Reduction of drug accumulation and DNA topoisomerase II activity in acquired teniposide-resistant human cancer KB cell lines

Abstract

We have isolated stable teniposide (VM26)-resistant cell lines from human cancer KB cells by stepwise exposure to increasing doses of the drug. At each step, we have purified VM26-resistant cell lines. KB/VM-a, KB/VM-b, KB/VM-1, KB/VM-2, KB/VM-3, and KB/VM-4 showed 3-, 6-, 12-, 16-, 74-, and 95-fold higher resistance to VM26 than did KB. We have further characterized KB/VM-2 and KB/VM-4 which showed about 15- and 100-fold higher resistance to VM26 or etoposide (VP16) than did KB. Both VM26-resistant cell lines showed 4- to 11-fold higher relative resistance to daunomycin and Adriamycin than did KB. Steady-state levels of the cellular accumulation of radioactive VP16 in KB/VM-2 and KB/VM-4 cells were about 40% of that of KB cells, whereas similar levels of radioactive daunomycin accumulation were observed in KB/VM-2 and KB/VM-4 cells as KB cells. Topoisomerase II activity of nuclear extracts of both KB/VM-2 and KB/VM-4 assayed by decatenation of kinetoplast DNA was consistently two-thirds or less the activity of KB cells. A similar reduction was seen in both immunoblot assays with specific anti-topoisomerase II antibody and Northern blot analysis with specific human DNA topoisomerase II complementary DNA. DNA topoisomerase I activity, however, was similar between the mutants and their parent. Furthermore, cell growth of KB/VM-2 and KB/VM-4 was more thermolabile than that of KB, while KB/VM-b already showed temperature-sensitive growth. KB/VM-1 did show reduced accumulation of VP16 as in KB/VM-2 or KB/VM-4, but it had a normal level of topoisomerase II content as in KB cells. These data suggest that the reduced expression of DNA topoisomerase II, possibly combined with decreased permeability to the drugs, can account for the acquired VM26 resistance of KB/VM-2 and KB/VM-4 cells and also that the temperature-sensitive phenotype might not be obligatorily coupled with the reduced expression of topoisomerase II or the decreased permeability.