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Biomedical subjects

Y Pommier

Publications and source records attributed to Y Pommier.

At least 163 records · Page 9Linked to original sources
PubMed1994

Topoisomerase II inhibition and cytotoxicity of the anthrapyrazoles DuP 937 and DuP 941 (Losoxantrone) in the National Cancer Institute preclinical antitumor drug discovery screen.

BACKGROUND: The cumulative cardiotoxicity of anthracyclines is thought to result from the generation of free radicals. New DNA topoisomerase II inhibitors less prone to redox reactions, such as mitoxantrone and more recently the anthrapyrazoles, were developed to circumvent this toxicity. PURPOSE: Two anthrapyrazoles currently in clinical evaluation, DuP 941 (Losoxantrone) and DuP 937, were compared to other topoisomerase II inhibitors with respect to their cytotoxic potency and selectivity and with respect to topoisomerase II inhibition. METHODS: Cytotoxicity was tested in the 60 cell lines of the National Cancer Institute preclinical antitumor drug discovery screen (NCI screen). The potency of anthrapyrazoles to inhibit purified topoisomerase II was determined. The specificity of drug-induced topoisomerase II pattern of cleavage, one of the cellular determinants of cytotoxicity, was investigated in human c-myc DNA. RESULTS: Using the COMPARE analysis, we found that the most closely related cytotoxic profiles in the NCI screen were between the anthrapyrazoles and mitoxantrone. Among topoisomerase II inhibitors, the cytostatic potency was by decreasing order: mitoxantrone; doxorubicin, which was slightly greater than DuP 941, azatoxin; DuP 937; and amsacrine, which was much greater than VP-16. The potency of mitoxantrone and anthrapyrazoles to generate DNA double-strand breaks, by induction of the topoisomerase II cleavable complexes in nuclear extracts, was in agreement with cytotoxicity. Sequencing of drug-induced topoisomerase II cleavages in c-myc DNA showed a common cleavage pattern for anthrapyrazoles and mitoxantrone. This pattern was different from the patterns obtained with other topoisomerase II inhibitors. CONCLUSION: At the molecular and cellular levels, anthrapyrazoles are potent topoisomerase II inhibitors closely related to mitoxantrone. IMPLICATIONS: These results validate the COMPARE analysis using the NCI screen to predict molecular mechanisms of drug action. Anthrapyrazoles, which are unlikely to produce free radicals, might be useful in the same indications as mitoxantrone, especially for patients with cardiac risks, for pediatric patients, and for patients treated with intensified protocols.

Anthraquinones↗
PubMed1994

Inhibition of HIV-1 integrase by flavones, caffeic acid phenethyl ester (CAPE) and related compounds.

The inhibition of HIV-1 integrase by flavones and related compounds was investigated biochemically and by means of structure-activity relationships. Purified enzyme and synthetic oligonucleotides were used to assay for three reactions catalysed by integrase: (1) processing of 3' termini by cleavage of the terminal dinucleotide; (2) strand transfer, which models the integration step; and (3) "disintegration," which models the reversal of the strand transfer reaction. Inhibitions of all three reactions by flavones generally occurred in parallel, but caffeic acid phenethyl ester (CAPE) appeared to inhibit reaction 2 selectively. CAPE, however, inhibited reactions 1 and 3 effectively when preincubated with the enzyme, suggesting that this compound differs from the flavones primarily in requiring more time to block the enzyme. The core integrase fragment consisting of amino acids 50-212 retained the ability to catalyse reaction 3, and flavones and CAPE retained the ability to inhibit. Hence, the putative zinc-finger region that is deleted in this fragment is probably not the target of inhibition. Inhibition by flavones usually required the presence of at least one ortho pair of phenolic hydroxyl groups and at least one or two additional hydroxyl groups. Potency was enhanced by the presence of additional hydroxyl groups, especially when present in ortho pairs or in adjacent groups of three. Inhibitory activity was reduced or eliminated by methoxy or glycosidic substitutions or by saturation of the 2,3 double bond. These structure-activity findings for flavones were generally concordant with those previously reported for reverse transcriptase and topoisomerase II. These findings are discussed in the context of a review of the effects of flavones on various enzymes, the possible mechanisms of inhibition, and the potential for building upon a general pharmacophore to generate target specificity.

Base Sequence↗
PubMed1994

Hydroxyrubicin, a deaminated derivative of doxorubicin, inhibits mammalian DNA topoisomerase II and partially circumvents multidrug resistance.

In vivo effectiveness of doxorubicin remains restricted due to toxicity and drug resistance. Hydroxyrubicin is a synthetic analog of doxorubicin in which the basic amino group at the C-3' has been replaced by a hydroxyl group in order to overcome recognition by the multidrug resistant (MDR) P-glycoprotein and limit cardiotoxicity. The present study shows that hydroxyrubicin is a less potent intercalator than doxorubicin. Induction of topoisomerase II-mediated DNA cleavage in the human c-myc origin by the two drugs was similar, reaching a maximum at 0.5 microM. Results from the NCI Cell Screening program indicate a relatively good correlation between the cytotoxicity of the 2 drugs on 55 cell lines of various origins (r = 0.723). Using a clonogenic assay, we observed that hydroxyrubicin was 20-fold more cytotoxic against the MDR KB-V1 cell line than doxorubicin and was slightly more cytotoxic than doxorubicin in the sensitive KB3.1 cell line. Uptake studies showed that doxorubicin was retained up to 1 hr in KB3.1 cells and rapidly eliminated from resistant KB-V1 cells. In contrast, hydroxyrubicin was rapidly eliminated from both sensitive KB3.1 and MDR-positive KB-V1 cells. Both drugs induced protein-linked DNA single-strand breaks (SSBs) in both KB3.1 and KB-V1 cells, which is consistent with topoisomerase inhibition. However, the kinetics of DNA SSBs induced by both drugs was very different. DNA breaks disappeared quickly in both KB3.1 and KB-V1 cell lines after hydroxyrubicin removal while DNA breaks induced by doxorubicin disappeared very slowly in KB3.1 cells and rapidly in KB-V1 cells. We conclude that removal of the basic amino group at the C-3' of doxorubicin modifies drug transport and partially circumvents MDR without changing topoisomerase II inhibition when compared with doxorubicin.

Antibiotics, Antineoplastic↗
PubMed1994

Inhibition of human immunodeficiency virus type 1 integrase by 3'-azido-3'-deoxythymidylate.

The effects of 3'-azido-3'-deoxythymidine (AZT) and three of its intracellular metabolites, azido- thymidine mono-, di-, and triphosphates, on the human immunodeficiency virus type 1 integrase have been determined. AZT mono-, di-, and triphosphate have an IC50 for integration between 110 and 150 microM, whereas AZT does not inhibit the integrase. The inhibition by AZT monophosphate can be partially reversed by coincubation with either thymidine monophosphate or 2',3'-dideoxythymidine monophosphate, suggesting that either of these monophosphates can bind to the integrase but that the azido group at the 3' position could be responsible for the inhibition. Integrase inhibition is associated with reduced enzyme-DNA binding but does not appear to be competitive with respect to the DNA substrate. Inhibition of an integrase deletion mutant containing only amino acids 50-212 suggests that these nucleotides bind in the catalytic core. Concentrations up to 1 mM AZT monophosphate can accumulate in vivo, indicating that integrase inhibition may contribute to the antiviral effects of AZT. The increasing incidence of AZT-resistant virus strains may, therefore, be associated with mutations not only in the reverse transcriptase but also in the human immunodeficiency virus integrase. Finally, these observations suggest that additional strategies for antiviral drug development could be based upon nucleotide analogs as inhibitors of human immunodeficiency virus integrase.

Base Sequence↗
PubMed1994

Comparison of topoisomerase I inhibition, DNA damage, and cytotoxicity of camptothecin derivatives presently in clinical trials.

BACKGROUND: Camptothecins belong to a group of anticancer agents with a unique mechanism of action: poisoning of eukaryotic DNA topoisomerase I. Two camptothecin derivatives, topotecan (TPT) and CPT-11, are in clinical trials and their chemotherapeutic efficacy appears promising. PURPOSE: Our aim was to compare simultaneously the molecular and cellular pharmacology of the various camptothecin derivatives that are presently in clinical trials. METHODS: Cytotoxicity of drugs toward human colon carcinoma HT-29 cells was determined by colony-forming assays. DNA single-strand breaks (SSB) were measured by alkaline elution. Drug potency to induce topoisomerase 1-mediated DNA cleavage and the sequence selectivity of the breaks were determined by sequencing gel autoradiography. RESULTS: SN-38 and CPT were more cytotoxic than 9-AC and TPT, and CPT-11 was almost inactive toward HT-29 cells. IC50 values were 8.8 nM for SN-38, 10 nM for CPT, 19 nM for 9-AC, 33 nM for TPT, and greater than 100 nM for CPT-11. In drug-induced DNA damage measured by alkaline elution drug concentrations producing 1000-rad-equivalents (C1000), values were 0.037 microM for SN-38, 0.051 microM for CPT, 0.085 microM for 9-AC, 0.28 microM for TPT, and greater than 1 microM for CPT-11. SN-38 remained the most potent compound in isolated nuclei, and CPT-11 was still inactive. The potency ranking was the same as in whole cells, and the C1000 values were 0.0025 microM for SN-38, 0.012 microM for CPT, 0.021 microM for 9-AC, 0.44 microM for TPT, and greater than 0.1 microM for CPT-11. Potency difference between SN-38 and the other compounds was greater in isolated nuclei than in whole cells. CONCLUSIONS: Kinetics of the reversal of drug-induced SSB in isolated nuclei suggest that dissociation of SN-38 from cleavable complexes is much slower than that of CPT. Cleavage patterns of CPT and 9-AC were similar but differed from those of TPT and SN-38. Although in vitro analyses do not necessarily reflect chemotherapeutic efficacy, this study found that SN-38 is the most potent compound and that 9-AC and TPT are less active than CPT in this system. The effect of CPT-11 is minimal. Therefore, the clinical activity of CPT-11 may strongly depend on its hydrolysis to SN-38. Differences in DNA sequence selectivity and the stability of cleavable complexes induced by the drugs may also contribute to differences among CPT derivatives.

Camptothecin↗
PubMed1994

Intermolecular disintegration and intramolecular strand transfer activities of wild-type and mutant HIV-1 integrase.

We report the activities of HIV integrase protein on a novel DNA substrate, consisting of a pair of gapped duplex molecules. Integrase catalyzed an intermolecular disintegration reaction that requires positioning of a pair of the gapped duplexes in a configuration that resembles the intgration intermediate. However, the major reaction resulted from an intramolecular reaction involving a single gapped duplex, giving rise to a hairpin. Surprisingly, a deletion mutant of integrase that lacks both the amino and carboxyl terminal regions still catalyzed the intermolecular disintegration reaction, but supported only a very low level of the intramolecular reaction. The central core region of integrase is therefore sufficient to both bind the gapped duplex DNA and juxtapose a pair of such molecules through protein-protein interactions. We suggest that the branched DNA structures of the previously reported disintegration substrate, and the intermolecular disintegration substrate described here, assist in stabilizing protein-protein interactions that otherwise require the amino and carboxy terminal regions of integrase.

Base Sequence↗
PubMed1994

Effects of DNA methylation on topoisomerase I and II cleavage activities.

DNA methylation is deregulated during oncogenesis. Since several major anti-cancer drugs act on topoisomerases, we investigated the effects of cytosine methylation on topoisomerase cleavage activities. Both topoisomerase I and II cleavage patterns were modified by CpG methylation in c-myc gene DNA fragments. Topoisomerase II changes, mainly cleavage reduction, occurred for methylation sites within 7 base pairs from the topoisomerase II breaks and were different for VM-26 and azatoxin. For topoisomerase I, cleavage enhancement as well as suppression were observed. Using synthetic methylated oligonucleotides, we show that hemimethylation is sufficient to alter topoisomerase I activity. Cytosine methylation on the scissile strand within the topoisomerase I consensus sequence had strong effects. Cleavage was stimulated by methylation at position -4 and was strongly inhibited by methylation at position -3 (with position -1 being the enzyme-linked nucleotide). This inhibitory effect was attributed to the presence of a methyl group in the major groove, since the transition uracil to thymine also inhibited cleavage. Altogether these results suggest an interaction of topoisomerase I with the DNA major grove at positions -3 and -4. In addition, DNA methylation may have profound effects on the activity of topoisomerases and may alter the distribution of cleavage sites produced by anticancer drugs in chromatin.

Base Sequence↗
PubMed1994

Induction of a common pathway of apoptosis by staurosporine.

The present observations show that staurosporine can rapidly trigger both the morphological changes and intranucleosomal DNA fragmentation typical of apoptosis. This occurred in a number of cell lines from various origins regardless of the state of differentiation and cell cycle phase, suggesting the presence of a common inducible suicide pathway. The broad apoptotic activity of staurosporine appears to be unique among other protein kinase or phosphatase inhibitors we tested. Results obtained in a cell-free assay suggest that cytoplasmic proteins directly modulated by staurosporine may be involved in a ubiquitous signal for the induction of DNA fragmentation and apoptosis.

Alkaloids↗
PubMed1994

Induction of DNA double-strand breaks by 8-methoxycaffeine: cell cycle dependence and comparison with topoisomerase II inhibitors.

We have studied the ability of 8-methoxycaffeine (8-MOC)--one of the most effective caffeine derivatives in inducing chromosomal aberrations--to induce DNA double strand breaks (DSB) in purified human T lymphocytes during the cell cycle. Etoposide- or ellipticine-mediated DNA break frequency was used as a parameter of topoisomerase II activity. DNA-DSB induced by either 8-MOC or VP16 or ellipticine rose co-ordinately with the level of DNA topoisomerase II and with the onset of DNA replication. At concentrations between 10 and 50 microM 8-MOC was approximately 75% as active in terms of DSB as VP16 and ellipticine. By contrast with VP16 and ellipticine, 8-MOC was not cytotoxic. In conclusion, our data suggest that 8-MOC is an agent that efficiently induces DNA-DSB at non-toxic concentrations, and without direct inhibition of topoisomerase II.

Caffeine↗
PubMed1994

Apoptosis induced by DNA topoisomerase I and II inhibitors in human leukemic HL-60 cells.

The induction of apoptosis following topoisomerase inhibitors proceeds in at least three distinct steps: (1) induction of cleavable complexes (potentially lethal damage), (2) topoisomerase-induced DNA damage, and (3) a presently unknown sequence of events that must either lead to cell cycle arrest (G2-block, differentiation) or apoptosis. DNA degradation provides a convenient way to quantify apoptosis in HL-60 cells. Extensive apoptosis can be induced rapidly in undifferentiated HL-60 cells without prevention by cycloheximide or actinomycin D. Therefore, HL-60 cells appear to express constitutively the apoptotic machinery that may be kept under control of a yet unknown repressor. The absence of the tumor suppressor p53 and the presence of bcl-2 are in contrast with the sensitivity of these cells to apoptosis. Agents that modify chromatin structure (zinc, poly[ADPribose] inhibitors, spermine) can block DNA fragmentation without affecting cell survival. By contrast macrophage-like differentiation by phorbol esters suppresses apoptosis without affecting topoisomerase-induced DNA damage. Better understanding of the apoptotic regulation in the widely used and characterized HL-60 cell line should allow the identification of new mechanisms and parameters of cellular sensitivity and resistance to the cytotoxic activity of anticancer agents.

Antineoplastic Agents↗
PubMed1994

[Pleiotropic resistance associated with topoisomerases].

There are at least two well-characterized mechanism of resistance to Topo I and II inhibitors: modifications of intracellular accumulation and reduced formation of cleavable complexes. Limited drug accumulation is usually due to P-glycoproteinMDR or to Multidrug Resistance associated Protein (MRP). Reduction of Topo I (or II) cleavable complexes not related to drug transport can either be due to decreased enzyme levels or enzyme mutations. For Topo II inhibitors, differential expression of the Topo II isoforms alpha and beta and changes in Topo II phosphorylation may also contribute to resistance. For dual Topo I and II inhibitors, resistance mechanisms are more complex to analyze but may also involve dual Topo I and II alterations. Finally, in a given cell line, several mechanisms are commonly associated in pleiotropic resistance to Topo inhibitors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Apoptosis of human leukemic HL-60 cells induced to differentiate by phorbol ester treatment.

In the human leukemic HL-60 cell line, we have reported previously that monocytic/macrophage-like differentiation induced by TPA (12-O-tetradecanoylphorbol-13-acetate) was associated with a decreased sensitivity to various apoptosis-inducing stimuli (Solary, Bertrand, Pommier, Blood 1993; 81:1359-1368). In the present study, we studied further the effects of TPA alone on the induction of apoptosis in HL-60 cells. Based on morphology by electron microscopy, identification of internucleosomal DNA cleavage by gel electrophoresis and quantitation of DNA fragmentation by a filter binding assay, we observed that neither morphologic changes nor DNA damage were identified in TPA-differentiated HL-60 cells as long as they kept the adherent phenotype characteristic of this differentiation pathway. However, adherent TPA-treated HL-60 cells that secondarily detached from the flask demonstrated internucleosomal DNA fragmentation associated with morphologic changes characteristic of apoptosis. Similarly, HL-60 cells that never became adherent after TPA treatment underwent rapid apoptosis. Granulocytic differentiation by retinoic acid (RA) treatment also induced apoptosis although more slowly. Interestingly, in both TPA- and RA-treated cells, apoptotic bodies appeared to be phagocytosed by differentiated cells from the same lineage. Internucleosomal DNA fragmentation was also identified in HL-60 cells induced to differentiate by sodium butyrate and dimethylsulfoxide treatment, suggesting that apoptosis could be the common mode of death of terminally differentiated HL-60 cells.

Apoptosis↗
PubMed1993

Cloning of Chinese hamster DNA topoisomerase I cDNA and identification of a single point mutation responsible for camptothecin resistance.

A camptothecin-resistant (DC3F/C-10) Chinese hamster cell line that contains a catalytically altered and camptothecin (CPT)-resistant DNA topoisomerase I (top 1) (Tanizawa, A., and Pommier, Y. (1992) Cancer Res. 52, 1848-1854) and the parent cell line (DC3F) were used to compare top 1 mRNAs and cDNAs. Northern blot analysis showed a single 4.1-kilobase band without quantitative reduction between the two cell lines. We have cloned and sequenced top 1 cDNAs. DC3F and DC3F/C-10 top 1 c-DNA are 3591 and 3626 base pair long, respectively, and encode 767 amino acids. The homology of deduced amino acid sequences between Chinese hamster and mouse or human top 1 are 98.1 and 96.7, respectively. cDNAs from DC3F/C-10 and DC3F cells differ by a single base point mutation (G to A) which results in an amino acid change from Gly505 to Ser (Gly505-->Ser). G505 corresponds to Gly503 of human top 1 cDNA and is located 220 amino acids away from the presumed catalytic Tyr725. The point mutation in the Chinese hamster top 1 is located in a region that is highly conserved among all cloned top 1 cDNAs (plant ATH, vaccinia virus, Shope fibroma virus, Drosophila, Saccharomyces cerevisiae, Schizosaccharomyces pombe, mouse, and Human). A mutation of Asp533 to Gly in this same region has been shown to confer CPT resistance for human top 1. Chinese hamster top 1 protein with a Gly505-->Ser mutation that was expressed in bacteria was resistant to CPT, indicating that this single base mutation is involved in CPT resistance. Our results suggest that the highly conserved region around Gly505 plays an important role in the interactions among top 1, DNA, and CPT.

Amino Acid Sequence↗
PubMed1993

Induction of cleavage in topoisomerase I c-DNA by topoisomerase I enzymes from calf thymus and wheat germ in the presence and absence of camptothecin.

In this study, we further examined the sequence selectivity of camptothecin in mammalian topoisomerase I cDNA from human and Chinese hamster. In the absence of camptothecin, almost all the bases at the 3'-terminus of cleavage sites are T for calf thymus and wheat germ topoisomerase I. In addition, wheat germ topoisomerase I exhibits preference for C (or not T) at -3 and for T at -2 position. As for camptothecin-stimulated cleavage with topoisomerase I, G (or not T) at +1 is an additional strong preference. This sequence selectivity of camptothecin is similar to that previously found in SV40 DNA, suggesting that camptothecin preferentially interacts with topoisomerase I-mediated cleavage sites where G is the base at the 5'-terminus. These results support the stacking model of camptothecin (Jaxel et al. (1991) J. Biol. Chem. 266, 20418-20423). Comparison of calf thymus and wheat germ topoisomerase I-mediated cleavage sites in the presence of camptothecin shows that many major cleavage sites are similar. However, the relative intensities are often different. One of the differences was attributable to a bias at position -3 where calf thymus topoisomerase I prefers G and wheat germ topoisomerase I prefers C. This difference may explain the unique patterns of cleavage sites induced by the two enzymes. Sequencing analysis of camptothecin-stimulated cleavage sites in the surrounding regions of point mutations in topoisomerase I cDNA, which were found in camptothecin-resistant cell lines, reveals no direct relationship between DNA cleavage sites in vitro and mutation sites.

Animals↗
PubMed1993

bcl-2 protein inhibits etoposide-induced apoptosis through its effects on events subsequent to topoisomerase II-induced DNA strand breaks and their repair.

Previous studies have shown that bcl-2 overexpression can inhibit apoptosis induced by DNA-damaging agents widely used in cancer chemotherapy, including X-irradiation, alkylating agents (hydroperoxycyclophosphamide, etc.), and topoisomerase II inhibitors (etoposide, etc.). However, little is known about the mechanism by which bcl-2 overexpression inhibits apoptosis triggered by these agents. In this study, we examined whether bcl-2 overexpression could have effects on etoposide-induced DNA damage and its repair. For these experiments, we developed CH31 clones (mouse B-cells) stably transfected with human bcl-2 sense plasmids and compared these clones with a parental CH31 clone or CH31 clones with antisense plasmids. Overexpression of bcl-2 protein inhibited etoposide-induced apoptosis and cytotoxicity. However, there was no or little difference in the production and repair of DNA-protein cross-links, DNA single-strand breaks, and double-strand beaks among a parental CH31 clone and CH31 clones with human bcl-2 sense or antisense plasmids. These findings indicate that (a) apoptosis or cytotoxicity induced by etoposide can be separated into early events (formation of double-strand breaks, DNA single-strand breaks, and double-strand breaks) and later events (secondary DNA fragmentation or cell death) and (b) bcl-2 inhibits apoptosis and cytotoxicity induced by etoposide at some steps between these events.

Animals↗
PubMed1993

Specific interaction of camptothecin, a topoisomerase I inhibitor, with guanine residues of DNA detected by photoactivation at 365 nm.

Camptothecin-induced DNA photolesions were examined after UVA irradiation at 365 nm. DNA single-strand breaks were induced both in supercoiled and in relaxed SV40 DNA. In uniquely end-labeled human c-myc DNA, camptothecin-induced cleavage occurred exclusively at guanines and was markedly enhanced by hot piperidine treatment. Runs of polyguanines were the most cleaved, especially in their 5' flank. Primer extension experiments in the absence of piperidine treatment confirmed these results and did not show additional lesions. We found that synthetic single-stranded oligonucleotides were more reactive than duplex oligonucleotides. In addition, an excess of dideoxyguanosine triphosphates competed for camptothecin-induced DNA photolesions. Therefore, camptothecin stacking in DNA grooves is more likely than genuine drug intercalation. Groove shielding with sodium or magnesium reduced camptothecin-induced photodamage while minor groove occupancy with spermine extended damages. Photolesion mechanisms were investigated using scavengers. In aerobic conditions, the most effective scavengers were thiourea, sodium azide, and catalase. Protection by superoxide dismutase was weak, and mannitol was ineffective. In anaerobic conditions, lesions were more extensive. Taken together, these results show that photoactivated camptothecin interacts specifically and intimately with guanines. This finding is consistent with preferential stimulation of topoisomerase I cleavage at sites that bear a guanine at their 5'-DNA terminus [Jaxel, C., et al. (1991) J. Biol. Chem. 266, 1465-1469] and with the camptothecin stacking model at topoisomerase I DNA cleavage sites.

Animals↗