Effects of U-71,184 and Several Other CC-1065 Analogues on Cell Survival and Cell Cycle of Chinese Hamster Ovary Cells

CC-1065 is a very potent antitumor antibiotic which selectively binds in the minor groove of DNA with alkylation at N-3 of adenine. Since therapeutic doses of CC-1065 caused delayed deaths in mice, analogues were synthesized, some of which had significant antitumor activity. The effects of several of these analogues on inhibition of CHO cell survival, cell progression, and their phase-specific toxicity are reported. CC-1065, U-66,664, U-66,819, U-66,694, and U-71,184 all have a left hand segment with an intact cyclopropyl group but have different tail segments. Lethality of these compounds after 2 h drug exposure was in the following order (50% lethal dose in nm in parentheses): CC-1065 (0.06) > U-71,184 (1.3) > U-66,694 (3.2) > U-68,819 (171) > U-66,664 (>1200). In general, these compounds did not inhibit progression from G1 to S but slowed progression through S and blocked cells in G2-M. The phase-specific toxicity of U-71,184 and U-66,694 was different from that of CC-1065. CC-1065 was most cytotoxic to cells in M and early G1 and toxicity decreased as cells entered late G1 and S. In contrast, U-66,694 and U-71,184 were most toxic to cells in late G1. The biochemical and cellular effects of U-71,184 were then studied in detail since it was the most active among these analogues. After a 2-h exposure to 3 ng/ml U-71,184, 90% cell kill or growth inhibition was observed whereas 100 ng/ml was needed for similar inhibition of DNA and RNA synthesis. This discrepancy between the doses suggested that inhibition of nucleic acid synthesis may not be causally related to lethality. Further studies showed that when drug was removed after 2 h exposure, DNA synthesis continued to be inhibited whereas RNA and protein synthesis reached levels higher than the control. Therefore, it is likely that at cytotoxic doses the low level of inhibition of DNA synthesis combined with the stimulation of RNA and protein synthesis leads to unbalanced growth and cell death.