[In situ nick translation for detection of DNA damages in glioma cells].
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)
Taki
T
T
Department of Neurosurgery, Osaka University, Japan.
Arita
N
N
Hayakawa
T
T
Yamamoto
H
H
Izumoto
S
S
Oonisi
T
T
Mogami
H
H
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001345:Autoradiography; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000187:drug effects* / Q000235:genetics; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D005910:Glioma / Q000235:genetics*; D014176:Protein Biosynthesis; D051381:Rats; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000970:Antineoplastic Agents; D004273:DNA, Neoplasm