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Transfection of 9-Hydroxyellipticine-resistant Chinese Hamster Fibroblasts with Human Topoisomerase II cDNA

Selective Restoration of the Sensitivity to DNA Religation Inhibitors¹

UMR 8532 Centre National de la Recherche Scientifique, Physicochimie et Pharmacologie des Macromolécules Biologiques, Institut Gustave Roussy, 94805 Villejuif Cedex [T. K., B. R., S. L., B. L., J-M. S., J. M., A. J-S.], and UPR 9044 Centre National de la Recherche Scientifique, Génétique Moléculaire et Intégration des Fonctions Cellulaires, BP 8, 94801 Villejuif Cedex [H. J-S.], France

In the Chinese hamster lung cell line DC-3F/9-OH-E, selected for resistance to 9-OH-ellipticine and cross-resistant to other topoisomerase II inhibitors, the amount of topoisomerase II is 4–5-fold lower than in the parental DC-3F cells, whereas topoisomerase IIß is undetectable. Cloning and sequencing of topoisomerase II cDNAs from DC-3F and DC-3F/9-OH-E cells revealed an allele polymorphism, one allele differing from the other by the presence of seven silent mutations and three mutations in the noncoding region. In addition, the mutated allele contains three missense mutations located close to the ATP binding site (Thr371Ser) or to the catalytic site (Ala751Gly; Ile863Thr). To analyze the contribution of these topoisomerase II alterations to their resistance phenotype, DC-3F/9-OH-E cells were transfected with an eukaryotic expression vector containing the human topoisomerase II cDNA. In one transfected clone, the amount of topoisomerase II isoform and the catalytic activity were similar to that in the parental DC-3F cells. These cells, which contain only topoisomerase II, are then a unique mammalian cell line to analyze the physiological and pharmacological properties of this enzyme. However, the restoration of a nearly normal topoisomerase II activity in the DC-3F/9-OH-E cells did not have the same effect on their sensitivity to different enzyme inhibitors; a 75% reversion of the resistance, associated with a 2–3-fold increased stabilization of the cleavable complex, was observed with both etoposide and m-AMSA, two drugs that inhibit the DNA religation step in the enzyme catalytic cycle; in contrast, the transfected cells remained fully resistant to ellipticine derivatives that did not induce the stabilization of the cleavable complex. We hypothesized that a trans-acting factor, inhibiting the induction of cleavable complex formation by drugs that are not religation inhibitors, might be present in the resistant cells. However, such a factor was not detected in in vitro experiments, and other hypotheses are discussed.

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