A drug-resistant human small cell lung cancer cell line, H209/V6, selected in the presence of increasing concentrations of 9-(4,6-O-ethylidene-β-d-glucopyranosyl)-4′-demethylepipodophyllotoxin (VP-16) from parental H209 cells, is 22-, 9-, and 4-fold resistant to VP-16, 4′-(9-acridinylamino)methanesulfon-m-anisidide, and doxorubicin, respectively, but not cross-resistant to 1,4-dihydroxy-5,8-bis({2-[(2-hydroxyethyl)amino]ethyl}-amino)-9,10-anthracenedione. These cells do not overexpress P-glycoprotein or the multidrug resistance-associated protein. Immunoblotting demonstrates that H209 cells contain the Mr 170,000 isoform of topoisomerase II (topo II), while H209/V6 cells have a Mr 160,000 enzyme but none of the Mr 170,000 isoform. The cell lines have equal amounts of topo IIβ. The H209/V6 cells have a 5-fold decrease in total immunoreactive topo IIα. The catalytic and VP-16-induced DNA cleavage activities of the topo II present in 0.35 m NaCl nuclear extracts are decreased 2- to 3-fold in the drug-resistant cell line. This decrease in enzymatic activity is not consistent with either the 22-fold VP-16 resistance of the H209/V6 cell line or the approximately 5-fold decrease in immunoreactive topo IIα in the cells. The Mr 160,000 isoform from the H209/V6 cell line and the Mr 170,000 enzyme from the parental cell line were purified so that the enzymatic activity of the 2 isoforms could be evaluated. The catalytic activities of the purified isoforms were found to be very similar. The drug-induced DNA cleavage activity of the Mr 160,000 enzyme was reduced compared to the Mr 170,000 enzyme. However, as with the nuclear extracts, the differences in enzymatic activity of the purified enzymes are considerably less than the level of drug resistance. Investigations of the subcellular localization of topo II by immunocytochemical techniques and cytoplasm/nuclear fractionation studies demonstrated that the Mr 160,000 topo IIα-related enzyme is primarily localized in the cytoplasm, while the Mr 170,000 topo IIα enzyme and topo IIβ are located in the nucleus. These data imply that the deleted sequence in the Mr 160,000 enzyme is not necessary for catalytic activity but is required to facilitate nuclear localization.


This work was supported by National Institutes of Health Grant CA01124 and James Graham Brown Cancer Center Funds.

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