ALTERED SUBCELLULAR-DISTRIBUTION OF TOPOISOMERASE-II-ALPHA IN A DRUG-RESISTANT HUMAN SMALL-CELL LUNG-CANCER CELL-LINE

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-beta-D-glucopyranosyl)-4'-demethylepipodophyllotoxin (VP-16) from pat-ental 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(12-[(2-hydroxyethyl)aminolethyl}amino)-9,10-anthracenedione. These cells do not overexpress P-glycoprotein or the multidrug resistance-associated protein. Immunoblotting demonstrates that H209 cells contain the M(r) 170,000 isoform of topoisomerase II (topo II), while H209/V6 cells have a M(r) 160,000 enzyme but none of the M(r) 170,000 isoform. The cell lines have equal amounts of topo IIbeta. The H209/V6 cells have a 5-fold decrease in total immunoreactive topo IIalpha. 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 IIalpha in the cells. The M(r) 160,000 isoform from the H209/V6 cell line and the M(r) 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 M(r) 160,000 enzyme was reduced compared to the M(r) 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 M(r) 160,000 topo IIalpha-related enzyme is primarily localized in the cytoplasm, while the M(r) 170,000 topo IIalpha enzyme and topo IIbeta are located in the nucleus. These data imply that the deleted sequence in the M(r) 160,000 enzyme is not necessary for catalytic activity but is required to facilitate nuclear localization.