The morpholinyl analogues of
doxorubicin (DOX) have previously been reported to be non-cross-resistant in multidrug resistant (MDR) cells due to a lower affinity for
P-glycoprotein relative to the parent compound. In order to further investigate the mechanisms of action of these morpholinyl
anthracyclines, we examined their ability to cause
DNA single- and double-strand breaks (SSB,
DSB) and their interactions with topoisomerases. Alkaline elution curves were determined after 2-h
drug treatment at 0.5, 2 and 5 microM, while neutral elution was conducted at 5, 10 and 25 microM in a human ovarian cell line, ES-2. A pulse-field gel electrophoresis assay was used to confirm the neutral elution data under the same conditions. Further, K-SDS precipitation and topoisomerase
drug inhibition assays were used to determine the effects of DOX and the morpholinyl analogues on topoisomerase (
Topo) I and II. Under deproteinated elution conditions (pH 12.1), DOX, morpholinyl DOX (MRA), methoxy-morpholinyl DOX (
MMDX) and morpholinyl
oxaunomycin (MX2) were equipotent at causing SSB in the human ovarian
carcinoma cell line, ES-2. However, neutral elution (pH 9.6) under deproteinated conditions revealed marked differences in the degree of
DNA DSB. After 2-h
drug exposures
at 10 microM, DSBs were 3300 rad equivalents for MX2, 1500 for DOX and 400 for both MRA and
MMDX in the ES-2 cell line. Pulse-field data substantiated these differences in DSBs, with breaks easily detected after MX2 and DOX treatment, but not with MRA and
MMDX. DOX and MX2 thus cause
DNA strand breaks selectively through interaction with
Topo II, but not
Topo I. In contrast, MRA and
MMDX cause DNA breaks through interactions with both topoisomerases with a predominant inhibition of
Topo I.