Taki T, Arita N, Hayakawa T, Yamamoto H, Izumoto S, Oonisi T, Mogami H
Department of Neurosurgery, Osaka University, Japan.
No To Shinkei. 1989 Nov;41(11):1065-70.
DNA damaging agents such as nitrosoureas are widely used for the treatment of malignant gliomas. Therefore, quantitative measurement of DNA damages induced by antineoplastic drugs is useful to judge the efficacy of the drug and understand the pharmacological action of the drug. We have utilized in situ nick translation method to demonstrate "nicks" in DNA of glioma cells treated by various antineoplastic agents. Exponentially growing rat 9 L glioma cells (4 x 10(4] were seeded in the chamber slide. After fourty eight hours, the medium was changed to that containing various concentration of the drug (ACNU, cis-DDP, BLM, ADM and VP-16) and the cell was treated for 1 hour. Then, the cell was fixed for 10 minutes in methanol-acetic acid (v/v 3:1). Following fixation, the cell was incubated in the nick translation mixture containing E. coli DNA polymerase I, 3H-TTP, and 4 dNTP's (ATP, GTP, CTP, CTP and TTP) for 10 minutes at room temperature. The slide was dipped in the autoradiographic emulsion, exposed for 4 days at 4 degrees C, and then developed, the number of the silver grains over nuclei was counted under the microscope. For comparison of the effect of the drug to glioma cells, IC50 (inhibitory concentration of the drug for 50% cell kill) of each drug was determined by treating the cell for 48 hours at the various concentration of the drug. Small number of the silver grains was noted in cells with no treatment. Over IC50 as the concentration of the drug increased, the number of the nick increased in cells treated with bleomycin or adriamycin which are known to produce single strand breaks in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
亚硝基脲等DNA损伤剂被广泛用于治疗恶性胶质瘤。因此，定量测量抗肿瘤药物诱导的DNA损伤有助于判断药物疗效并了解其药理作用。我们利用原位缺口平移法来显示经各种抗肿瘤药物处理的胶质瘤细胞DNA中的“缺口”。将指数生长期的大鼠9L胶质瘤细胞（4×10⁴）接种于培养载玻片上。48小时后，更换培养基为含不同浓度药物（ACNU、顺铂、博来霉素、阿霉素和VP - 16）的培养基，细胞处理1小时。然后，细胞在甲醇 - 乙酸（体积比3:1）中固定10分钟。固定后，细胞在含大肠杆菌DNA聚合酶I、³H - TTP和4种脱氧核苷酸（ATP、GTP、CTP、CTP和TTP）的缺口平移混合物中于室温孵育10分钟。将玻片浸入放射自显影乳剂中，在4℃下曝光4天，然后显影，在显微镜下计数细胞核上的银颗粒数量。为比较药物对胶质瘤细胞的作用，通过在不同浓度药物下处理细胞48小时来确定每种药物的IC50（使50%细胞死亡的药物抑制浓度）。未处理的细胞中可见少量银颗粒。超过IC50时，随着药物浓度增加，用已知能在DNA中产生单链断裂的博来霉素或阿霉素处理的细胞中的缺口数量增加。（摘要截断于250字）
