The cyanomorpholino derivative of
doxorubicin (
MRA-CN) is
a DNA intercalator and
alkylator that is a highly potent
cytotoxin, non-cross-resistant in multidrug-resistant cells, and noncardiotoxic in comparison with
doxorubicin. To further examine mechanisms of action and resistance to
MRA-CN, a cell line resistant to
MRA-CN, ES-2R, was established by growing a human ovarian
carcinoma cell line, ES-2, in increasing concentrations of the
drug. The resistant subline was 4-fold resistant to
MRA-CN and cross-resistant to other
DNA cross-linking agents,
cisplatin (7-fold) and
carmustine (3-fold), as well as to the
DNA strand-breaking agents
etoposide (6-fold),
doxorubicin (2-fold),
bleomycin (5-fold), and ionizing radiation (2-fold). In contrast, ES-2R cells were not cross-resistant to
vinblastine. Several months of additional growth of ES-2R cells in
MRA-CN did not yield higher, stable levels of drug resistance. A low level of
P-glycoprotein was detectable in the ES-2R cells. However, the extent of intracellular accumulation of [3H]
MRA-CN by this resistant cell line was identical to that of the sensitive line. The number of
DNA cross-links formed by
cisplatin in ES-2R was only 50% of that of the ES-2 cells and was associated with a 50% increase in the rate of repair of these cross-links in the resistant cells. Ionizing radiation induced similar amounts of single- and double-strand breaks in the ES-2 line as well as in the ES-2R cells. There was no apparent difference between the two cell lines in the rate and extent of repair of these DNA breaks. Thus, enhanced DNA repair cannot explain the phenomenon of cross-resistance to radiation. Comparisons of
glutathione (GSH) content and the
enzymes involved in GSH homeostasis showed significant differences. Resistant cells contained 1.5-fold more GSH, a 2.2-fold increase in gamma-glutamyltranspeptidase activity, and a 2.4-fold increase in GSH
reductase compared with ES-2 cells (all P less than 0.05). Total
glutathione-S-transferase (GST) activity was 2.6-fold higher (P less than 0.01) in the ES-2R line. The pi-class GST subunit by Western blotting and GST activity toward
ethacrynic acid were increased 2-fold in the resistant cells. Depletion of GSH levels in ES-2R cells by
buthionine sulfoximine restored the sensitivity of ES-2R to
MRA-CN. These findings implicate a role for GSH metabolism in the resistance phenotype of ES-2R cells. We have previously reported that these cells have an increased generation time and decreased
topoisomerase II content. Thus, the ES-2R cell line exhibits a complex phenotype of broad cross-resistance, which is likely to involve multiple mechanisms, and includes enhanced DNA repair and increased GSH content and GST activity.