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, sele cted in the presence of increasing concentrations of ta-D-glucopyranos yl)-4'-demethylepipodophyllotoxin (VP-16) from pat-ental H209 cells, i s 22-, 9-, and 4-fold resistant to VP-16, 4'-(9-acridinylamino) methan esulfon-m-anisidide, and doxorubicin, respectively, but not cross-resi stant to roxyethyl)aminolethyl}amino)-9,10-anthracenedione. These cell s do not overexpress P-glycoprotein or the multidrug resistance-associ ated protein. Immunoblotting demonstrates that H209 cells contain the M(r) 170,000 isoform of topoisomerase II (topo II), while H209/V6 cell s 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-resi stant 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 th e 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 t he enzymatic activity of the 2 isoforms could be evaluated. The cataly tic 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 nuc lear extracts, the differences in enzymatic activity of the purified e nzymes are considerably less than the level of drug resistance. Invest igations of the subcellular localization of, topo II by immunocytochem ical techniques and cytoplasm/nuclear fractionation studies demonstrat ed that the M(r) 160,000 topo IIalpha-related enzyme is primarily loca lized 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 del eted sequence in the M(r) 160,000 enzyme is not necessary for catalyti c activity but is required to facilitate nuclear localization.