通过凋亡小体的Hoechst 33342染色和DNA梯状条带形成的琼脂糖凝胶电泳分析凋亡诱导情况。
发现:(1)对于LET大于30 KeV·μm⁻¹的辐射,SAS/neo和SAS/mp53细胞的细胞敏感性无显著差异,尽管SAS/neo细胞对X射线的放射敏感性比SAS/mp53细胞高(1.2倍);(2)在LET低于70 KeV·μm⁻¹时,SAS/neo细胞的放射敏感性存在热增强的相互作用,尽管在所有检测的LET水平下,SAS/mp53细胞仅观察到热增强的相加作用;(3)低LET辐射仅在SAS/neo细胞中诱导凋亡;(4)在等存活剂量下,高LET辐射诱导SAS/neo细胞凋亡频率高于低LET辐射;(5)高LET辐射在SAS/mp53细胞中诱导p53非依赖性凋亡;(6)细胞对X射线的热增强敏感性是由于p53依赖性凋亡的诱导。
这些发现表明,放射敏感性的热增强可能是由于通过调节细胞周期或诱导DNA修复抑制p53依赖性细胞存活系统而诱导的p53依赖性凋亡。还表明,分析癌细胞的p53基因状态可能预测对低LET辐射和热疗联合治疗的反应。