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Y Pommier

Publications and source records attributed to Y Pommier.

At least 217 records · Page 12Linked to original sources
PubMed1989

Differential requirement of DNA replication for the cytotoxicity of DNA topoisomerase I and II inhibitors in Chinese hamster DC3F cells.

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.

Amsacrine↗
PubMed1989

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

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.

Animals↗
PubMed1989

Mammalian topoisomerase II activity is modulated by the DNA minor groove binder distamycin in simian virus 40 DNA.

DNA topoisomerases II are nuclear enzymes that have been identified recently as targets for some of the most active anticancer drugs. Antitumor topoisomerase II inhibitors such as teniposide (VM-26) produce enzyme-induced DNA cleavage and inhibition of enzyme activity. By adding to such reactions distamycin, a compound whose effects on DNA have been extensively characterized, we investigated the effects of drug binding upon topoisomerase II-mediated DNA cleavage induced by VM-26. We have found a correspondence between distamycin binding (determined by footprinting analysis) and topoisomerase II-mediated cleavage of SV40 DNA (determined by sequencing gel analysis). Distamycin binding potentiated the cleavage of specific sites in the near proximity of distamycin-binding sites (within at least 25 base pairs), which indicates that DNA secondary structure is involved in topoisomerase II-DNA interactions. That distamycin potentiated cleavage only at sites that were recognized in the absence of distamycin and suppressed cleavage directly at distamycin-binding sites indicates that topoisomerase II recognizes DNA on the basis of primary sequence. In addition, distamycin stimulated topoisomerase II-mediated DNA relaxation and antagonized the inhibitory effect of VM-26. These results show that the DNA sequence-specific binding of distamycin produces local and propagated effects in the DNA which markedly affect topoisomerase II activity.

Animals↗
PubMed1989

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.

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.

Animals↗
PubMed1989

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

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.

Amsacrine↗
PubMed1989

Progressive resistance to doxorubicin in mouse leukemia L1210 cells with multidrug resistance phenotype: reductions in drug-induced topoisomerase II-mediated DNA cleavage.

Cells selected for resistance to doxorubicin (DOX) express the multidrug resistance (MDR) phenotype, and resistance has been suggested to be due primarily to enhanced cellular efflux of drug. A progressively DOX-resistant (10- and 40-fold) L1210 mouse leukemia model system, which does not exhibit enhanced DOX efflux as a primary mechanism of resistance, was found to display the MDR phenotype, based on overexpression of P-glycoprotein in western blots and cross-resistance to vinca alkaloids. Cross-resistance to another topoisomerase II inhibitor, etoposide (VP-16), was similar to that of DOX (10- and 40-fold), whereas resistance to N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulfonamide (m-AMSA) was 5-fold lower. In contrast, no cross-resistance to camptothecin, an inhibitor of topoisomerase I, was observed. Topoisomerase II decatenation activity in nuclear extracts from 10- and 40-fold DOX-resistant cells was 2- and 4-fold lower, respectively, when compared to sensitive cells. In these cells, however, marked reductions in m-AMSA- and VP-16-induced topoisomerase II mediated DNA cleavage were found to exceed decreases in the catalytic activity of the enzyme. Results from this study demonstrated that, in progressively DOX-resistant L1210 mouse leukemia cells with the MDR phenotype, a better relation existed between the degree of resistance and reduced VP-16- and m-AMSA-induced topoisomerase II mediated DNA cleavage, than between increases in P-glycoprotein and concomitant reduction in DOX accumulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Topoisomerase I interaction with SV40 DNA in the presence and absence of camptothecin.

Camptothecin is an antitumor drug, which is a specific inhibitor of eukaryotic topoisomerase I. Enzyme inhibition is related to the stabilization of cleavable complexes between topoisomerase I and DNA. The genomic and DNA sequence localization of L1210 topoisomerase I-mediated DNA breaks produced by camptothecin were determined in the SV40 genome. DNA cleavage was predominantly single-stranded and localized in selective regions of the DNA. A major cleavage site was found at nucleotide 4995 on the coding strand in the early transcription region. The DNA sequence was determined at prominent cleavage sites (nucleotides 127 and 199 in the two 72 bp repeats and nucleotide 4955). A DNA consensus sequence 5'-GATG-3' was found in SV40 DNA. Cleavage occurred between the T and the G and topoisomerase I was linked to the 3'-DNA terminus at the T position. The sequence GATG is more frequent in the non transcribed strand of the early and late transcription of SV40 than in the transcribed strands. This finding is consistent with the role of topoisomerase I in transcription.

Base Sequence↗
PubMed1988

DNA strand breaks produced by etoposide (VP-16,213) in sensitive and resistant human breast tumor cells: implications for the mechanism of action.

Pleotropic resistant human breast cancer cells (MCF-7), selected for resistance to Adriamycin, were used to study the production of DNA strand breaks by etoposide (VP-16) and its relationship to drug cytotoxicity. It was shown that the resistant MCF-7 cell line was cross-resistant to VP-16, and the degree of resistance was found to be 125-200-fold. Alkaline elution studies indicated that the parental cell line was very sensitive to VP-16 which caused extensive DNA strand breakage. In contrast, little DNA strand breakage was detected in the resistant MCF-7 cells, even at very high drug concentrations, indicating a good agreement between strand breaks and cytotoxicity. Further studies indicated that the nuclei isolated from the parental cell line were more resistant to VP-16-induced DNA strand breaks than the intact cells, while the opposite was found in the resistant cell line. In addition, the alkaline elution studies in isolated nuclei showed only a 2-fold reduction of VP-16-induced DNA breaks in nuclei from the resistant cells. In agreement with this result, it was found that nuclear extract from the resistant cells produced 2-3-fold less VP-16-induced DNA breaks than that from the sensitive cells in 32P-end-labeled SV40 DNA. VP-16 uptake and efflux studies indicated that there was a 2-3-fold decrease in net cellular accumulation of VP-16 in the resistant cells. Although the reduced uptake of VP-16 and decreased drug sensitivity of topoisomerase II appear to contribute to the mechanism of action and the development of resistance to VP-16, they do not completely explain the degree of resistance to VP-16 in this multidrug-resistant MCF-7 cell line indicating that other biochemical factors, such as activation of VP-16, are also involved in drug resistance and suggesting that the resistance is multifactorial.

Breast Neoplasms↗
PubMed1988

Production of protein-associated DNA breaks by 10-[diethylaminopropylamino]-6-methyl-5H-pyrido[3',4':4,5]pyrrolo [2,3-g]isoquinoline in cultured L1210 cells and in isolated nuclei: comparison with other topoisomerase II inhibitors.

10-[3-Diethylaminopropylamino]-6-methyl-5H-pyrido[3',4':4,5] pyrrolo[2,3-g]isoquinoline (PZE) is an ellipticine derivative currently in clinical trials. PZE has been postulated to produce cellular DNA lesions by an uncommon mechanism. PZE-induced DNA damage was further investigated in L1210 cells in culture. PZE was highly cytotoxic for these cells (90% inhibitory concentration = 3.1 microM). The effects of PZE on cellular DNA were studied first by alkaline sucrose sedimentation, in comparison with those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). Like m-AMSA, PZE induced DNA strand breaks which were detected without a proteolytic treatment of the cell lysate. This result rules out the existence of covalent protein bridges sealing DNA termini at the break sites. PZE was less active than m-AMSA. DNA fragmentation was maximum at 5 microM and was lower at higher concentrations. The DNA effects of PZE were also studied by alkaline elution, and compared with those of Adriamycin and m-AMSA. Like Adriamycin, PZE induced single-strand breaks (SSBs) in a bell-shaped manner with respect to drug concentration. The maximum SSB frequency [1784 +/- 370 (SEM) rad equivalents)] was obtained at 16 microM. The kinetics of SSB reversion after drug removal was slower than in the case of m-AMSA. Similar bell-shaped curves were obtained for PZE-induced double-strand breaks and DNA-protein cross-links. PZE induced more double-strand breaks per SSB than did m-AMSA. However, as in the case of m-AMSA, PZE induced equal SSB and DNA-protein cross-link frequencies. These results suggest that PZE induces DNA breaks by inhibiting topoisomerase II as do other antitumor intercalators.

Animals↗
PubMed1988

Topoisomerase II-mediated DNA damage produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide and related acridines in L1210 cells and isolated nuclei: relation to cytotoxicity.

4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) is a DNA intercalating 9-aminoacridine with clinical activity in adult acute leukemia. m-AMSA has been shown to produce protein-linked DNA strand breaks in mammalian cells through an interaction with the nuclear enzyme DNA topoisomerase II. We have compared the effects of m-AMSA and several acridine analogues (9-aminoacridine; A, NSC 343499; B, SN 16507; C, NSC 140701; D, SN 13553) on DNA integrity and cell survival in L1210 leukemia in vitro. Cells (or isolated nuclei) were treated with drugs (0.1-50 microM) for 0.5-1.0 h and subsequently analyzed using the alkaline elution technique. All drugs, except Compound D, produced DNA-protein cross-links (DPC) in L1210 cells. At 1 microM, potency was in the order, C greater than m-AMSA greater than B greater than A much greater than 9-aminoacridine. In isolated nuclei, DPC and single-strand breaks were produced in essentially a 1:1 ratio, which is consistent with topoisomerase II-mediated protein-linked DNA breaks. Potency differences were less pronounced in nuclei than in cells. In isolated nuclei, Compound D produced extensive DPC not associated with single-strand breaks, which suggests a more complex activity for this compound. Colony formation assays demonstrated the cytotoxicity of most of these acridine analogues (C greater than B greater than A approximately equal to m-AMSA much greater than D = 9-aminoacridine). Correlation of DPC with cell kill gave similar curves for each compound. These results are evidence for a causal relationship between drug-induced topoisomerase II-mediated DNA breaks and cytotoxicity.

Aminoacridines↗
PubMed1988

Sister chromatid exchanges, chromosomal aberrations, and cytotoxicity produced by antitumor topoisomerase II inhibitors in sensitive (DC3F) and resistant (DC3F/9-OHE) Chinese hamster cells.

4'-(9-Acridinylamino)methanesulfon-m-anisidide, etoposide, and 2-methyl-9-hydroxyellipticinium are antitumor topoisomerase II (topo II) inhibitors. The relationship between drug-induced sister chromatid exchanges (SCEs) or chromosomal aberrations and cytotoxicity was investigated in Chinese hamster cells sensitive (DC3F) and resistant (DC3F/9-OHE) to topo II inhibitors. Thirty-min drug treatments produced SCEs and chromosomal aberrations in sensitive (DC3F) cells, 4'-(9-acridinylamino)methanesulfon-m-anisidide being more potent than etoposide or 2-methyl-9-hydroxyellipticinium at equimolar concentrations. Comparable treatments of resistant (DC3F/9-OHE) cells did not produce chromosomal damage. The cytotoxicity of 4'-(9-Acridinylamino)-methanesulfon-m-anisidide was also greater than that of etoposide or 2-methyl-9-hydroxyellipticinium in DC3F cells, and no cytotoxicity was observed in DC3F/9-OHE at drug concentrations that produced more than two logs of cell kill in DC3F cells. A plot of cytotoxicity versus SCEs showed a good correlation between the two parameters. Therefore, short treatments of mammalian cells with topo II inhibitors produce reversible topo II-mediated DNA breaks which are associated with chromosomal aberrations and SCEs whose number correlates with cytotoxicity. In addition, topo II mutant DC3F/9-OHE cells were more sensitive than DC3F cells to the chromosomal, DNA cross-linking and cytotoxic effects of mitomycin C and were equally sensitive to the cytotoxic effect of camptothecin.

Amsacrine↗
PubMed1987

Role of DNA intercalation in the inhibition of purified mouse leukemia (L1210) DNA topoisomerase II by 9-aminoacridines.

An attempt was made to analyze the mechanism by which 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) inhibits mammalian DNA topoisomerase II. The effects of various 9-aminoacridine derivatives differing by their DNA affinities and DNA sequence selectivity of binding were compared in the presence of purified mouse leukemia L1210 DNA topoisomerase II. No correlation was found between DNA unwinding and topoisomerase II inhibition. 9-Aminoacridine was inactive as a topoisomerase II inhibitor and o-AMSA was only weakly active. The location of L1210 topoisomerase II mediated DNA breaks produced in the absence or presence of 9-aminoacridines were studied in [32P]-end-labeled pBR 322 DNA. All 9-aminoacridines, even those differing by their DNA sequence selectivity of binding, produced similar DNA cleavage patterns. Most drug-induced topoisomerase II mediated DNA breaks appeared at sites that were already cleaved by the enzyme in the absence of drug. The present results suggest that 9-aminoacridines inhibit L1210 DNA topoisomerase II by interacting at or near enzyme-DNA complexes by some unknown DNA effect or by direct protein interaction.

Aminoacridines↗
PubMed1987

DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.

Aminacrine↗
PubMed1987

Topoisomerase II-mediated DNA breaks and cytotoxicity in relation to cell proliferation and the cell cycle in NIH 3T3 fibroblasts and L1210 leukemia cells.

The DNA intercalator, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and the nonintercalator, etoposide (VP-16) produce topoisomerase II-mediated protein-linked DNA strand breaks. This function of topoisomerase II was investigated in relation to cell proliferation and cell cycle. Mouse fibroblasts NIH 3T3 and mouse leukemia L1210 cells stop proliferation when they reach a certain density. Nuclei were isolated from proliferative or quiescent cells and then treated with drug for 30 min. DNA modifications were assayed by alkaline elution. We found that the frequencies of m-AMSA- or VP-16-induced DNA-protein links were higher in nuclei from exponentially growing than in those from quiescent cells in both the 3T3 and the L1210 lines. Drug-induced protein-associated DNA breaks were also studied as a function of the cell cycle in 3T3 cells that had been arrested by contact inhibition in medium containing 1% calf serum and then stimulated to proliferate by raplating at a lower cell density in medium containing 10% serum. In these synchronized cells, a large peak of [3H]thymidine incorporation occurred 18-30 h after replating. The yield of DNA-protein cross-links produced by 30-min drug treatments of nuclei isolated at various times after growth initiation increased concomitantly with the peak of the DNA synthesis. The topoisomerase II activity of nuclear extracts, as measured by kinetoplast DNA decatenation followed a similar pattern. Using colony-forming assays, we also observed that m-AMSA and VP-16 were most cytotoxic in proliferative cells and during DNA synthesis. These results suggest that alkaline elution measurement of m-AMSA- or VP-16-induced protein-linked DNA breaks reflects the association of topoisomerase II with DNA. This association is increased during DNA replication, making the cells more vulnerable to m-AMSA and VP-16 at this time.

Amsacrine↗
PubMed1987

Protein-linked DNA strand breaks produced by etoposide and teniposide in mouse L1210 and human VA-13 and HT-29 cell lines: relationship to cytotoxicity.

The two demethylepipodophyllotoxin glycosides, teniposide (VM-26) and etoposide (VP-16), have previously been reported to interact with DNA topoisomerase II by stabilizing a topoisomerase II-DNA covalent intermediate. This study examined the protein-associated aspect of the topoisomerase II-DNA-epipodophyllotoxin lesion. We found that in mouse (L1210) and human (VA-13 and HT-29) log-phase cell cultures, all DNA strand breaks produced by VP-16 or VM-26 were protein-associated. We found also that these protein-associated breaks occurred with a frequency which correlated with cytotoxicity in all three cell lines. For all three cell lines and for both compounds the regression lines were similar. Therefore, for a given class of topoisomerase II inhibitors, it may be possible to generate a characteristic line from which DNA-protein crosslink frequency predicts cytotoxicity.

Animals↗