Lin X, Gately D P, Hom D, Mishima M, Los G, Howell S B
Department of Medicine and the Cancer Center, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0058, USA.
Int J Cancer. 2001 Feb 15;91(4):555-62. doi: 10.1002/1097-0215(200002)9999:9999<::aid-ijc1083>3.0.co;2-q.
The GADD153 gene is strongly transcriptionally activated by many types of cellular injury and the magnitude of the change in GADD153 expression is proportional to the extent of damage. We developed a novel reporter system in which a chimeric gene containing the GADD153 promoter linked to the coding region of an enhanced green fluorescent protein (EGFP) gene was stably integrated into the genome of a clone of UMSCC10b head and neck carcinoma cells. Activation of the exogenous GADD153 promoter was quantified using flow cytometric measurement of EGFP expression following drug exposure. The exogenous GADD153 promoter in this clone was activated by N-methl-N'-nitro-N-nitrosoguanidine (MNNG) in a concentration-dependent manner with kinetics that closely paralleled perturbation of cell cycle phase distribution. EGFP expression was strongly activated by a variety of genotoxic agents including DNA cross-linking and methylating agents, oxygen free radicals, DNA intercalator, UV and gamma-radiation and hypoxia. When grown as a xenograft in nude mice, the stably transfected clone also demonstrated dose-dependent EGFP expression when measured 4 days after cisplatin treatment. The reporter system accurately categorized the relative potency of adducts produced by 6 related platinum-containing drugs. In conclusion, this reporter system can facilitate in vitro and in vivo screening for agents capable of producing cytotoxicity via a wide variety of different mechanisms, and can be utilized to investigate the relative potency of structurally related DNA adducts.
GADD153基因可被多种类型的细胞损伤强烈转录激活，且GADD153表达变化的幅度与损伤程度成正比。我们开发了一种新型报告系统，其中一个包含与增强型绿色荧光蛋白（EGFP）基因编码区相连的GADD153启动子的嵌合基因被稳定整合到UMSCC10b头颈癌细胞克隆的基因组中。在药物暴露后，通过流式细胞术测量EGFP表达来定量外源性GADD153启动子的激活情况。该克隆中的外源性GADD153启动子被N-甲基-N'-硝基-N-亚硝基胍（MNNG）以浓度依赖的方式激活，其动力学与细胞周期阶段分布的扰动密切平行。EGFP表达被多种遗传毒性剂强烈激活，包括DNA交联剂和甲基化剂、氧自由基、DNA嵌入剂、紫外线和γ射线以及缺氧。当作为异种移植物在裸鼠中生长时，在顺铂治疗4天后测量，稳定转染的克隆也显示出剂量依赖性的EGFP表达。该报告系统准确地对6种相关含铂药物产生的加合物的相对效力进行了分类。总之，该报告系统可促进体外和体内筛选能够通过多种不同机制产生细胞毒性的药物，并可用于研究结构相关DNA加合物的相对效力。
