Showing papers by "Yves Pommier published in 1986"

Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors

Abstract: Most DNA intercalators and epipodophyllotoxins inhibit mammalian topoisomerase II by trapping the enzyme within DNA cleavage complexes that can be detected in cells as protein-associated DNA strand breaks. We have characterized previously a line of Chinese hamster cells (DC3F/9-OHE cells) the resistance of which to the cytotoxic effect of intercalators and etoposide is associated with a reduced formation of protein-associated DNA strand breaks. In the present study, topoisomerases of these cells were compared to those of the parental sensitive cells (DC3F). NaCl extracts (0.35 m) of isolated DC3F/9-OHE nuclei did not form 4′-(9-acridinylamino)methanesulfon- m -anisidide-induced DNA-protein linking, whereas DC3F nuclear extracts did. In addition, DC3F/9-OHE nuclear extract had an unusually high level of DNA linking activity in the absence of 4′-(9-acridinylamino)methanesulfon- m -anisidide. Topoisomerases II from DC3F/9-OHE and DC3F nuclei appeared similar qualitatively. DC3F/9-OHE nuclear extract had approximately twice less topoisomerase II molecules than did DC3F nuclear extract but similar topoisomerase II activity. Topoisomerase I activities appeared also similar in sensitive and resistant cells. However, part of DC3F/9-OHE topoisomerase I copurified with a DNA linking activity which was not present in DC3F nuclei. This unusual DNA linking activity was not sensitive to the stimulatory effect of 4′-(9-acridinylamino)methanesulfon- m -anisidide.

Isolation of intercalator-dependent protein-linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase II

Abstract: DNA intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) have previously been found to induce in mammalian cells the formation of protein-associated DNA single- and double-strand breaks. In the current work, an activity characterized by the production of DNA-protein links associated with DNA strand breaks and by stimulation by m-AMSA was isolated from L1210 cell nuclei and was shown to be due to topoisomerase II. Nuclei were extracted with 0.35 M NaCl, and the extract was fractionated by gel filtration, DNA-cellulose chromatography, and glycerol gradient centrifugation. A rapid filter binding assay was devised to monitor the fractionation procedure on the basis of DNA-protein linking activity. The active DNA-cellulose fraction contained both topoisomerase I and topoisomerase II whereas the glycerol gradient purified material contained only topoisomerase II activity. The properties of the active material were studied at both stages of purification. m-AMSA enhanced the formation of complexes between purified topoisomerase II and SV40 DNA in which the DNA sustained a single- or double-strand cut and the enzyme was covalently linked to the 5' terminus of the DNA. This action was further enhanced by ATP, as well as by nonhydrolyzable ATP analogues. m-AMSA inhibited the topoisomerization and catenation reactions of topoisomerase II, probably because of trapping of the enzyme-DNA complexes. The activity showed a dependence on the type of DNA intercalators used, analogous to what was previously observed in intact cells. m-AMSA had no effect on topoisomerase I.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced Formation of Protein-associated DNA Strand Breaks in Chinese Hamster Cells Resistant to Topoisomerase II Inhibitors

Abstract: DNA intercalating drugs and the epipodophyllotoxins etoposide and teniposide interfere with the action of mammalian DNA topoisomerase II by trapping an intermediate complex of the enzyme covalently linked to the 5′-termini of DNA breaks. This effect can be observed in intact cells by alkaline elution measurement of protein-associated DNA strand breaks. To assess the cytotoxic role of this effect, we have studied a subline of DC3F Chinese hamster lung cells selected for resistance to the intercalating agent 9-hydroxyellipticine. This subline (DC3F/9-OHE) was cross-resistant to other intercalators as well as to etoposide. Resistance to Adriamycin was associated with reduced uptake. However, resistance to 4′-(9-acridinylamino)methanesulfon- m -aniside and 2-methyl-9-hydroxyellipticinium was observed in the absence of changes in drug uptake, suggesting a second mode of resistance. DC3F/9-OHE cells formed fewer protein-associated DNA strand breaks in response to 4′-(9-acridinylamino)methanesulfon- m -aniside, 2-methyl-9-hydroxyellipticinium, or etoposide than did the sensitive parental cells. The same was true for isolated nuclei from these cells, which is consistent with a mode of resistance unrelated to drug uptake through the plasma membrane. These data suggest that resistance to DNA topoisomerase II inhibitors exhibited by DC3F/9-OHE cells is due in part to a modification of topoisomerase II activity.

Effects of the Bifunctional Antitumor Intercalator Ditercalinium on DNA in Mouse Leukemia L1210 Cells and DNA Topoisomerase II

Abstract: Ditercalinium, a 7H-pyridocarbazole dimer (bisintercalator) belongs to a new class of antineoplastic intercalating agents. To investigate its mechanism of cytotoxicity, the effects of ditercalinium on DNA were assessed using normal (L1210) and drug-resistant (L1210/PyDi1) mouse leukemia cells. Alkaline elution assays demonstrated that ditercalinium produced no DNA strand breaks, DNA-protein cross-links, or DNA-DNA cross-links, eliminating these effects as cytotoxic lesions. This result sets ditercalinium apart from other intercalating agents with respect to its interaction with DNA. Nucleoids (histone-depleted chromatin) from ditercalinium-treated L1210 cells were considerably more compact than those from untreated cells, as determined by sedimentation in neutral sucrose gradients. In contrast, nucleoids from ditercalinium-treated L1210/PyDi1 (resistant) cells were similar in compactness to those from control cells. Thus, ditercalinium altered chromatin structure in vivo. The effect of the bisintercalator on purified DNA topoisomerase II, an intracellular target of monointercalators, was measured in vitro. Ditercalinium (5 X 10(-7) M) completely inhibited both the formation of covalent complexes between this enzyme and simian virus 40 DNA and the enzyme-induced DNA cleavage. In addition, ditercalinium induced DNA catenation in the presence of topoisomerase II and adenosine triphosphate. Thus, the cytotoxicity of ditercalinium may derive from a mechanism that, although involving topoisomerase II, is manifested by condensation of DNA rather than by the induction of protein-associated DNA strand breaks.