Jiang Yan Lin, Escaño Michael F T, Sasaki Ryohei, Fujii Shigeki, Kusuhara Sentaro, Matsumoto Akira, Sugimura Kazuro, Negi Akira
Department of Organ Therapeutics, Kobe University Graduate School of Medicine, Kobe, Japan.
Jpn J Ophthalmol. 2004 Mar-Apr;48(2):106-14. doi: 10.1007/s10384-003-0043-x.
To investigate the molecular mechanisms for cell growth inhibition or apoptosis in human retinal pigment epithelium (RPE) cells after ionizing radiation.
Cell survival studies, a TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay, and a caspase-3 immunocytochemical analysis were performed on irradiated ARPE-19 cell cultures at different time periods. Transcriptional levels of p53, p21, Bax, Fas/Fas-L, vascular endothelial growth factor ( VEGF), and pigment epithelium-derived growth factor ( PEDF) were evaluated by semiquantitative reverse transcriptional polymerase chain reaction. Mutations in the p53 gene were analyzed by DNA sequencing. Protein levels of p53, VEGF, and PEDF were evaluated by Western blot.
Cell viability was inversely related to radiation dose. TUNEL-positive cells were detected 6 h after radiation exposure. Caspase-3 immunocytochemical analysis revealed increased immunoreactivity in the TUNEL-positive cells. Levels of p53, p21, and Bax mRNA were greatest at the 2-h postradiation period. VEGF and PEDF mRNA and protein levels were constant. Protein levels of p53 were increased at the 4- and 6-h postradiation period.
Ionizing radiation induces apoptosis in normal proliferating RPE cells through p53 activation, without affecting expression of VEGF or PEDF. We documented a molecular basis for explaining the decrease in effectiveness of radiation therapy, particularly for age-related macular degeneration. In the clinical setting, selection of appropriate radiation therapy methods and the doses for specific diseases need careful evaluation.
研究电离辐射后人类视网膜色素上皮（RPE）细胞生长抑制或凋亡的分子机制。
对不同时间段照射后的ARPE - 19细胞培养物进行细胞存活研究、TdT介导的dUTP生物素缺口末端标记（TUNEL）检测及半胱天冬酶 - 3免疫细胞化学分析。通过半定量逆转录聚合酶链反应评估p53、p21、Bax、Fas/Fas - L、血管内皮生长因子（VEGF）和色素上皮衍生生长因子（PEDF）的转录水平。通过DNA测序分析p53基因的突变。通过蛋白质印迹法评估p53、VEGF和PEDF的蛋白质水平。
细胞活力与辐射剂量呈负相关。辐射暴露6小时后检测到TUNEL阳性细胞。半胱天冬酶 - 3免疫细胞化学分析显示TUNEL阳性细胞中的免疫反应性增加。p53、p21和Bax mRNA水平在辐射后2小时最高。VEGF和PEDF mRNA及蛋白质水平保持恒定。p53蛋白质水平在辐射后4小时和6小时升高。
电离辐射通过激活p53诱导正常增殖的RPE细胞凋亡，而不影响VEGF或PEDF的表达。我们记录了一个分子基础来解释放射治疗效果的下降，特别是对于年龄相关性黄斑变性。在临床环境中，选择合适的放射治疗方法和针对特定疾病的剂量需要仔细评估。
