Mode of inhibition of DNA replication in neocarzinostatin-treated HeLa cells

Abstract

The effect of antitumor antibiotic neocarzinostatin on DNA replication in HeLa cells was studied by pulse-labeling of DNA with [ 3H]thymidine and sedimentation analysis of the DNA with alkaline sucrose gradients. The drug, which produced DNA damage, primarily inhibited the replicon initiation in the cells at low doses (⩽ 0.1 μg/ml), and at high doses (⩾ 0.5 μg/ml) inhibited the DNA chain elongation. An analysis of the number of single-strand breaks of parental DNA, induced by neocarzinostatin, indicated that inhibition of the initiation occurred with introduction of single-strand breaks of less than 1.5·10 4/cell, while inhibition of the elongation occurred with introduction of single-strand breaks of more than 7.5·10 4/cell. Assuming that the relative molecular mass of DNA/HeLa cell was about 10 13 Da, the target size of DNA for inhibition of replicon initiation was calculated to be about 10 9 Da, such being close to an average size of loop DNA in the cell and for inhibition of chain elongation, 1–2·10 8 Da which was of the same order of magnitude as the size of replicons. Recovery of inhibited DNA replication by neocarzinostatin occurred during post-incubation of the cells and seemed to correlate with the degree of rejoining of the single-strand breaks of parental DNA. Caffeine and theophylline enhanced the recovery of the inhibited replicon initiation, but did not aid in the repair of the breaks in parental DNA.