Showing papers by "Yves Pommier published in 1989"

Structure-Activity Study of the Actions of Camptothecin Derivatives on Mammalian Topoisomerase I: Evidence for a Specific Receptor Site and a Relation to Antitumor Activity

Abstract: Twenty-two compounds related to camptothecin, a known inhibitor of eukaryotic topoisomerase I, were studied. The following effects on the actions of topoisomerase I were observed and were well correlated among most of the compounds studied: (a) inhibition of the first-order rate of relaxation of supercoiled DNA; (b) conversion of supercoiled DNA to nicked circles; and (c) single-strand cleavage of linear DNA at specific sites. The locations of the stimulated cleavage sites were the same for all of the active derivatives. Stereochemistry and the positions of substituents were found to be crucial for the presence or absence of effects on topoisomerase I, indicating that the compounds interact with an asymmetrical receptor site on the enzyme or enzyme-DNA complex. From the structure-activity relations, the regions of interaction between the camptothecin ring system and the receptor site were inferred. Striking correlations were observed between activity against topoisomerase I and reported activity against murine leukemias, indicating that an action on topoisomerase I is responsible for the antitumor activity of the camptothecins.

Differential Requirement of DNA Replication for the Cytotoxicity of DNA Topoisomerase I and II Inhibitors in Chinese Hamster DC3F Cells

Abstract: The cytotoxicity of topoisomerase inhibitors is thought to result from the induction of enzyme-mediated DNA breaks. The fact that these breaks reverse rapidly in cells programmed to die, led us to investigate further the cytotoxic mechanisms of topoisomerase I (camptothecin) and topoisomerase II inhibitors (VP-16 and amsacrine) in Chinese Hamster lung fibroblasts (DC3F). Exposures (30 min) to camptothecin produced limited cell killing with approximately 20% of the cells naturally resistant. This resistance was overcome by increasing the drug exposure time. Inhibition of DNA synthesis by 5-min pretreatments with aphidicolin or hydroxyurea abolished the cytotoxicity of camptothecin without changing the level of camptothecin-induced DNA breaks. A good correlation was found between the degree of DNA synthesis inhibition by aphidicolin and the reduction of camptothecin cytotoxicity. In similar experiments performed with topoisomerase II inhibitors, aphidicolin prevented only partially against VP-16- and amsacrine-induced cytotoxicities, yet had no effect upon drug-induced DNA breaks. These results indicate that the production of topoisomerase-mediated DNA breaks by antitumor drugs is not sufficient for cell killing. Instead, an interference of moving DNA replication forks with drug-stabilized topoisomerase-DNA complexes is critical for cell death. The cytotoxicity of camptothecin seemed to be completely related to this process, while that of topoisomerase II inhibitors seemed to involve additional mechanisms in DC3F cells.

Protein-linked DNA strand breaks induced in mammalian cells by camptothecin, an inhibitor of topoisomerase I.

Abstract: Camptothecin was recently identified as an inhibitor of mammalian topoisomerase I. Similar to inhibitors of topoisomerase II, camptothecin produces DNA single-strand breaks (SSB) and DNA-protein cross-links (DPC) in mammalian cells. However, their one-to-one association, expected for trapped topoisomerase complexes, has not previously been demonstrated. We have studied camptothecin-induced SSB and DPC in Chinese hamster DC3F cells and their isolated nuclei, using the DNA alkaline elution technique. It was found that the SSB and DPC frequencies detected following camptothecin treatment depend upon the conditions used for lysis. When lysis was with sodium dodecyl sulfate, the observed frequencies of SSB and DPC were 2- to 3-fold greater than when sodium dodecyl sarkosinate (Sarkosyl) was used. In either case, the SSB:DPC ratio was close to 1. All of the camptothecin-induced SSB were protein linked, as indicated by the absence of DNA elution under nondeproteinizing conditions. DNA cleavage assays with purified topoisomerase I also indicated that the weaker Sarkosyl detergent fails to trap all of the enzyme-DNA complexes. In contrast, lysis conditions had little effect on levels of SSB or DPC produced by 4'-(9-acridinylamino)-methanesulfon-m-anisidide, suggesting that trapping of topoisomerase II complexes occurs equally well with either detergent. In experiments using isolated nuclei, it was found that the camptothecin-induced SSB, in contrast to trapped topoisomerase II complexes, can form and reverse within minutes at 4 degrees C. The activity of camptothecin at low temperature was also seen with purified topoisomerase I. These results support the hypothesis that the SSB and DPC induced by camptothecin in mammalian cells are due to an action on topoisomerase I.

Topological complexes between DNA and topoisomerase II and the effects of polyamines

Abstract: The polyamines spermine and spermidine were found to enhance the formation of a stable noncovalent complex between mammalian topoisomerase II and DNA. This complex is not associated with DNA strand breaks and forms to a greater extent with supercoiled than with relaxed circular or with linear DNA. Polyamine-induced complex formation is associated with a stimulation of the enzymatic relaxation of DNA supercoils. In these respects, the polyamine-enhanced complex differs from the covalent cleavable complexes stabilized by DNA intercalators such as amsacrine (m-AMSA) or epipodophylotoxins such as teniposide (VM-26). In the polyamine-enhanced complex, the topoisomerase II may be a donutlike structure topologically bound to the DNA and able to migrate and dissociate from the ends of linear DNA molecules. At relatively high concentrations, spermine (1 mM) enhances topoisomerase II induced cleavage at certain sites on the SV40 genome that could have regulatory significance.

Antineoplastic Agents Inhibitor of Topoisomerase II

Abstract: The extreme length of the DNA molecules in the nuclei of eukaryotic cells, as well as the possible attachment of the DNA to a structural matrix or scaffold in the nuclei, constrains the topology of the DNA helices. The topological constraint is that the two strands of a DNA helix are unable to change the number of turns of one strand about the other (the “linking number”, Lk), unless one or both strands are temporarily cut to form a gap through which other parts of the strands can pass. The topoisomerases provide mechanisms for such cutting and passing of strands, without which the DNA helix cannot unwind and replicated chromosomes cannot segregate.