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1. Purification and characterization of human DNA topoisomerase IIIalpha.

Author

Goulaouic, H, Roulon, T, Flamand, O, Grondard, L, Lavelle, F, and Riou, J F

Abstract

Human topoisomerase IIIalpha (hTopo IIIalpha), the recently identified first member of the topoisomerase IA subfamily in humans, has a central domain which is highly homologous to the yeast topoisomerase III, but an overall organization closer to that of Escherichia coli DNA topoisomerase I. In order to determine the properties of hTopo IIIalpha, compared to those of other topoisomerase IA subfamily members, we purified this enzyme to near homogeneity, together with an active site-mutant Y337F. We show that hTopo IIIalpha is able to relax negatively supercoiled DNA in a distributive manner, leading to the total disappearance of the initial substrate and the appearance of intermediate topoisomers. This DNA relaxation activity is magnesium-dependent, although a low concentration of MgCl2is sufficient to obtain efficient catalysis. 32P-transfer experiments demonstrated that hTopo IIIalpha is able to cleave a single-stranded oligonucleotide and to bind covalently to the 5'-end of the cleaved DNA. Addition of 0.5 M NaCl reverses the reaction, leading to the religation of the oligo-nucleotide. Experiments utilizing several different single-stranded oligonucleotides permitted us to map several cleavage sites and to deduce a consensus sequence for DNA cleavage (CANNN downward arrow), which is different from that for other members of the Topo IA subfamily.

Published
1999
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2. Intoplicine (RP 60475) and its derivatives, a new class of antitumor agents inhibiting both topoisomerase I and II activities.

Author

Riou JF, Fossé P, Nguyen CH, Larsen AK, Bissery MC, Grondard L, Saucier JM, Bisagni E, and Lavelle F

Subjects
Animals, DNA drug effects, DNA metabolism, Female, Indoles therapeutic use, Male, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Neoplasms, Experimental drug therapy, Pyridines therapeutic use, Structure-Activity Relationship, Antineoplastic Agents pharmacology, Indoles pharmacology, Pyridines pharmacology, Topoisomerase I Inhibitors, Topoisomerase II Inhibitors
Abstract

Intoplicine (RP 60475, NSC 645008) is an antitumor derivative in the 7H-benzo[e]pyrido[4,3-b]indole series which is now being tested in clinical trials. Intoplicine strongly binds DNA (KA = 2 x 10(5) M-1) and thereby increases the length of linear DNA. These properties are consistent with DNA unwinding by intoplicine. Intoplicine was found to be a dual topoisomerase I and II inhibitor, with DNA sites of enzyme inhibition being different for these two enzymes. In this study, 22 analogues of intoplicine were evaluated for their effects on topoisomerase I- and II-mediated DNA cleavage reactions by using enzymes purified from calf thymus. Site-specific DNA cleavage mediated by topoisomerase I was observed with 7H-benzo[e]pyrido[4,3-b]indole derivatives but not with 11H-benzo[g]-pyrido[4,3-b]indole derivatives. Site-specific DNA cleavage mediated by topoisomerase II occurred with derivatives having hydroxyl groups at the 3-position on the 7H-benzo[e]pyrido[4,3-b]indole ring or at the 4-position on the 11H-benzo[g]pyrido[4,3-b]indole ring. Study of the relationships between the in vivo antitumor activity on P388 leukemia and the topoisomerase I- and/or II-mediated DNA cleavage activity revealed that the most highly active antitumor compounds possessed both topoisomerase I-and II-inhibitory properties. Compounds selectively inhibiting either topoisomerase I or II were less active. These results suggest that dual topoisomerase I and II inhibition is critical for the antitumor activity of this new series of antitumor compounds.

Published
1993

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