Detection of DNA strand breaks in HeLa cells in vitro and in mouse sarcoma 180 cells in vivo induced by an alkylating agent, carboquone, using in situ nick translation.
We determined the correlation between DNA strand breaks and the toxicity of carboquone (CQ) in HeLa cells in vitro and in mouse sarcoma 180 (S-180) cells in vivo, using in situ nick translation. The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and [3-H]-labelled dTTP, and sites in the DNA were visualized by autoradiographic observation of grains in the nuclei. These breaks appeared as early as 5 min in the CQ-treated HeLa cells and increased in a dose- and time-dependent manner compared to findings in the control cells, i.e., 10.2-fold at 3 x 10(-6) M in 60 min. Strand breaks in the S-180 cells appeared in a dose- and time-dependent manner, i.e., 6.1-fold after the mice had been exposed to CQ (3.6 mg/kg) for 2 h. This level correlated with the increase in host life span. Our findings show that the survival response of cells decreases, while the level of DNA strand breaks increases following exposure to CQ. The nick translation method is a rapid in situ assay for determining drug-induced DNA damage of tumor cells, under in vitro and in vivo conditions and in a semiquantitative manner.
Maehara
Y
Y
Cancer Center, Kyushu University Hospital, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Anai
H
H
Sakaguchi
Y
Y
Kusumoto
T
T
Emi
Y
Y
Sugimachi
K
K
D000818:Animals; D001345:Autoradiography; D001389:Azirines / Q000494:pharmacology*; D002225:Carbazilquinone / Q000494:pharmacology*; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D008297:Male; D051379:Mice; D012510:Sarcoma 180 / Q000235:genetics; D013942:Thymine Nucleotides / Q000378:metabolism
D001389:Azirines; D013942:Thymine Nucleotides; D002225:Carbazilquinone; C024157:thymidine 5'-triphosphate