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 cells than in sensitive ones, resistant cells being able to tolerate more DNA damage than sensitive cells. We have then quantified the doxorubicin- 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 higher in resistant cells at equitoxicity, just as was DNA damage. In contrast, doxorubicin-induced topoII-DNA complexes, which were much less abundant than those induced by etoposide, were not differently produced in sensitive and resistant cells. This indicates that the DNA damage occurring in resistant cells at high doxorubicin concentrations might originate from source other than topoII-DNA complex formation. When verapamil was added during drug exposure, it restored doxorubicin intracellular accumulation to the level reached in sensitive cells, partially reversed both doxorubicin and etoposide resistance, increased the formation of etoposide-induced topoII-DNA complexes, but not those induced by doxorubicin. Immunoblot analysis of topoII as well as the measure of its catalytic activity in nuclear extracts revealed a quantitative defect of this enzyme in the resistant line. When inhibiting this activity by doxorubicin and etoposide, we observed that the concentrations of etoposide required for a given inhibition of kinetoplast DNA decatenation are much higher that those of doxorubicin. The topoII extracted from both cell lines is, therefore, much more sensitive to doxorubicin than to etoposide, but no difference in drug sensitivity was evident between sensitive and resistant cells, indicating that no qualitative alteration in topoII catalytic activity was likely to occur.
Montaudon
D
D
Department of Medical Biochemistry and Molecular Biology, Université Victor Segalen Bordeaux 2 and Institut Bergonié, France.
Pourquier
P
P
Denois
F
F
de Tinguy-Moreaud
E
E
Lagarde
P
P
Robert
J
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects / Q000378:metabolism*; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000187:drug effects / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D004795:Enzyme Stability; D005047:Etoposide / Q000494:pharmacology*; D005909:Glioblastoma; D051381:Rats; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D005047:Etoposide; D004317:Doxorubicin; D004247:DNA; D014700:Verapamil; D004250:DNA Topoisomerases, Type II