Comparative effects of adriamycin and DNA-non-binding analogues on DNA, RNA, and protein synthesis in vitro

Abstract

Drug-DNA binding is claimed to be the basis by which the antitumor antibiotic adriamycin (doxorubicin) inhibits DNA and RNA synthesis in vitro. However, in preliminary studies the DNA-non-binding adriamycin analogue N-trifluoroacetyladriamycin-14-valerate (AD 32) showed somewhat greater inhibition of DNA and RNA synthesis than adriamycin under identical conditions. The kinetics of macromolecule synthesis inhibition induced by adriamycin and AD 32, and the two principal DNA-non-binding metabolites of AD 32, N-trifluoroacetyladriamycin (AD 41) and N-trifluoroacetyladriamycinol (AD 92), have now been subjected to comparative study in cultured CEM (human leukemic lymphoblastic) cells. At equimolar concentrations (10 microM), or at concentrations related to their 50% growth-inhibitory values vs CEM cells, AD 32 was consistently found to be more inhibitory than adriamycin of DNA and RNA synthesis, as measured by the incorporation of tritiated thymidine and uridine, respectively, into acid-precipitable fractions relative to untreated controls. Marked inhibitory activity was apparent with 10 microM AD 32 even at the earliest sampling time (15 min); with adriamycin at the same concentration the maximal effect was not achieved until 3 h. AD 32 at 4.8 microM concentration continued to show strong inhibition of nucleic acid synthesis, whereas adriamycin at 1.0 microM was essentially inactive. Like AD 32, AD 41 and AD 92 showed greater inhibition than adriamycin of DNA and RNA synthesis at the early sampling times, although in all instances the effects of AD 32 were more profound. AD 32 at 10 microM concentration produced a moderate but significant inhibition of the incorporation of tritiated methionine into protein compared with adriamycin, which at this concentration was not active. Parallel HPLC analytical studies with similar drug-treated cultures indicated that, while small amounts of adriamycin were found in cells treated with 10 microM AD 32, the amount of adriamycin present at 15 min was only a small fraction (less than 5%) of the amount of adriamycin achieved at 3 h in cultures treated with 1.0 microM adriamycin, a concentration already shown to be only slightly inhibitory of nucleic acid synthesis under the culture conditions. The present study thus confirms the marked DNA and RNA synthesis-inhibitory effects of AD 32, and establishes that this inhibitory activity is not due to conversion of AD 32 into adriamycin. These findings accordingly call into question the validity of the drug-DNA binding mechanism as the explanation for the nucleic acid synthesis inhibitory effects seen with ADR.