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.
CNRS URA 147, Laboratoire de Pharmacologie Moléculaire, Institut Gustave Roussy, Villejuif, France.
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Benchokroun
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Couprie
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Larsen
D000255:Adenosine Triphosphate / Q000378:metabolism; D000677:Amsacrine / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001312:Aurintricarboxylic Acid / Q000494:pharmacology*; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000302:isolation & purification; D011485:Protein Binding / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D059005:Topoisomerase II Inhibitors; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology
D059005:Topoisomerase II Inhibitors; D000677:Amsacrine; D001312:Aurintricarboxylic Acid; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II