The cyanomorpholino analog of
doxorubicin (
MRA-CN) is a potent
cytotoxic agent which is known to cross-link
DNA. A human ovarian
carcinoma cell line, ES-2, was grown in increasing concentrations of
MRA-CN from 0.1 to 0.5 nM. The resultant resistant subline, ES-2R, was 4-fold resistant to
MRA-CN. DNA damage and repair in response to
MRA-CN were compared in the parental and resistant cell lines using alkaline elution.
DNA cross-links were detectable after 3-h incubation of the cells at 37 degrees C in
MRA-CN at concentrations greater than or equal to 1.0 nM. Paradoxically, 2-fold more cross-links were detected in the ES-2R cells as compared with the ES-2 cells. This paradoxical difference in cross-links between the 2 cell lines was observed to increase with time of exposure to 2.5 nM of
MRA-CN. Non-
protein-associated
DNA strand breaks were also detected in the 2 cell lines after exposure to 2.5 nM of the
drug. The ES-2 cells consistently showed twice as many breaks as the ES-2R cells, which could explain the paradoxical higher apparent
DNA cross-linking observed with the ES-2R cells after exposure to
MRA-CN. Studies of the time course of cross-link repair after exposure to
MRA-CN revealed that 75% of the
DNA cross-links disappeared in the ES-2R cells by the end of 8 h in
drug-free medium. In contrast, cross-links in the ES-2 cells were undetectable after 4 h, which coincided with a progressive increase in
DNA strand breaks. The
topoisomerase II level in the ES-2 cells was 2- to 4-fold higher than that in the ES-2R cells. However,
proteinase K treatment of the lysed cells did not increase the number of apparent strand breaks produced by
MRA-CN, suggesting that
topoisomerase II may not be involved. These findings indicate that, in addition to
DNA cross-linking,
MRA-CN causes
DNA strand breakage. Resistance to
MRA-CN in the ES-2R cells is associated with more apparent
DNA cross-linking and less
DNA strand breakage, which may be a consequence of differences in DNA repair and/or nonspecific
DNA degradation between the resistant and the sensitive cell lines.