Conditional expression of wild-type topoisomerase II complements a mutant enzyme in mammalian cells.
Alterations in the amino acid composition, phosphorylation pattern, or intracellular levels of topoisomerase II have been associated with resistance to antineoplastic agents whose effects are mediated through interactions with this enzyme. To develop a model system with which to investigate the determinants of topoisomerase II sensitivity or resistance to antineoplastic agents that target this enzyme, a cDNA encoding the wild-type Drosophila melanogaster topoisomerase II was ligated into a mammalian expression vector containing a glucocorticoid-inducible mouse mammary tumor virus promoter and transfected into an epipodophyllotoxin-resistant Chinese hamster ovary cell line (VPM(r)-5). In two transfectants carrying an intact, full-length Drosophila topoisomerase II cDNA, exposure to the inducing agent, dexamethasone (10 microM), resulted in complementation of the endogenous mutant topoisomerase II and phenotypic reversion to etoposide sensitivity. In the presence of glucocorticoid, etoposide-induced cytotoxicity increased 20-fold, despite the fact that Drosophila topoisomerase II mRNA expression was only 0.1% of that of the endogenous mammalian topoisomerase II. Induced cells demonstrated a marked increase in DNA single strand breaks compared with uninduced resistant cells, thereby providing biochemical evidence supporting increased DNA strand cleavage due to activation of the Drosophila enzyme. These observations demonstrate the ability of a wild-type Drosophila topoisomerase II to complement a mutant mammalian enzyme and suggest that transfectants capable of conditional topoisomerase II expression represent a useful model for studies of the biochemical pharmacology and structure-function relationships of normal and mutant enzymes.
Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215.
J P
JP
Eder
V T
VT
Chan
E
E
Niemierko
B A
BA
Teicher
L E
LE
Schnipper
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology; D002999:Clone Cells; D006224:Cricetinae; D004247:DNA; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis* / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005047:Etoposide / Q000494:pharmacology*; D007700:Kinetics; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; D005047:Etoposide; D003907:Dexamethasone; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D004250:DNA Topoisomerases, Type II; D002945:Cisplatin