Transfection of 9-hydroxyellipticine-resistant Chinese hamster fibroblasts with human topoisomerase IIα cDNA : Selective restoration of the sensitivity to DNA religation inhibitors

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 IIα is 4–5-fold lower than in the parental DC-3F cells, whereas topoisomerase IIβ is undetectable. Cloning and sequencing of topoisomerase IIα 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 IIα alterations to their resistance phenotype, DC-3F/9-OH-E cells were transfected with an eukaryotic expression vector containing the human topoisomerase IIα cDNA. In one transfected clone, the amount of topoisomerase IIα isoform and the catalytic activity were similar to that in the parental DC-3F cells. These cells, which contain only topoisomerase IIα, 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 IIα 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.