Transfection of 9-hydroxyellipticine-resistant Chinese hamster fibroblastswith human topoisomerase II alpha cDNA: Selective restoration of the sensitivity to DNA religation inhibitors
T. Khelifa et al., Transfection of 9-hydroxyellipticine-resistant Chinese hamster fibroblastswith human topoisomerase II alpha cDNA: Selective restoration of the sensitivity to DNA religation inhibitors, CANCER RES, 59(19), 1999, pp. 4927-4936
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 inhibito
rs, the amount of topoisomerase II alpha is 4-5-fold lower than in the pare
ntal DC-3F cells, whereas topoisomerase II beta is undetectable. Cloning an
d sequencing of topoisomerase II alpha cDNAs from DC-3F and DC-3F/9-OH-E ce
lls revealed an allele polymorphism, one allele differing from the other by
the presence of seven silent mutations and three mutations in the noncodin
g region. In addition, the mutated allele contains three missense mutations
located close to the ATP binding site (Thr371Ser) or to the catalytic site
(Ala751Gly; IIe863Thr), To analyze the contribution of these topoisomerase
II alpha alterations to their resistance phenotype, DC-3F/9-OH-E cells wer
e transfected with an eukaryotic expression vector containing the human top
oisomerase II alpha cDNA, In one transfected clone, the amount of topoisome
rase IIa isoform and the catalytic activity were similar to that in the par
ental DC-3F cells. These cells, which contain only topoisomerase II alpha,
are then a unique mammalian cell line to analyze the physiological and phar
macological properties of this enzyme. However, the restoration of a nearly
normal topoisomerase II alpha activity in the DC-3F/9-OH-E cells did not h
ave the same effect on their sensitivity to different enzyme inhibitors; a
75% reversion of the resistance, associated with a 2-3-fold increased stabi
lization of the cleavable complex, was observed with both etoposide and m-A
MSA, two drugs that inhibit the DNA religation step in the enzyme catalytic
cycle; in contrast, the transfected cells remained fully resistant to elli
pticine derivatives that did not induce the stabilization of the cleavable
complex. We hypothesized that a trans-acting factor, inhibiting the inducti
on of cleavable complex formation by drugs that are not religation inhibito
rs, might be present in the resistant cells. However, such a factor was not
detected in in vitro experiments, and other hypotheses are discussed.