Analysis of comptothecin resistance in yeast: relevance to cancer therapy.

The budding yeast Saccharomyces cerevisiae is a well defined genetic system to investigate various aspects of camptothecin (Cpt)-induced cytotoxicity. This antineoplastic agent and its derivatives specifically poison eukaryotic DNA topoisomerase I, the product of the TOP1 gene, by stabilizing a covalent enzyme-DNA intermediate. Analyses of various yeast and human top1 mutants in yeast strains deleted for TOP1 (top1Delta) have defined amino acid residues critical for enzyme function and Cpt sensitivity. Cpt cytotoxicity is also mediated by the pleiotropic drug resistance network, primarily through the action of an ABC transporter. The potential clinical relevance of these and related studies are discussed.

Altered topoisomerase I and II activities in suramin-resistant lung fibrosarcoma cells.

To better understand the molecular basis for the cytotoxic effects of suramin, we have developed suramin-resistant DC-3F/SU 1000 cells by continuous exposure of fibrosarcoma cells to increasing concentrations of suramin. The suramin resistance (approximately 10-fold) is not associated with changes in uptake or intracellular distribution of the drug. The sensitivity to actinomycin D, cytarabine, aphidicolin, hydroxyurea, vincristine, and 5-fluorouracil is unaltered. In contrast, DC-3F/SU 1000 cells are about 2-fold resistant to classical DNA topoisomerase II inhibitors such as doxorubicin, amsacrine, and etoposide, whereas the cells are 1.5-fold more sensitive to the topoisomerase I inhibitor camptothecin. The cross-resistance to topoisomerase II inhibitors occurred earlier than the collateral sensitivity to camptothecin. Amsacrine- and etoposide-induced DNA-protein complex formation is reduced about 2-fold in DC-3F/SU 1000 cells, compared with DC-3F cells, whereas camptothecin-induced DNA-protein complex formation is increased 1.5-fold. Western blot analysis of cellular lysates from the two cell lines shows no significant differences in the level of topoisomerase II, whereas the level of topoisomerase I is increased 2.5-fold in DC-3F/SU 1000 cells. The catalytic activities of topoisomerases I and II in nuclear extracts from DC-3F/SU 1000 cells are both about 2-fold higher than those in extracts from DC-3F cells, whereas amsacrine- and etoposide-induced DNA-protein complex formation is comparable between the two cell lines. Taken together, our results support the involvement of DNA topoisomerases in the cytotoxic activity of suramin. We further believe that the DC-3F/SU 1000 cells may be a useful model for the elucidation of factors that lead to low, clinically relevant, levels of resistance to topoisomerase II inhibitors.

Aurintricarboxylic acid, a putative inhibitor of apoptosis, is a potent inhibitor of DNA topoisomerase II in vitro and in Chinese hamster fibrosarcoma cells.

Aurintricarboxylic acid (ATA) is a polyanionic, polyaromatic compound which has been shown to inhibit apoptotic cell death in various cell types induced by a variety of factors. Since ATA is known to be a general inhibitor of nuclease activities in vitro (ID50S ranging from 2 to 50 microM), the in vivo effects are usually attributed to inhibition of endogenous endonuclease activities. We show herein that ATA is a potent inhibitor of the nuclear enzyme DNA topoisomerase II. ATA inhibits the catalytic activity of purified yeast topoisomerase II with an ID50 of approx. 75nM as measured by relaxation assays. ATA does not stabilize the covalent DNA-topoisomerase II reaction intermediate ("cleavable complex") as do other inhibitors of this enzyme such as 4'-(9-acridinylamino)-methane sulfon-m-anisidide (amsacrime), 4'-demethyl-epipodophyllotoxin-9-(4,6-O-ethylidine-beta-D-gluco pyr anoside) (etoposide) and ellipticines. In contrast, cleavable complex formation induced by amsacrine and etoposide is trongly inhibited in the presence of ATA. ATA also prevents the binding of topoisomerase II to DNA and inhibits topoisomerase II-catalysed ATP hydrolysis. The ability of ATA to interfere with more than one step in t he catalytic cycle of DNA topoisomerase II may explain its unusual potency as an inhibitor of this enzyme. ATA reduces the number of amsacrine-induced DNA-protein complexes in intact DC-3F Chinese hamster fibrosarcoma cells and protects these cells from the cytotoxic action of amsacrine. The effects of ATA on DNA-protein complex formation in living cells appear to be due to the direct interaction of the drug with topoisomerase II, since similar results are found when nuclei from untreated DC-3F cells are exposed to amsacrine after a short preincubation with ATA. Cells resistant to 9-hydroxyellipticine, which have been shown to possess altered topoisomerase II activity, are approx. 5-fold more resistant to ATA than the sensitive parental cells as shown by colony formation essays. We conclude that ATA is a potent inhibitor of topoisomerase II and that the drug interacts with topoisomerase II in living cells. Our findings raise the possibility that the protective effects of ATA towards apoptotic cell death might, at least in part, involve DNA topoisomerase II.