Altered Topoisomerase IIα in a Drug-resistant Small Cell Lung Cancer Cell Line Selected in VP-16

Abstract

The H209/V6 cell line was derived from the H209 small cell lung cancer cell line by selection in etoposide (VP-16). Cytogenetic analysis indicates that the sensitive and resistant cell lines share 20 marker chromosomes and thus are clearly related. However, the H209/V6 cell line has four additional structurally altered chromosomes and a 2 N-modal chromosome number, while the H209 cell line is hypotetraploid (4 N-). H209/V6 cells are cross-resistant to some drugs that interact with topoisomerase II but not mitoxantrone. H209/V6 cells are also not cross-resistant to vincristine, trimetrexate, or cisplatin. The rates of VP-16 efflux are the same in the resistant and sensitive cell lines, which is consistent with the observation that P-glycoprotein mRNA is not detectable in either cell line. Fewer VP-16-induced DNA-protein complexes are observed in H209/V6 cells, and immunoblot analysis shows that levels of topoisomerase IIα are reduced in H209/V6 cells compared to the sensitive H209 cells. Furthermore, the topoisomerase IIα-related protein in H209/V6 cells has an increased electrophoretic mobility, with an apparent M_r of 160,000. The levels of the topoisomerase IIα 6.1-kilobase mRNA in H209/V6 cells are reduced >10-fold. In addition, a second topoisomerase IIα-related mRNA of approximately 4.8 kilobases is observed in H209/V6 cells but not in H209 cells. The quantity and electrophoretic mobility of the M_r 180,000 topoisomerase IIβ protein and its 6.1-kilobase mRNA are the same in the sensitive and resistant cell lines. The topoisomerase II strand-passing activity in H209/V6 nuclear extracts is reduced about 2-fold, but this activity is not more resistant to inhibition by VP-16 than the activity in H209 cells. However, band depletion immunoblot experiments show that the topoisomerase IIα-related M_r 160,000 protein in H209/V6 cells is not bound to DNA in the presence of concentrations of VP-16 that deplete the M_r 170,000 topoisomerase IIα in H209 cells and the M_r 180,000 topoisomerase IIβ in both the resistant and sensitive cells. We conclude that quantitative and qualitative alterations in topoisomerase IIα have occurred in H209/V6 cells and are likely to contribute to its resistance phenotype.