Characterization of a mammalian mutant with a camptothecin-resistant DNA topoisomerase I.

Abstract

DNA topoisomerase I was purified to near homogeneity from a clonal line of human lymphoblastic leukemia cells, RPMI 8402, that is resistant to camptothecin, a cytotoxic alkaloid from Camptotheca acuminata, and compared with that of the parent wild-type cells. As assayed by relaxation of the supercoiled plasmid DNA and by formation of enzymelinked DNA breaks, the purified enzyme from the resistant cells was shown to be >125-fold as resistant to camptothecin as the wild-type enzyme, comparable to a cellular resistance index of about 300. Therefore, the cellular resistance appears to be due to the resistance of the enzyme. The amount of the immunoreactive enzyme protein in whole extract appeared to be reduced to less than half that of the wild-type enzyme. These results establish that DNA topoisomerase I is the cellular target of camptothecin and that DNA topoisomerase I is essential for the survival of mammalian cells.The DNA topoisomerases are enzymes that catalyze the concerted breakage and rejoining of the DNA backbone and thereby are presumed to participate in various genetic processes (1-5). The type I topoisomerases transiently cut and reseal one DNA strand so that the linking number changes by steps of one. Genetic and functional studies of the enzymes have been largely limited to prokaryotes and the lower eukaryote yeast. Viable mutants ofEscherichia coli defective in the topA gene encoding topoisomerase I were isolated (6, 7), but these proved to have mutations in DNA gyrase genes that compensated for the mutation in topA (8-10). This finding suggested an essential role for the enzyme in regulating the degree of supercoiling of DNA by counteracting the activity of the type II enzyme. In contrast, however, topoisomerase I-deficient mutants of yeast were isolated and shown to be viable, although they possessed the wild-type allele of topoisomerase II (11,12). The effect of the topoisomerase I mutation, however, was manifested by an additional mutation in topoisomerase II, implicating the complementary role of the latter (12).Topoisomerase I was previously found associated with transcriptionally active chromatin in mammalian cells (13,14). It also appears to be catalytically active on transcriptionally active genes in Drosophila polytene chromosomes (15) as well as on nucleolus-associated ribosomal genes (16-19). These experiments suggest a functional role for the enzyme in transcriptional events involving either RNA polymerase I or II.The availability of mutants and specific inhibitors of this enzyme as was the case in prokaryotes might help dissect and establish the role of this enzyme in DNA metabolism. We previously reported that heparin is a potent inhibitor of a mammalian DNA topoisomerase I (20, 21), but its broad specificity limits its usefulness for this purpose. Camptothecin (CPT) is a cytotoxic alkaloid isolated from Camptotheca acuminata (22, 23), which has a strong antitumor activity against a wide range of experimental tumors. CPT inhibits RNA and DNA synthesis and causes rapid and rev...

Combination chemotherapy of irinotecan plus cisplatin for advanced gastric cancer: efficacy and feasibility in clinical practice

“…Irinotecan hydrochloride (CPT-11) is a water-soluble, semisynthetic derivative of camptothecin (CPT) that retains the original antitumor activity of CPT, but has lower toxicity. 9 Its antitumor effects are due to the inhibition of DNA topoisomerase, 10,11 and a response rate of 18% for gastric cancer patients has been reported in a phase II study, regardless of prior chemotherapy. 12 Combination chemotherapy of CPT-11 with CDDP was reported to be effective for metastatic gastric cancers, with a response rate of 48% (21/44) for all patients and 59% (17/29) for chemotherapy-naive patients in a phase II study.…”

A phase II trial of combination of CPT-11 and cisplatin for advanced non-small-cell lung cancer

“…Both CPT-11 and cisplatin are active against NSCLC. Because of the differences in mechanisms of action (Andoh et al, 1987;Zwelling and Kohn, 1979) and toxicity profiles (Gottlieb and Drewinko, 1975;Negoro et al, 1991a), and with limited cross-resistance Noda et al, 1991;Masuda et al, 1992a) between the two drugs, the synergism between CPT-11 and its major active metabolite, in combination with cisplatin seems to have enormous clinical potential. Based on these reports, we conducted a phase I trial of the combination of CPT-11 (escalating doses) with cisplatin in advanced NSCLC (Masuda et al, 1992b).…”

Low-level resistance to camptothecin in a human small-cell lung cancer cell line without reduction in DNA topoisomerase I or drug-induced cleavable complex formation

“…To date, acquired resistance to CPT analogues has been associated with reduced formation of drug-induced DNA SSBs (reviewed . This can be due to (1) decreased enzyme levels (Eng et al, 1990), (2) resistant forms of enzyme due to mutations (Andoh et al, 1987;Tamura et al, 1991) or (3) decreased accumulation of drug in rare cases (Chang et al, 1992). It is still an open question whether these observations translate to the clinical situation.…”