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Duocarmycin A (DUMA) and DU‐86, a semisynthetic derivative of duocarmycins (DUMs) and a possible active form of KW‐2189, both showed potent cell growth‐inhibitory and cell‐killing activities against human uterine cervix carcinoma HeLa 83 cells. Both drugs showed similar profiles of inhibition of macromolecular synthesis and influence on cell‐cycle distribution. Namely, they inhibited [3H]thymidine uptake at lower concentrations than [3H]uridine or [3H]Ieucine uptake, suggesting that the inhibition of DNA synthesis is the primary site of their actions. Furthermore, they induced the accumulation of cells in early S phase. However, a significant difference was observed between these drugs in terms of DNA‐fragmentation activity against HeLa S3 cells by using two independent methods, pulse‐field gel electrophoresis and alkaline elution. DNA fragmentation was insignificant in the cells treated with DU‐86, in contrast to the cells treated with DUMA. The analysis of DNA adducts in the cells revealed that DU‐86 alkylated adenine quite selectively, while DUMA alkylated both adenine and guanine. These results suggest that the pyrrolidone ring of DUMA is responsible for its adduct formation with guanine and the subsequent DNA‐fragmentation and inhibition of DNA synthesis, while DU‐86 alkylated adenine and inhibited DNA synthesis through mechanisms other than DNA‐fragmentation.

Pulse field gel electrophoresis using a contour-clamped homogeneous electric field was applied for the analysis of DNA-fragmenting activity of antitumor agents towards human uterine cervix carcinoma HeLa S3 cells. Duocarmycins (DUMs), novel antitumor antibiotics with ultrapotent cell growth-inhibitory activities, caused DNA fragmentation at 10 times their IC50 values at 2 h exposure. At 100 times their IC50 values, the size of the smallest fragments was about 245 kilobase pairs (kbp). DUMA, DUMB1 and DUMB2 exhibited similar DNA fragmentation patterns, suggesting similar action mechanisms. DNA fragmentation was also detected in cells treated with radical producers, intercalators and topoisomerase inhibitors. Two bands of about 1800 and 1500 kbp were commonly detected in the cells treated with DUMs and these agents. In addition, fragments of about 900 kbp were detected in the cells treated with a topoisomerase inhibitor, 4'-(9-acridinylamino)methane-sulfon-m-anisidine, and fragments in the broad size range between 700 and 245 kbp in the cells treated with radical producers, bleomycin and neocarzinostatin. DUMs showed a characteristic DNA fragmentation pattern, since both types of fragments induced by the topoisomerase inhibitor and the radical producers were simultaneously detected, suggesting a novel mode of interaction with DNA. DNA-crosslinking agents and mitotic inhibitors did not induce DNA fragmentation under these conditions. The pulse field gel electrophoresis is potentially useful for characterizing DNA-cleaving activity of various antitumor agents at the cellular level.

The anticellular and antitumor activities of novel antitumor antibiotics, duocarmycins (DUMs), were examined against human and murine tumor cells. DUMs consist of live compounds, A, B1, B2, C1, and C2, which possess a pharmacophore similar to that of CC‐1065, a previously isolated antibiotic. Among them, DUMA exhibited ultrapotent growth‐inhibitory activity with an IC50 value of 6 pM against human uterine cervix carcinoma HeLa S3 cells. DUMA and DUMB1 also inhibited the growth of adriamycin (ADM)‐resistant lines of human nasopharynx carcinoma KB cells and breast carcinoma MCF‐7 cells as well as their sensitive lines. DUMs inhibited the growth of s.c.‐inoculated murine tumors such as B16 melanoma, sarcoma 180, M5076 sarcoma and colon 26. DUMs were also significantly effective in increasing the lifespan of i.p.‐inoculated B16 melanoma‐bearing mice, although their effect was marginal against other i.p.‐inoculated tumors. As a whole, DUMB1 exhibited superior activity to the other four compounds. DUMB1 rapidly inhibited the incorporation of [3H]‐TdR into macromolecules of HeLa S3 cells as compared with that of [3H]UR or [3H]leucine. DNA strand breaks were detected in DUMB1 ‐treated HeLa S3 cells by agarose gel electrophoresis with a contour‐clamped homogeneous electric field apparatus. These results indicate that DUMs possess interesting biological activities as DNA‐targeting antitumor antibiotics.