Comparative studies on the action of 7-N-[2-[[2-(γ-l-glutamylamino)ethyl]dithio]ethyl]mitomycin C and of mitomycin C on cultured HL-60 cells and isolated phage and plasmid DNA

Abstract

The mechanism of action of a new mitomycin C (MMC) derivative, KT6149, was studied in human leukemia HL-60 cells and in isolated phage and plasmid DNA, and its effects were compared with those of MMC. Cell growth was markedly inhibited by KT6149, with an IC50 of 2 x 10(-9) M, that for MMC being 2 x 10(-8) M. DNA synthesis of HL-60 cells as determined by incorporation of [3H]-thymidine was also inhibited by KT6149, with an IC50 of 2 x 10(-7) M as compared with 2 x 10(-6) M for MMC. RNA and protein synthesis were less markedly inhibited at low concentrations. Alkaline sucrose density-gradient centrifugation revealed a significant decrease in sedimentation velocity for cellular DNA of the cells after 1 h treatment with KT6149 at concentrations higher than 10(-7) M. In contrast, no such change was observed for DNA of cells treated with MMC, even at a concentration of 10(-5) M. In a cell-free system, analysis by agarose gel electrophoresis patterns showed that the drug induced a decrease in the amount of covalently closed circular DNA of phage PM2 and an increase in that of open circular DNA in the presence of dithiothreitol (DTT), whereas MMC did not cause any change in DNA subfraction amounts. Furthermore, the electrophoretic mobility of linearized pBR322 DNA in alkaline agarose gel was significantly decreased by KT6149 in the presence of DTT and FeSO4, no such change being observed in the case of MMC. The results clearly indicate that the inhibitory effects of KT6149 on the growth and DNA synthesis of HL-60 cells are more potent than those of MMC and that KT6149-induced DNA damage is due to single-strand scission and to cross-linking of DNA, suggesting a mode of activation different from that of MMC.