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Departments of Pathology [M. S., I. W., B. H., R. S. O.] and Oncology [I. W., L. H. J., B. H., R. S. O., P. B. J.], Rigshospitalet, DK-2100 Copenhagen, Denmark; Cellular Pharmacology and Antitumour Chemistry Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom [S. K., A. M. C.]; and Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 [L. H. J., J. L. N.]
Anticancer drugs targeted to the nuclear enzyme DNA topoisomerase II are classified as poisons that lead to DNA breaks or catalytic inhibitors that appear to completely block enzyme activity. To examine the effects of the bisdioxopiperazine class of catalytic inhibitors to topoisomerase II, we investigated a Chinese hamster ovary (CHO) subline selected for resistance to ICRF-159 (CHO/159-1). Topoisomerase II
content in CHO/159-1 cells was reduced by 40–50%, compared to wild-type CHO cells, whereas the ß isoform was increased by 10–20% in CHO/159-1 cells. However, the catalytic activity of topoisomerase II in nuclear extracts from CHO/159-1 cells was unchanged, as was its inhibition by the topoisomerase II poison etoposide (VP-16). No inhibition of topoisomerase II catalytic activity by ICRF-187 was seen in CHO/159-1 cells up to 500 µM, whereas inhibition was evident at 50 µM in wild-type CHO cells. VP-16-mediated DNA single-strand breaks and cytotoxicity were similar in the two sublines. ICRF-187 could abrogate these VP-16 effects in the wild-type line but had no effect in CHO/159-1 cells. Western blots of topoisomerase II after incubation of CHO cells with ICRF-187 demonstrated a marked band depletion, whereas this effect was completely lacking in CHO/159-1 cells, and an equal effect of VP-16 was observed in both lines. These data imply that the CHO/159-1 topoisomerase II lacks sensitivity to bisdioxopiperazines and that the mechanism of resistance in this cell line does not confer cross-resistance to topoisomerase II poisons, suggesting that mutations conferring resistance to bisdioxopiperazines can occur at sites distinct from those responsible for resistance to complex stabilizing agents. Accordingly, CHO/159-1 cDNA showed two heterozygous mutations in the proximal NH2-terminal part of topoisomerase II (Tyr49Phe and 
309Gln-Gln-Ile-Ser-Phe313), which is in contrast to those induced by topoisomerase II poisons, which cluster further downstream. Site-directed mutagenesis and transformation of the homologous Tyr50Phe coding mutation in human topoisomerase II in a temperature-conditional yeast system demonstrated a high-level resistance to ICRF-193, compared to cells expressing wild-type cDNA, but none toward the poisons VP-16 or amsacrine, thus confirming that the Tyr50Phe mutation confers specific resistance to bisdioxopiperazines. Thus, these results indicate that the region of the protein involved in ATP-binding also plays a critical role in sensitivity to bisdioxopiperazines, a result consistent with the known requirement for the formation of an ATP-bound closed clamp for bisdioxopiperazine activity. These results may enable a more precise understanding of the interaction of topoisomerase II-directed drugs with their target enzyme.
1 This work was supported by the Danish Cancer Society, the Novo Nordisk Foundation, the H:S Research Foundation, Fonden til Laegevidenskabens Fremme, The Hojmosegaard Legacy, National Cancer Institute Grants CA52814 and CA21765, and the American Lebanese Syrian Associated Charities (ALSAC).
2 To whom requests for reprints should be addressed, at Department of Pathology, Rigshospitalet 5444, DK-2100 Copenhagen, Denmark. Phone: (45) 3545 5432; Fax: (45) 3545 5414; E-mail: maxwell@rh.dk.
3 Present address: Department of Medicine, Addenbrookes Hospital, Cambridge CB2 2QQ, United Kingdom.
4 Present address: Department of Reproductive Physiology, St. Bartholomew's Hospital Medical College, London EC1A 7BE, United Kingdom.
Received 12/ 3/97. Accepted 2/17/98.
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