title: Paradoxical increase in DNA cross-linking in a human ovarian carcinoma cell line resistant to cyanomorpholino doxorubicin.
abstract: 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.
authors.affiliation: Department of Medicine, Stanford University School of Medicine, California 94305-5306.
authors.forename: D H
authors.initials: DH
authors.lastname: Lau
authors.forename: K L
authors.initials: KL
authors.lastname: Ross
authors.forename: B I
authors.initials: BI
authors.lastname: Sikic
mesh_terms: D003432:Cross-Linking Reagents / Q000378:metabolism*; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000032:analysis; D004273:DNA, Neoplasm / Q000378:metabolism*; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000378:metabolism; D004351:Drug Resistance; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D004317:Doxorubicin; C038555:3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin; D004250:DNA Topoisomerases, Type II