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Biomedical subjects

Y Pommier

Publications and source records attributed to Y Pommier.

At least 19 recordsLinked to original sources
PubMed2002

Induction of breast cancer resistance protein by the camptothecin derivative DX-8951f is associated with minor reduction of antitumour activity.

DX-8951f (exatecan mesylate), a new water-soluble derivative of camptothecin, is currently being evaluated in phase II clinical trials. Resistance may be acquired when treating cancer patients with DX-8951f. Therefore, we selected a subline of the human ovarian cancer cell line A2780 for resistance against DX-8951f to investigate possible mechanisms of resistance. This DX-8951f-resistant subline, designated 2780DX8 (resistance factor=9.3), displayed a typical cross-resistance pattern including compounds, such as topotecan (resistance factor =34), SN-38 (resistance factor =47), mitoxantrone (resistance factor =59) and doxorubicin (resistance factor =2.9), which have previously been associated with the expression of breast cancer resistance protein. 2780DX8 cells did not show changes in the topoisomerase I gene, in topoisomerase I protein levels or catalytic activity. Overexpression of breast cancer resistance protein could be detected, both at the mRNA and protein level, while staining for Pgp, MRP1, or LRP was negative. GF120918, an inhibitor of breast cancer resistance protein, was able to reverse the DX-8951f-induced resistance in 2780DX8 cells. In vivo experiments in well-established 2780DX8 human tumour xenografts demonstrated that the growth inhibition induced by CPT-11 was more affected by breast cancer resistance protein expression than that of DX-8951f. These data indicate for the first time that DX-8951f is able to induce breast cancer resistance protein as a mechanism of resistance. Breast cancer resistance protein, however, results in only minor reduction of antitumour activity of DX-8951f which is an advantage over topotecan and CPT-11/SN-38.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Loss of heterozygosity of nucleotide excision repair factors in sporadic ovarian, colon and lung carcinomas: implication for their roles of carcinogenesis in human solid tumors.

The deficiencies of nucleotide excision repair (NER) factors are genetic diseases, xeroderma pigmentosum (XP) increasing risk of developing cancer on sun-exposed areas of the skin. However, the abnormality of NER factors in human sporadic carcinoma remains unclear. Loss of heterozygosity (LOH) analysis for the XP, XPA, XPB, XPC, XPD, XPE, XPF, XPG and the transcription-coupled repair factor, Cockayne syndrome B (CSB) revealed that NER factors were abnormal in 62.1 % of ovarian tumors (18/29), 16.7% of colon (2/12) and 22.2% lung (2/9) carcinomas. Furthermore, 13.8% of ovarian, 8.3% of colon and 22% of lung carcinomas exhibited LOH for NER factors without LOH for tumor suppressor genes such as p53, FHIT, APC, BRCAI, BRCA2 and DCC. Although both microsatellite instability and LOH of NER factors were observed in some cases, there was no strong association between them in the present study. These observations raise the possibility that alterations of NER factors may be frequent in human sporadic carcinomas. Further study should be needed to find the direct evidence of NER gene abnormalities in human sporadic carcinoma tissues.

Chromosome Aberrations↗
PubMed2001

Quantitative structure-antitumor activity relationships of camptothecin analogues: cluster analysis and genetic algorithm-based studies.

Topoisomerase 1 (top1) inhibitors are proving useful against a range of refractory tumors, and there is considerable interest in the development of additional top1 agents. Despite crystallographic studies, the binding site and ligand properties that lead to activity are poorly understood. Here we report a unique approach to quantitative structure-activity relationship (QSAR) analysis based on the National Cancer Institute's (NCI) drug databases. In 1990, the NCI established a drug discovery program in which compounds are tested for their ability to inhibit the growth of 60 different human cancer cell lines in culture. More than 70 000 compounds have been screened, and patterns of activity against the 60 cell lines have been found to encode rich information on mechanisms of drug action and drug resistance. Here, we use hierarchical clustering to define antitumor activity patterns in a data set of 167 tested camptothecins (CPTs) in the NCI drug database. The average pairwise Pearson correlation coefficient between activity patterns for the CPT set was 0.70. Coherence between chemical structures and their activity patterns was observed. QSAR studies were carried out using the mean 50% growth inhibitory concentrations (GI(50)) for 60 cell lines as the dependent variables. Different statistical methods, including stepwise linear regression, principal component regression (PCR), partial least-squares regression (PLS), and fully cross-validated genetic function approximation (GFA) were applied to construct quantitative structure-antitumor relationship models. For our data set, the GFA method performed better in terms of correlation coefficients and cross-validation analysis. A number of molecular descriptors were identified as being correlated with antitumor activity. Included were partial atomic charges and three interatomic distances that define the relative spatial dispositions of three significant atoms (the hydroxyl hydrogen of the E-ring, the lactone carbonyl oxygen of the E-ring, and the carbonyl oxygen of the D-ring). The cross-validated r(2) for the final GFA model was 0.783, indicating a predictive QSAR model.

Algorithms↗
PubMed2001

Antisense inhibition of Chk2/hCds1 expression attenuates DNA damage-induced S and G2 checkpoints and enhances apoptotic activity in HEK-293 cells.

The cellular response to DNA damage involves checkpoint controls that delay cell cycle progression in order to provide time for repair of damaged DNA. Chk2/hCds1 is a recently identified homolog of the yeast Cds1 kinase that is involved in cell cycle checkpoint response to DNA damage. To investigate the functions of Chk2/hCds1 in response to DNA damage in mammalian cells, we established a stable human kidney embryonic cell line (HEK-293) that expresses antisense Chk2/hCds1 (Chk2AS) under the control of an inducible promoter. Cells that express Chk2AS display defective S-phase delay in response to DNA replication-mediated DNA damage induced by the topoisomerase I inhibitor camptothecin. The defective G2 checkpoint was also observed in Chk2AS cells exposed to the DNA damaging agent VP-16 or gamma-radiation. Enhanced apoptosis was observed in Chk2AS cells after exposure to gamma-radiation or camptothecin. No p53 activation was observed after DNA damage in HEK-293 or Chk2AS cells. Our results indicate that perturbation of Chk2/hCds1 expression adversely affects the S- and G2-phase checkpoints following DNA damage or DNA replication block, and suggest that reduced expression of Chk2/hCds1 might promote a p53-independent apoptotic response.

Apoptosis↗
PubMed2001

Human mitochondrial topoisomerase I.

Tension generated in the circular mitochondrial genome during replication and transcription points to the need for mtDNA topoisomerase activity. Here we report a 601-aa polypeptide highly homologous to nuclear topoisomerase I. The N-terminal domain of this novel topoisomerase contains a mitochondrial localization sequence and lacks a nuclear localization signal. Therefore, we refer to this polypeptide as top1mt. The pattern of top1mt expression matches the requirement for high mitochondrial activity in specific tissues. top1mt is a type IB topoisomerase that requires divalent metal (Ca(2+) or Mg(2+)) and alkaline pH for optimum activity. The TOP1mt gene is highly homologous to the nuclear TOP1 gene and consists of 14 exons. It is localized on human chromosome 8q24.3.

Amino Acid Sequence↗
PubMed2001

Discovery of a nuclease-resistant, non-natural dinucleotide that inhibits HIV-1 integrase.

Integration of HIV viral DNA into human chromosomal DNA catalyzed by HIV integrase is essential for the replication of HIV. Discovery of novel inhibitors of HIV integrase is of considerable significance in approaches to the development of therapeutic agents against AIDS. We have synthesized a new dinucleotide 1 with an internucleotide phosphate bond that is unusually resistant to exonucleases. This compound exhibits potent anti-HIV-1 integrase activity.

DNA, Viral↗
PubMed2001

Activation of the Fas pathway independently of Fas ligand during apoptosis induced by camptothecin in p53 mutant human colon carcinoma cells.

The present study explored the role of the cell surface receptor Fas (CD95/APO-1) in apoptosis induced by camptothecin (CPT) in the HT29 colon carcinoma cell line. CPT-induced apoptosis was associated with high molecular weight DNA fragmentation as measured by filter elution. This fragmentation was inhibited by the caspase inhibitor, z-VAD-fmk and by cycloheximide, which also prevented proteolytic activation of caspase-3 and poly(ADP-ribose)polymerase cleavage. Under such conditions, Fas, Fas ligand, Bax, and p21 expression were increased and Fas recruited the FADD adaptor. Fas expression increase was blocked by cycloheximide but not by z-VAD-fmk, consistent with caspase activation downstream from Fas. Treatment of HT29 cells with FasL or with the CH-11 agonistic anti-Fas antibody potentiated the apoptotic response of cells treated with CPT. The anti-Fas blocking antibody ZB4 and the Fas-ligand inhibitor failed to protect HT29 cells from CPT-induced apoptosis. Such a protection was obtained by transient expression of constructs encoding a dominant-negative mutant of FADD, FADD in an antisense orientation and E8 or MC159 viral proteins that inhibit Fas-induced apoptosis at the level of FADD and procaspase-8, respectively. Together, these data show that topoisomerase I-mediated DNA damage-induced apoptosis involves activation of the Fas pathway without detectable Fas-ligand requirement in CPT-treated cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Characterization of a novel topoisomerase I mutation from a camptothecin-resistant human prostate cancer cell line.

In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.

Amino Acid Sequence↗
PubMed2001

Use of camptothecin-resistant mammalian cell lines to evaluate the role of topoisomerase I in the antiproliferative activity of the indolocarbazole, NB-506, and its topoisomerase I binding site.

NB-506 is a topoisomerase I (top1) inhibitor in clinical trials. In this study, we used a series of camptothecin (CPT)-resistant cell lines with known top1 alterations. We show that three mutations in different domains of the top1 enzyme that confer CPT resistance also confer cross-resistance to NB-506. The CPT-resistant cell lines and corresponding mutations were: human prostate carcinoma cells DU-145/RC1 (mutation R364H), Chinese hamster fibroblasts DC3F/C10 (mutation G503S), and human leukemia CEM/C2 cells (N722S). This result suggests that NB-506 and CPT share a common binding site in the top1-DNA complex. We next used these three cell lines and their parental cells to study the relationship between top1 poisoning by NB-506 and antiproliferative activity. We found that the CPT-resistant cells were only 2-10-fold resistant to NB-506, which suggests that NB-506 targets other cellular processes/pathways besides top1. This conclusion was further supported by the limited cross-resistance of top1-deficient murine leukemia P388/CPT45 cells (2-fold). Cross-resistance was also limited for J-109,382, an isomer of NB-506 that does not intercalate into DNA, indicating that the non-top1-mediated antiproliferative activity of NB-506 is not attributable to DNA intercalation. Together, these data indicate that NB-506 and indolocarbazoles are promising agents to overcome CPT resistance.

Antineoplastic Agents↗
PubMed2001

Topoisomerase I-mediated cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine: trapping of topoisomerase I by the O6-methylguanine.

Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are known to covalently link alkyl groups at the position 6 of guanines (O6MG) in DNA. O6-alkylguanine-DNA alkyltransferase (AGT) specifically removes the methyl group of the O6MG. Using purified human topoisomerase I (Top1), we found an 8-10-fold enhancement of Top1 cleavage complexes when O6MG is incorporated in oligonucleotides at the +1 position relative to a unique Top1 cleavage site. Top1 poisoning by O6MG is attributable to a decrease of the Top1-mediated DNA religation as well as an increase in the enzyme cleavage step. Increased cleavage is probably linked to a change in the hydrogen bonding pattern, such as in the case of the 8-oxoguanine, whereas inhibition of religation could be attributed to altered base pairing, such as abasic sites or base mismatches, because incorporation of a 6-thioguanine did not affect Top1 activity. Top1-DNA covalent complexes are also induced in MNNG-treated CHO cells constitutively lacking the AGT enzyme. Conversely, no increase could be detected in CHO cells transfected with the wild-type human AGT. Moreover, we show that yeasts overexpressing the human Top1 are more sensitive to MNNG, whereas knock-out Top1 strain cells display some resistance to the drug. Altogether, these results suggest a role for Top1 poisoning by alkylated bases in the antiproliferative activity of alkylating agents as well as in the DNA lesions resulting from endogenous and carcinogenic DNA modifications.

Alkylating Agents↗
PubMed2001

Topoisomerase I-mediated DNA damage.

Topoisomerase I is a ubiquitous and essential enzyme in multicellular organisms. It is involved in multiple DNA transactions including DNA replication, transcription, chromosome condensation and decondensation, and probably DNA recombination. Besides its activity of DNA relaxation necessary to eliminate torsional stresses associated with these processes, topoisomerase I may have other functions related to its interaction with other cellular proteins. Topoisomerase I is the target of the novel anticancer drugs, the camptothecins. Recently a broad range of physiological and environmentally-induced DNA modifications have also been shown to poison topoisomerases. This review summarizes the various factors that enhance or suppress top1 cleavage complexes and discusses the significance of such effects. We also review the different mechanisms that have been proposed for the repair of topoisomerase I-mediated DNA lesions.

Animals↗
PubMed2001

Arylisothiocyanate-containing esters of caffeic acid designed as affinity ligands for HIV-1 integrase.

Integrase is an enzyme found in human immunodeficiency virus, which is required for the viral life cycle, yet has no human cellular homologue. For this reason, HIV integrase (IN) has become an important target for the development of new AIDS therapeutics. Irreversible affinity ligands have proven to be valuable tools for studying a number of enzyme and protein systems, yet to date there have been no reports of such affinity ligands for the study of IN. As an initial approach toward irreversible ligand design directed against IN, we appended isothiocyanate functionality onto caffeic acid phenethyl ester (CAPE), a known HIV integrase inhibitor. The choice of isothiocyanate as the reactive functionality, was based on its demonstrated utility in the preparation of affinity ligands directed against a number of other protein targets. Several isomeric CAPE isothiocyanates were prepared to explore the enzyme topography for reactive nitrogen and sulfur nucleophiles vicinal to the enzyme-bound CAPE. The preparation of these CAPE isothiocyanates, required development of new synthetic methodology which employed phenyl thiocarbamates as latent isothiocyanates which could be unmasked near the end of the synthetic sequence. When it was observed that beta-mercaptoethanol (beta-ME), which is required to maintain the catalytic activity of soluble IN (a F185KC280S mutant), reacted with CAPE isothiocyanate functionality to form the corresponding hydroxyethylthiocarbamate, a variety of mutant IN were examined which did not require the presence of beta-ME for catalytic activity. Although in these latter enzymes, CAPE isothiocyanate functionality was presumed to be present and available for acylation by IN nucleophiles, they were equally effective against Cys to Ser mutants. One conclusion of these studies, is that upon binding of CAPE to the integrase, nitrogen or sulfur nucleophiles may not be properly situated in the vicinity of the phenethyl aryl ring to allow reaction with and covalent modification of reactive functionality, such as isothiocyanate groups. The fact that introduction of the isothiocyanate group onto various positions of the phenethyl ring or replacement of the phenyl ring with naphthyl rings, failed to significantly affect inhibitory potency, indicates a degree of insensitivity of this region of the molecule toward structural modification. These findings may be useful in future studies concerned with the development and use of HIV-1 integrase affinity ligands.

Amino Acid Substitution↗
PubMed2001

Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair.

While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

Animals↗
PubMed2001

Structure-activity of inhibition of HIV-1 integrase and virus replication by G-quartet oligonucleotides.

As novel anti-HIV agents, the G-tetrad-forming oligonucleotides have been explored for their structure-activity relations with regard to inhibition of integrase (IN) (N. Jing, Expert Opin. Investig. Drugs (2000) 9, 1777-1785). We have now developed two families of G-quartet oligonucleotides: T40217-T40222, with potential formation of a tail-to-tail G-quartet dimer, and T40224-T40227, with phosphorothioate (PT) linkages in the guanine loops. The results obtained from biophysical measurements and the assays of the inhibition of HIV-1 IN and virus replication demonstrated that an increase in the length of the G-quartet structure from a monomer (15A) to a tail-to-tail dimer (47A) does not distinctly disrupt the inhibition of HIV-1 IN activity or the inhibition of HIV-1 replication in cell cultures. G-quartet oligonucleotides were observed to induce molecular aggregation of HIV-1 IN and interrupt the binding of viral DNA to HIV-1 IN. Also, PT substitutions did not confer any advantages compared with the regular phosphodiesters for the inhibition of HIV-1 replication by intramolecular G-quartets. The G-quartet motif is the primary requirement for the remarkable nuclease resistance and pronounced biological efficacy of these oligonucleotides.

Cell Line↗
PubMed2001

Ecteinascidin 743 induces protein-linked DNA breaks in human colon carcinoma HCT116 cells and is cytotoxic independently of topoisomerase I expression.

Ecteinascidin 743 (Et743; NSC 648766) is a potent antitumor agent presently in clinical trials. Et743 selectively alkylates guanine N2 from the minor groove of duplex DNA and bends the DNA toward the major groove. This differentiates Et743 from other DNA-alkylating agents presently in the clinic. To date, the cellular effects of Et743 have not been elucidated. Recently, Et743 DNA adducts have been found to suppress gene expression selectively and to induce topoisomerase I (top1) cleavage complexes in vitro and top1-DNA complexes in cell culture. In the present study, we characterized the DNA damage and the cell cycle response induced by Et743 in human colon carcinoma HCT116 cells. Alkaline elution experiments demonstrated that micromolar concentrations of Et743 produced comparable frequencies of DNA-protein cross-links and DNA single-strand breaks. The single-strand breaks were protein-cross-linked and were not associated with detectable DNA double-strand breaks. By contrast with camptothecin, these lesions persisted for several hours after drug removal and were not formed at 4 degrees C. Et743 treatment induced transient p53 elevation, dose-dependent cell cycle accumulation in G2-M and in G1- and S-phase, and inhibition of DNA synthesis. The sensitivity of camptothecin-resistant mouse leukemia P388/ CPT45 cells, which fail to express detectable top1, was similar to the sensitivity of wild-type P388 cells, suggesting that top1 is not a critical target for the antiproliferative activity of Et743.

Antineoplastic Agents, Alkylating↗
PubMed2001

Dual role of glutathione in modulating camptothecin activity: depletion potentiates activity, but conjugation enhances the stability of the topoisomerase I-DNA cleavage complex.

Depletion of glutathione (GSH) in MCF-7 and MDA-MB-231 cell lines by pretreatment with the GSH synthesis inhibitor buthionine sulfoximine potentiated the activity of 10,11-methylenedioxy-20(S)-camptothecin, SN-38 [7-ethyl-10-hydroxy-20(S)-camptothecin], topotecan, and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin (CMMDC). The greatest potentiation was observed with the alkylating camptothecin CMMDC. Buthionine sulfoximine pretreatment also increased the number of camptothecin-induced DNA-protein crosslinks, indicating that GSH affects the mechanism of action of camptothecin. We also report that GSH interacts with CMMDC to form a stable conjugate, 7-(glutathionylmethyl)-10,11-methylenedioxy-20(S)-camptothecin (GSMMDC), which is formed spontaneously in buffered solutions and in MCF-7 cells treated with CMMDC. GSMMDC was synthesized and found to be nearly as active as 10,11-methylenedioxy-20(S)-camptothecin in a topoisomerase (topo) I-mediated DNA nicking assay. The resulting topo I cleavage complexes were remarkably stable. In cell culture, GSMMDC displayed potent growth-inhibitory activity against U937 and P388 leukemia cell lines. GSMMDC was not active against a topo I-deficient P388 cell line, indicating that topo I is its cellular target. Peptide-truncated analogues of GSMMDC were prepared and evaluated. All three derivatives [7-(gamma-glutamylcysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, 7-(cysteinylglycylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, and 7-(cysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin] displayed topo I and cell growth-inhibitory activity. These results suggest that 7-peptidyl derivatives represent a new class of camptothecin analogues.

Antineoplastic Agents, Phytogenic↗