The potential mechanisms of the extremely potent anthracycline analogue 3′-deamino-3′-(3-cyano-4-morpholinyl)doxorubicin (MRA-CN) have been compared with those of doxorubicin (DOX) by examination of drug effects on colony formation, macromolecular synthesis, DNA integrity, and ultrastructure of human leukemia cells in vitro. Following a 1-h exposure, MRA-CN was found to be 1400-fold more cytocidal than DOX which correlated with the drugs' inhibitory effects on DNA and total RNA synthesis. Treatment with MRA-CN resulted in a dosedependent production of DNA interstrand cross-links as quantified by alkaline elution. One-h treatments with DOX or 3′-deamino-3′-(4-morpholinyl)doxorubicin (the non-cyano-containing analogue of MRA-CN) produced no DNA-DNA cross-links; rather they produced protein-concealed DNA single-strand breaks. After removal of MRA-CN, the DNA of KBM-3 cells displayed time-dependent fragmentation as indicated by rapid DNA filter elution during the pH 10 lysis step which preceded pH 12 elution. Within 4 h of MRA-CN exposure (10 nm, 1 h), 50% of the cellular DNA was in the lysis fraction. By 24 h, all the cellular DNA was in this fraction. MRA-CN (10 nm), 3′-deamino-3′-(4-morpholinyl)doxorubicin (1 µm), and actinomycin D (1 µm), but not DOX (3 µm), each produced distinctive nucleolar macrosegregation, indicating an effect on rRNA synthesis. The α-CN substituent on the morpholinyl moiety of MRA-CN appears to be responsible for the unique antitumor potency of this anthracycline. Nucleolar macrosegregation is probably associated with the morpholinyl moiety and is independent of the α-CN substituent.


Supported in part by a National Cancer Institute Career Development Award (K04 CA 01135) to R. A. N.

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