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Altered Topoisomerase II in a Drug-resistant Small Cell Lung Cancer Cell Line Selected in VP-16¹

Cancer Research Laboratories [S. E. L. M., K. C. A., S. P. C. C.] and Department of Pharmacology and Toxicology [C. D. E., S. P. C. C.], Queen's University, Kingston, Ontario K7L 3N6, Canada, and Department of Cytogenetics, City of Hope National Medical Center, Duarte, California 91010-0269 [M. L. S.]

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 II are reduced in H209/V6 cells compared to the sensitive H209 cells. Furthermore, the topoisomerase II-related protein in H209/V6 cells has an increased electrophoretic mobility, with an apparent M_r of 160,000. The levels of the topoisomerase II 6.1-kilobase mRNA in H209/V6 cells are reduced >10-fold. In addition, a second topoisomerase II-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 IIß 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 II-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 II in H209 cells and the M_r 180,000 topoisomerase IIß in both the resistant and sensitive cells. We conclude that quantitative and qualitative alterations in topoisomerase II have occurred in H209/V6 cells and are likely to contribute to its resistance phenotype.

¹ Supported by Grant 2049 from the National Cancer Institute of Canada (S. P. C. C.). M. L. S. is a member of the City of Hope Cancer Research Center supported by Grant CA33572 from the National Cancer Institute. C. D. E. was supported in part by a Queen's University graduate student award. S. P. C. C. is a Career Scientist of the Ontario Cancer Treatment and Research Foundation.

² To whom requests for reprints should be addressed, at Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6 Canada.

Received 11/30/92. Accepted 8/11/93.

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