培養glioma細胞におけるDNA損傷の判定—In situ nick translation法による

IN SITU NICK TRANSLATION FOR DETECTION OF DNA DAMAGES IN GLIOMA CELLS

　抄録　In situ nick translation法を用いて，培養glioma細胞における抗癌剤によるDNA損傷の判定を試みた。すなわち，ラット9L glioma cellを各種抗癌剤で1時間治療後，細胞核DNAに生じた切れ目"nick"を，アイソトープでラベルしたヌクレオシド（³H-TTP）を用いたmicroautoradiography法で観察した。DNAにsingle strand breakを生じるとされるbleomycin治療群では，細胞増殖抑制効果を示す程度以上の薬剤濃度で，濃度依存性にgrain数の増加を認めた。ADM, Cis-DDP治療群でも，同様の傾向を認めた。しかし，抗腫瘍効果の機構がDNA鎖間のcross linking形成によるとされるACNU，あるいはDNA topoisomerase II　阻害によるとされるVP−16では，低濃度でもgrainが出現し，grain数は必ずしも薬剤濃度に比例しなかった。本法は，約1週間という短期間で，抗癌剤によるDNA損傷をin situにおいて定量的に判定できるという利点を持つ。また本法は，培養細胞のみならず臨床組織切片を用いて行うこともできるため，抗癌剤の感受性，薬剤の作用機序の評価など，応用範囲が広いと考えられる。

DNA damaging agents such as nitrosoureas are widely used for the treatment of malignant glio-mas. 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 demon-strate "nicks" in DNA of glioma cells treated by various antineoplastic agents. Exponentially gro-wing rat 9L glioma cells (4x10⁴) 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 mix-ture containing E. coli DNA polymerase I, 3H-TTP, and 4 dNTP's (ATP, GTP, CTP, CTP and TTP) for 10 minutes at room temparature. The slide was dipped in the autoradiographic emulsion, exposed for 4 days at 4°C, and then developed, the number of the silver grains over nuclei was coun-ted 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 concen-tration 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. However, no such correla-tion between the number of nicks and drug concen-tration was observed in cells treated by either ACNU or VP-16. These results demonstrated that the DNA damage in cells produced by antineopla-stic agents is quantitatively evaluated by in situ nick translation method. Furthermore, the in situ pharmacological action of these agents is able to be assessed in the cells. Evidence obtained would indi-cate a possible clinical application of the present method to drug sensitivity test for gliomas.