DIFFERENTIAL STABILIZATION OF TOPOISOMERASE-II-DNA CLEAVABLE COMPLEXES BY DOXORUBICIN AND ETOPOSIDE IN DOXORUBICIN-RESISTANT RAT GLIOBLASTOMA CELLS

Using the technique of alkaline filter elution, we have evaluated the DNA damage induced by doxorubicin and etoposide in a rat glioblastoma cell line, C6, and its doxorubicin-selected resistant variant, C6 0.5. DNA damage paralleled drug-induced cytotoxicity, but it appeared that the same DNA damage generated much less cytotoxicity in resistant cel ls than in sensitive ones, resistant cells being able to tolerate more DNA damage than sensitive cells. We have then quantified the doxorubi cin- and etoposide-induced complexes between topoisomerase II (topoII) DNA with the technique of SDS/KCl precipitation. Etoposide produced a concentration-dependent increase in topoII-DNA complexes, which was h igher in resistant cells at equitoxicity, just as was DNA damage. In c ontrast, doxorubicin-induced topoII-DNA complexes, which were much les s abundant than those induced by etoposide, were not differently produ ced in sensitive and resistant cells. This indicates that the DNA dama ge occurring in resistant cells at high doxorubicin concentrations mig ht originate from source other than topoII-DNA complex formation. When verapamil was added during drug exposure, it restored doxorubicin int racellular accumulation to the level reached in sensitive cells, parti ally reversed both doxorubicin and etoposide resistance, increased the formation of etoposide-induced topoII-DNA complexes, but not those in duced by doxorubicin. Immunoblot analysis of topoII as well as the mea sure of its catalytic activity in nuclear extracts revealed a quantita tive defect of this enzyme in the resistant line. When inhibiting this activity by doxorubicin and etoposide, we observed that the concentra tions of etoposide required for a given inhibition of kinetoplast DNA decatenation are much higher that those of doxorubicin. The topoII ext racted from both cell lines is, therefore, much more sensitive to doxo rubicin than to etoposide, but no difference in drug sensitivity was e vident between sensitive and resistant cells, indicating that no quali tative alteration in topoII catalytic activity was likely to occur.