The mechanism of inhibition of eukaryotic by itself has no effect on the binding of various forms of DNA to yeast DNA topoisomerase II. When both and ATP are present, however, the enzyme is converted to the closed form, as evidenced by the SV8 endoproteinase cleavage pattern as well as the characteristics of the DNAenzyme complexes. These results suggest that bis(2,6-dioxopiperazines) act by stabilizing eukaryotic DNA topoisomerase II in the closed-clamp form and preventing it from opening again. MATERIALS AND METHODS 1781The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Abstract. , a novel noncleavable, complexstabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T. Miki, T. Narita, R. Yui, S. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Cancer Res. 51:4909--4916). With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated. Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes. In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by . Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases. In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of ode2 kinase, spindle apparatus reorganization and disassembly and re, assembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase. Cells then continued through further cell cycle rounds, becoming polyploid and losing viability. This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast. The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes. In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes. topoisomerases (topo) t have been implicated in e maintenance of genetic processes such as repliation, transcription, and recombination by controlling the higher order chromosomal structure through the cell cycle (for review, see Wang, , 1987Cozzarelli and Address all correspondence to Ryoji Ishida,

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...

The sialyl Lewis a antigen is a well known tumor marker, CA19-9, which is frequently elevated in the serum in gastrointestinal and pancreatic cancers. UDPgalactose:N-acetylglucosamine ␤1,3-galactosyltransferase(s) (␤3Gal-Ts) are required for the synthesis of the sialyl Lewis a epitope. In the present study, a novel ␤3Gal-T, named ␤3Gal-T5, was isolated from a Colo205 cDNA library using a degenerate primer strategy based on the amino acid sequences of the four human ␤3Gal-T genes cloned to date. Transfection experiments demonstrated that HCT-15 cells transfected with the ␤3Gal-T5 gene expressed all the type 1 Lewis antigens. In gastrointestinal and pancreatic cancer cell lines, the amounts of ␤3Gal-T5 transcripts were quite well correlated with the amounts of the sialyl Lewis a antigens. The ␤1,3Gal-T activity toward agalacto-lacto-N-neotetraose was also well correlated with the amounts of ␤3Gal-T5 transcripts in a series of cultured cancer cells, and in Namalwa and HCT-15 cells transfected with the ␤3Gal-T5 gene. Thus, the ␤3Gal-T5 gene is the most probable candidate responsible for the synthesis of the type 1 Lewis antigens in gastrointestinal and pancreatic epithelia and tumor cells derived therefrom. In addition, ␤3Gal-T5 is a key enzyme that determines the amounts of the type 1 Lewis antigens including the sialyl Lewis a antigen.