Combined modalities of resistance in etoposide-resistant human KB cell lines

The alkaloid derivative 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene)-beta-D-glucopyranoside (etoposide, VP-16) is believed to exert cytotoxicity by causing double-stranded DNA breaks through interruption of the breaking-resealing reaction of topoisomerase II (topo II). Thus it was conceivable that cells could become resistant to VP-16 by a decrease in topo II enzyme level, since this would lead to fewer DNA breaks. As well, given the structure of VP-16, it was also possible that a pleiotropic mechanism of resistance could decrease sensitivity to this drug. To study these possibilities, a series of VP-16-resistant human KB cell lines was established by stepwise selection. The concentrations of VP-16 required to inhibit cell proliferation by 50% in the parent line and KB/1c, KB/7d, KB/20a, and KB/40a lines were, respectively, 0.16, 4.7, 24, 31, and 47 microM. These cell lines expressed cross-resistance to 4'-(9-acridinylamino)methanesulfon-m-anisidide, doxorubicin, vincristine, and methotrexate, although the pattern of relative drug sensitivity was quite different from that of pleiotropic resistant cell lines reported elsewhere. The resistance to vincristine and methotrexate did not increase above the level of the KB/1c cells, and resistance to VP-16, doxorubicin, and especially vincristine was unstable in VP-16-resistant cells cultured in the absence of drug. Although the drug resistance marker Mr 180,000 glycoprotein could not be detected in any of our cell lines, cellular accumulation of [3H]VP-16 was reduced 50-75% in the resistant lines compared with parent KB. With increasing VP-16 resistance, the level of topo II protein, detected by antibody staining, decreased at each step of selection, concomitant with a general decrease in topo II unknotting activity. Sensitivity of the topo II unknotting assay to inhibition by VP-16 was the same for the parent and all resistant cell lines. The level of topo I activity and enzyme increased slightly in the resistant cells. Thus, these cell lines are resistant to VP-16 by virtue of at least two mechanisms: (a) reduced levels of topo II, which confers cross-resistance to other compounds which are topo II-dependent cytotoxic agents; and (b) reduced accumulation of drug, which is likely also responsible for vincristine and methotrexate resistance. However, the possible existence of other mechanisms of resistance cannot be ruled out.