ABSTRACT
Extracts of K21 murine mastocytoma cells contain a factor that enhances formation of amsacrine-induced topoisomerase II-DNA complexes (PDCs) when added to isolated K21 nuclei. The PDC-enhancing activity is reduced in extracts from 2 or 6 h cycloheximide or cordycepin-treated cells, implying that continuous protein synthesis is required to maintain the factor. The factor is heat-labile, proteinase-sensitive and has other properties that distinguish it from the two known classes of topoisomerases. The data suggest that the factor is a labile protein with a molecular weight in excess of 50,000. This appears to be the first direct evidence of a protein factor that modulates drug-induced topoisomerase II action.
Subject(s)
Amsacrine/pharmacology , DNA, Neoplasm/metabolism , Mast-Cell Sarcoma/physiopathology , Topoisomerase II Inhibitors , Animals , Cell Nucleus/physiology , Cell-Free System , Cytoplasm/physiology , Hot Temperature , Macromolecular Substances , Mice , Peptide Hydrolases/pharmacology , Protein Binding/drug effectsABSTRACT
The cold-sensitive (proliferating at 39.5 degrees C, reversibly arrested in GI-phase at 33 degrees C) cell-cycle mutant 21-Fb of the murine mastocytoma cell line P815 was used to study the effect of amsacrine on non-cycling cells. The sensitivity of arrested 21-Fb cells decreased less than 2-fold in cell survival experiments when compared to proliferating cells. In contrast, DNA breakage and stimulation of protein-DNA complex formation in intact or lysed cells was reduced approx. 10-fold in arrested cells and DNA topoisomerase II activity in arrested cells was only 5% of the activity in proliferating cells. Thus, there was no correlation between cell survival and DNA damage or DNA topoisomerase II activity in drug-treated cells.
Subject(s)
Amsacrine/pharmacology , Cell Cycle , DNA Damage , DNA Topoisomerases, Type II/metabolism , Animals , Cell Division , Cell Survival/drug effects , Cold Temperature , DNA/drug effects , DNA/metabolism , Mast-Cell Sarcoma , Mice , Mutation , Proteins/metabolism , Tumor Cells, CulturedABSTRACT
N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (NSC 601316) is a DNA intercalating experimental antitumour agent which is curative against the Lewis lung carcinoma in mice. Its action has been compared with amsacrine, its inactive isomer oAMSA, the solid tumour active derivative CI-921 (NSC 343499), a C-6 methylene chain-linked bisacridine (NSC 210733), 9-aminoacridine and quinacrine. All compounds inhibited the unknotting of phage P4 DNA by topoisomerase II in nuclear extracts prepared from L1210 cells. NSC 601316 inhibited growth of cultured L1210, P388, P/AMSA (P388 resistant to amsacrine) and P/ACTD (resistant to actinomycin D) cell lines at concentrations of 87, 150, 2020 and 150 nM respectively. A 1 h drug exposure to 0.85 microM NSC 601316 killed 50% of L1210 cells. L1210 cells treated for 1 h with NSC 601316 accumulated DNA breaks and protein-DNA cross-links. There was a good correlation between DNA breakage and cytotoxicity, but the relationship between drug concentration and number of protein-DNA cross-links was non-linear and differed from that of amsacrine and CI-921. There was also a positive correlation between the degree of cross-resistance of P/AMSA cells (which have altered topoisomerase II function) and ability to induce DNA breakage or protein-DNA complexes. The results suggest that topoisomerase II is the target of action of NSC 601316.
Subject(s)
Acridines , Aminoacridines/pharmacology , Antineoplastic Agents/pharmacology , Leukemia P388/drug therapy , Leukemia, Experimental/drug therapy , Aminacrine/pharmacology , Amsacrine/analogs & derivatives , Amsacrine/pharmacology , Animals , Cell Survival/drug effects , DNA Damage , DNA Topoisomerases, Type II/physiology , Dose-Response Relationship, Drug , Leukemia L1210/drug therapy , Mice , Neoplasm Proteins , Quinacrine/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Stem Cell AssayABSTRACT
CHO-AA8 cells were used as a model system to study the role of DNA topoisomerase II in the resistance of non-cycling cells to amsacrine. Plateau-phase AA8 cells have previously been shown to be resistant to amsacrine and to contain fewer DNA breaks than log-phase cells after drug treatment (Robbie, M.A., Baguley, B.C., Denny, W.A., Gavin, J.R. and Wilson, W.R. (1988) Cancer Res., in press). The phage P4-unknotting activity of nuclear extracts decreased 2-fold when AA8 cells entered into the non-cycling state, but there was no difference in sensitivity to amsacrine between log- and plateau-phase nuclear extracts. Drug stimulation of protein-DNA complex formation was similar in whole cells, isolated nuclei and nuclear extracts from either log- or plateau-phase cells. However, stimulation of complex formation in cells, nuclei or nuclear extracts was approx. 4-fold lower in plateau-phase than in log-phase. The data presented suggested that drug-enzyme interaction was altered in plateau-phase cells.
