Algan O, Stobbe C C, Helt A M, Hanks G E, Chapman J D
Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Radiat Res. 1996 Sep;146(3):267-75.
Radiation-induced apoptosis detected by gel electrophoresis was measured in cells of three human prostate carcinoma cell lines (TSU, PC-3 and DU-145) and compared to their intrinsic radiosensitivities as measured by clonogenic assays. The intrinsic radiosensitivities of each cell line were defined by their alpha and beta coefficients and their surviving fraction at 2 Gy, derived from complete survival curves. The temporal expression and kinetics of radiation-induced apoptosis for DU-145 cells, the human prostate carcinoma cell line which expressed the highest rate of radiation-induced apoptosis, was characterized further by differential sedimentation and the immunofluorescence assay (Apoptag) which was specific for 3'-OH ends in cellular DNA. Cell viability was measured microscopically with trypan blue staining. Cell survival after various doses was computer-fitted to either a simple linear or a linear-quadratic equation. Twenty-four hours after a 10-Gy dose of 137Cs gamma rays, DNA fragmentation to nucleosome multimers was strongly expressed in only DU-145 cells. In this cell line, when centrifugation at 12,000g for 10 min was used to separate fragmented from large molecular weight DNA, the proportion of DNA in the supernatant increased to a maximum of approximately 17% of the total by 10-12 h after radiation treatment. Cell death 24 h after irradiation measured by trypan blue exclusion assays followed single-hit kinetics up to 80 Gy. The proportion of cells which were labeled with Apoptag displayed single-hit kinetics and yielded the same inactivation coefficient as measured by trypan blue. Together, these data indicate that the rapid (24 h) inactivation of irradiated DU-145 cells results from apoptosis and accounts for about 5% of the single-hit killing measured by clonogenic assay. Temporal studies of radiation-induced killing of DU-145 cells distinguished this rapid mechanism of cell death from the major mechanism (72-144 h). These may correlate with apoptosis and proliferative cell death, respectively. Of the three prostate cancer cell lines investigated, only DU-145 cells displayed significant levels of radiation-induced DNA fragmentation and rapid cell death, with characteristics of apoptosis. This mechanism of cell death was complete by 24 h after irradiation and was well separated in time from the death of cells by the major mechanisms which occurred after 72 h, and accounted for about 5% of cell inactivation by a single-hit mechanism.
通过凝胶电泳检测辐射诱导的凋亡，在三种人前列腺癌细胞系（TSU、PC - 3和DU - 145）的细胞中进行测量，并与通过克隆形成试验测量的它们的内在放射敏感性进行比较。每个细胞系的内在放射敏感性由其α和β系数以及在2 Gy时的存活分数定义，这些数据来自完整的存活曲线。DU - 145细胞是辐射诱导凋亡率最高的人前列腺癌细胞系，通过差异沉降和针对细胞DNA中3'-OH末端的免疫荧光测定法（Apoptag）进一步表征其辐射诱导凋亡的时间表达和动力学。用台盼蓝染色在显微镜下测量细胞活力。将不同剂量后的细胞存活情况用计算机拟合到简单线性或线性二次方程。在10 Gy的137Csγ射线照射24小时后，DNA片段化形成核小体多聚体仅在DU - 145细胞中强烈表达。在该细胞系中，当以12,000g离心10分钟用于分离片段化DNA与大分子质量DNA时，辐射处理后10 - 12小时，上清液中DNA的比例增加到最大约占总量的17%。通过台盼蓝排斥试验测量，照射后24小时的细胞死亡遵循单靶动力学直至80 Gy。用Apoptag标记的细胞比例显示单靶动力学，并产生与台盼蓝测量相同的失活系数。总之，这些数据表明，照射后DU - 145细胞的快速（24小时）失活是由凋亡引起的，约占克隆形成试验测量的单靶杀伤的5%。对DU - 145细胞辐射诱导杀伤的时间研究区分了这种快速细胞死亡机制与主要机制（72 - 144小时）。这些机制可能分别与凋亡和增殖性细胞死亡相关。在所研究的三种前列腺癌细胞系中，只有DU - 145细胞表现出显著水平的辐射诱导DNA片段化和快速细胞死亡，具有凋亡特征。这种细胞死亡机制在照射后24小时完成，并且在时间上与72小时后发生的主要机制导致的细胞死亡很好地分开，并且占单靶机制导致细胞失活的约5%。
