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Biomedical subjects

Y Pommier

Publications and source records attributed to Y Pommier.

At least 37 records · Page 2Linked to original sources
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↗
PubMed2000

Activity of a novel camptothecin analogue, homocamptothecin, in camptothecin-resistant cell lines with topoisomerase I alterations.

Homocamptothecin (hCPT), which differs from camptothecin (CPT) by the presence of an additional methylene group in the E-ring, was evaluated in CPT-resistant cell lines. Topoisomerase I (top1)-deficient leukemia P388/CPT45 cells were highly resistant to hCPT, which demonstrates that top1 is the primary target of hCPT. Three CPT-resistant cell lines with top1 point mutations (Chinese hamster lung fibroblast DC3F/C10, human prostate carcinoma DU-145/RC1, and human leukemia CEM/C2) and their top1 enzymes were cross-resistant to hCPT. The antiproliferative activity of hCPT was greater than that of CPT in both parental and CPT-resistant cell lines, particularly in the prostate cell lines. The top1 cleavage complexes formed in the presence of hCPT appear to be more stable than those induced by CPT. Together, these data indicate that hCPT is a specific top1 inhibitor, which shares a common binding site with CPT in the topl-DNA cleavage complexes. Because of its potency, hCPT might overcome resistance to CPT in some cancer cells.

Animals↗
PubMed2000

The novel silatecan 7-tert-butyldimethylsilyl-10-hydroxycamptothecin displays high lipophilicity, improved human blood stability, and potent anticancer activity.

We describe the rational design and synthesis of B- and A, B-ring-modified camptothecins. The key alpha-hydroxy-delta-lactone pharmacophore in 7-tert-butyldimethylsilyl-10-hydroxycamptothecin (DB-67, 14) displays superior stability in human blood when compared with clinically relevant camptothecin analogues. In human blood 14 displayed a t(1/2) of 130 min and a percent lactone at equilibrium value of 30%. The tert-butyldimethylsilyl group renders the new agent 25-times more lipophilic than camptothecin, and 14 is readily incorporated, as its active lactone form, into cellular and liposomal bilayers. In addition, the dual 7-alkylsilyl and 10-hydroxy substitution in 14 enhances drug stability in the presence of human serum albumin. Thus, the net lipophilicity and the altered human serum albumin interactions together function to promote the enhanced blood stability. In vitro cytotoxicity assays using multiple different cell lines derived from eight distinct tumor types indicate that 14 is of comparable potency to camptothecin and 10-hydroxycamptothecin, as well as the FDA-approved camptothecin analogues topotecan and CPT-11. In addition, cell-free cleavage assays reveal that 14 is highly active and forms more stable top1 cleavage complexes than camptothecin or SN-38. The impressive blood stability and cytotoxicity profiles for 14 strongly suggest that it is an excellent candidate for additional in vivo pharmacological and efficacy studies.

Animals↗
PubMed2000

Synthesis of new indeno[1,2-c]isoquinolines: cytotoxic non-camptothecin topoisomerase I inhibitors.

In an attempt to design and synthesize potential anticancer agents acting by inhibition of topoisomerase I (top1), a new series of indenoisoquinolines was prepared and tested for cytotoxicity in human cancer cell cultures and for activity against top1. The synthesis relied on the condensation of substituted Schiff bases with homophthalic anhydrides to produce cis-3-aryl-4-carboxyisoquinolones that were cyclized to indenoisoquinolines in the presence of thionyl chloride. Both top1 inhibitory activity and cytotoxicity maximized in a single compound, 6-[3-(2-hydroxyethyl)aminopropyl]-5,6-dihydro-2,3-dimethoxy-8, 9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline hydrochloride (19a), which proved to be a very potent top1 inhibitor having a 110 nM mean graph midpoint (MGM) when tested for cytotoxicity in 55 human cancer cell cultures. A number of structurally related indenoisoquinolines were also obtained that had both potent cytotoxicity as well as top1 inhibitory activity. The key feature of the more potent compounds was the presence of an aminoalkyl side chain on the indenoisoquinoline nitrogen atom. The DNA cleavage patterns induced by top1 in the presence of the indenoisoquinolines were different from those seen with camptothecin. Some of the cleavage sites induced by the indenoisoquinolines were different from those seen with camptothecin, and conversely, camptothecin induced unique cleavage sites not apparent with the indenoisoquinolines. However, both camptothecin and the indenoisoquinolines also induced DNA cleavage sites that were the same in both series but varied in intensity. In addition, some of the DNA cleavages seen with the free base of 19a (compound 18c) in the presence of top1 were inhibited at higher drug concentrations, suggesting either a direct inhibition of the enzyme or an alternative mechanism involving DNA intercalation. Consistent with intercalation, compound 18c did unwind DNA.

Antineoplastic Agents↗
PubMed2000

Position-specific trapping of topoisomerase I-DNA cleavage complexes by intercalated benzo[a]- pyrene diol epoxide adducts at the 6-amino group of adenine.

DNA topoisomerase I (top1) is the target of potent anticancer agents, including camptothecins and DNA intercalators, which reversibly stabilize (trap) top1 catalytic intermediates (cleavage complexes). The aim of the present study was to define the structural relationship between the site(s) of covalently bound intercalating agents, whose solution conformations in DNA are known, and the site(s) of top1 cleavage. Two diastereomeric pairs of oligonucleotide 22-mers, derived from a sequence used to determine the crystal structure of top1-DNA complexes, were synthesized. One pair contained either a trans-opened 10R- or 10S-benzo[a]pyrene 7, 8-diol 9,10-epoxide adduct at the N(6)-amino group of a central 2'-deoxyadenosine residue in the scissile strand, and the other pair contained the same two adducts in the nonscissile strand. These adducts were derived from the (+)-(7R,8S,9S,10R)- and (-)-(7S,8R,9R, 10S)-7,8-diol 9,10-epoxides in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans. On the basis of analogy with known solution conformations of duplex oligonucleotides containing these adducts, we conclude that top1 cleavage complexes are trapped when the hydrocarbon adduct is intercalated between the base pairs flanking a preexisting top1 cleavage site, or between the base pairs immediately downstream (3' relative to the scissile strand) from this site. We propose a model with the +1 base rotated out of the duplex, and in which the intercalated adduct prevents religation of the corresponding nucleotide at the 5' end of the cleaved DNA. These results suggest mechanisms whereby intercalating agents interfere with the normal function of human top1.

Adenine↗
PubMed2000

Mutations in the HIV type 1 integrase of patients receiving long-term dideoxynucleoside therapy do not confer resistance to zidovudine.

Metabolites of AZT can inhibit HIV-1 integrase in vitro (Mazumder A, et al., Proc Natl Acad Sci USA 1994;91:5771-5775). To determine if long-term dideoxynucleoside therapy can lead to the emergence of HIV-1 AZT-resistant variants containing mutations in the integrase, we have sequenced the proviral DNA encoding the HIV-1 integrase of nine HIV-1-infected patients at different time points during treatment. Four of the nine patients developed mutations during the course of treatment. Although most mutations occurred at nonconserved amino acids, one patient developed a mutation at codon (R166T), a residue that is conserved among all integrases from known HIV-1 isolates. This mutation was introduced in the recombinant HIV-1 integrase protein to determine if it could confer resistance to AZT in vitro. We show that the R166T integrase mutant is still proficient at carrying 3'-processing and 3' end-joining but that the enzyme is not resistant to AZT-TP. Our results suggest that it is unlikely that integrase inhibition contributes to the antiviral activity of AZT.

Amino Acid Sequence↗
PubMed2000

Mechanism of inhibition of HIV-1 integrase by G-tetrad-forming oligonucleotides in Vitro.

The G-tetrad-forming oligonucleotides and have been identified as potent inhibitors of human immunodeficiency virus type 1 integrase (HIV-1 IN) activity (Rando, R. F., Ojwang, J., Elbaggari, A., Reyes, G. R., Tinder, R., McGrath, M. S., and Hogan, M. E. (1995) J. Biol. Chem. 270, 1754-1760; Mazumder, A., Neamati, N., Ojwang, J. O., Sunder, S., Rando, R. F., and Pommier, Y. (1996) Biochemistry 35, 13762-13771; Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). To understand the inhibition of HIV-1 IN activity by the G-quartet inhibitors, we have designed the oligonucleotides and, composed of three and four G-quartets with stem lengths of 19 and 24 A, respectively. The fact that increasing the G-quartet stem length from 15 to 24 A kept inhibition of HIV-1 IN activity unchanged suggests that the binding interaction occurs between a GTGT loop domain of the G-quartet inhibitors and a catalytic site of HIV-1 IN, referred to as a face-to-face interaction. Docking the NMR structure of (Jing and Hogan (1998)) into the x-ray structure of the core domain of HIV-1 IN, HIV-1 IN-(51-209) (Maignan, S., Guilloteau, J.-P. , Qing, Z.-L., Clement-Mella, C., and Mikol, V. (1998) J. Mol. Biol. 282, 359-368), was performed using the GRAMM program. The statistical distributions of hydrogen bonding between HIV-1 IN and were obtained from the analyses of 1000 random docking structures. The docking results show a high probability of interaction between the GTGT loop residues of the G-quartet inhibitors and the catalytic site of HIV-1 IN, in agreement with the experimental observation.

Base Composition↗
PubMed2000

Substitutions of Asn-726 in the active site of yeast DNA topoisomerase I define novel mechanisms of stabilizing the covalent enzyme-DNA intermediate.

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology and is the cellular target of camptothecin. Recent reports of enzyme structure highlight the importance of conserved amino acids N-terminal to the active site tyrosine and the involvement of Asn-726 in mediating Top1p sensitivity to camptothecin. To investigate the contribution of this residue to enzyme catalysis, we evaluated the effect of substituting His, Asp, or Ser for Asn-726 on yeast Top1p. Top1N726S and Top1N726D mutant proteins were resistant to camptothecin, although the Ser mutant was distinguished by a lack of detectable changes in activity. Thus, a basic residue immediately N-terminal to the active site tyrosine is required for camptothecin cytotoxicity. However, replacing Asn-726 with Asp or His interfered with distinct aspects of the catalytic cycle, resulting in cell lethality. In contrast to camptothecin, which inhibits enzyme-catalyzed religation of DNA, the His substituent enhanced the rate of DNA scission, whereas the Asp mutation diminished the enzyme binding of DNA. Yet, these effects on enzyme catalysis were not mutually exclusive as the His mutant was hypersensitive to camptothecin. These results suggest distinct mechanisms of poisoning DNA topoisomerase I may be explored in the development of antitumor agents capable of targeting different aspects of the Top1p catalytic cycle.

Amino Acid Substitution↗
PubMed2000

Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139.

Histone H2AX is a ubiquitous member of the H2A histone family that differs from the other H2A histones by the presence of an evolutionarily conserved C-terminal motif, -KKATQASQEY. The serine residue in this motif becomes rapidly phosphorylated in cells and animals when DNA double-stranded breaks are introduced into their chromatin by various physical and chemical means. In the present communication we show that this phosphorylated form of H2AX, referred to as gamma-H2AX, appears during apoptosis concurrently with the initial appearance of high molecular weight DNA fragments. gamma-H2AX forms before the appearance of internucleosomal DNA fragments and the externalization of phosphatidylserine to the outer membrane leaflet. gamma-H2AX formation is inhibited by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the inhibitor of caspase-activated DNase, and it is induced when DNase I and restriction enzymes are introduced into cells, suggesting that any apoptotic endonuclease is sufficient to induce gamma-H2AX formation. These results indicate that gamma-H2AX formation is an early chromatin modification following initiation of DNA fragmentation during apoptosis.

Apoptosis↗
PubMed2000

Benzo[a]pyrene diol epoxide adducts in DNA are potent suppressors of a normal topoisomerase I cleavage site and powerful inducers of other topoisomerase I cleavages.

The catalytic intermediates of DNA topoisomerase I (top1) are cleavage complexes that can relax DNA supercoiling (intramolecular reaction) or mediate recombinations (intermolecular religation). We report here that DNA adducts formed from benzo[a]pyrene bay-region diol epoxides can markedly affect top1 activity. Four oligonucleotide 22-mers of the same sequence were synthesized, each of which contained a stereoisomerically unique benzo[a]pyrene 7, 8-diol 9,10-epoxide adduct at the 2-amino group of a central 2'-deoxyguanosine residue. These four adducts correspond to either cis or trans opening at C-10 of the (+)-(7R, 8S, 9S, 10R)- or (-)-(7S, 8R, 9R, 10S)-7,8-diol 9,10-epoxides. Their solution conformations in duplex DNA (intercalated and minor-groove bound for the cis and trans opened adducts respectively) can be deduced from previous NMR studies. All four adducts completely suppress top1 cleavage activity at the alkylation site and induce the formation of new top1cleavage complexes on both strands of the DNA 3-6 bases away from the alkylation site. The trans opened adduct from the highly carcinogenic (+)-diol epoxide is the most active in inducing top1 cleavage independently of camptothecin, demonstrating that minor groove alkylation can efficiently poison top1. We also found that this isomer of the diol epoxide induces the formation of top1-DNA complexes in mammalian cells, which suggests a possible relationship between induction of top1 cleavage complexes and carcinogenic activity of benzo[a]pyrene diol epoxides.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2000

Induction of topoisomerase I cleavage complexes by 1-beta -D-arabinofuranosylcytosine (ara-C) in vitro and in ara-C-treated cells.

1-beta-d-Arabinofuranosylcytosine (Ara-C) is a nucleoside analog commonly used in the treatment of leukemias. Ara-C inhibits DNA polymerases and can be incorporated into DNA. Its mechanism of cytotoxicity is not fully understood. Using oligonucleotides and purified human topoisomerase I (top1), we found a 4- to 6-fold enhancement of top1 cleavage complexes when ara-C was incorporated at the +1 position (immediately 3') relative to a unique top1 cleavage site. This enhancement was primarily due to a reversible inhibition of top1-mediated DNA religation. Because ara-C incorporation is known to alter base stacking and sugar puckering at the misincorporation site and at the neighboring base pairs, the observed inhibition of religation at the ara-C site suggests the importance of the alignment of the 5'-hydroxyl end for religation with the phosphate group of the top1 phosphotyrosine bond. This study also demonstrates that ara-C treatment and DNA incorporation trap top1 cleavage complexes in human leukemia cells. Finally, we report that camptothecin-resistant mouse P388/CPT45 cells with no detectable top1 are crossresistant to ara-C, which suggests that top1 poisoning is a potential mechanism for ara-C cytotoxicity.

Animals↗
PubMed2000

Recognition and inhibition of HIV integrase by a novel dinucleotide.

The viral enzyme, HIV integrase, is involved in the integration of viral DNA into host cell DNA. In the quest for a small nucleotide system with nuclease stability of the internucleotide phosphate bond and critical structural features for recognition and inhibition of HIV-1 integrase, we have discovered a conceptually novel dinucleotide, pIsodApdC, which is a potent inhibitor of this key viral enzyme.

HIV Integrase↗
PubMed2000

Identification and proposed mechanism of action of thymidine kinase inhibition associated with cellular exposure to camptothecin analogs.

PURPOSE: To investigate the effects of several camptothecin analogs including 9-aminocamptothecin (9-AC), SN38, topotecan, and irinotecan (CPT-11) on the enzymes involved in the pyrimidine salvage pathway including thymidylate synthase (TS). A COMPARE analysis using the NCI 60 cell line drug-screening panel suggested that there were similarities in the mechanisms of action of camptothecin analogs and TS inhibitors. METHODS: TS enzymatic activity was measured by both an in situ tritium release assay using both the H630 colon cancer cell line and the CEM human leukemia cell line, and by a radiolabelled in vitro assay using partially purified human TS as the enzyme source. Thymidine kinase (TK) activity was measure by a radiolabelled in vitro assay using H630 colon cancer cell lysates as the enzyme source. RESULTS: In vitro studies indicated that none of the analogs directly inhibited TS enzymatic activity; however, utilization of a coupled TS/TK in situ assay with radiolabelled deoxyuridine as the precursor revealed marked inhibition by the camptothecin analogs. 9-AC, SN38, and topotecan yielded IC50 values of 1.3, 1.6, and 1.1 microM respectively. In contrast, there was no inhibition detected when deoxycytidine was used as the radiolabelled nucleoside precursor, suggesting that the drug effect was through inhibition of TK, rather than inhibition of TS. In vitro studies using cell lysates from H630 human colon cancer cells to measure TK activity showed no decrease in TK activity after 9-AC treatment. In addition, no changes were detected in the dATP and dTTP nucleotide pools. Permeabilizing the cell membranes with saponin did not abolish the inhibitory effect of the camptothecins indicating that altered cell transport was not responsible for the decreased activity in the in situ assay in intact cells. CONCLUSION: These studies suggest that there is inhibition of TK in intact cells associated with topoisomerase I inhibition by camptothecin analogs, and the inhibition of TK is the result of an indirect effect not related to feedback inhibition by changes in dTTP pools.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Retroviral integrase inhibitors year 2000: update and perspectives.

HIV-1 integrase is an essential enzyme for retroviral replication and a rational target for the design of anti-AIDS drugs. A number of inhibitors have been reported in the past 8 years. This review focuses on the recent developments in the past 2 years. There are now several inhibitors with known sites of actions and antiviral activity. The challenge is to convert these leads into drugs that will selectively target integrase in vivo, and can be added to our antiviral armamentarium.

Anti-HIV Agents↗
PubMed2000

Identification of HIV-1 integrase inhibitors via three-dimensional database searching using ASV and HIV-1 integrases as targets.

Integration of viral DNA into the host cell genome is a critical step in the life cycle of HIV. This essential reaction is catalyzed by integrase (IN) through two steps, 3'-processing and DNA strand transfer. Integrase is an attractive target for drug design because there is no known cellular analogue and integration is essential for successful replication of HIV. A computational three-dimensional (3-D) database search was used to identify novel HIV-1 integrase inhibitors. Starting from the previously identified Y3 (4-acetylamino-5-hydroxynaphthalene-2,7-disulfonic acid) binding site on the avian sarcoma virus integrase (ASV IN), a preliminary search of all compounds in the nonproprietary, open part of the National Cancer Institute 3-D database yielded a collection of 3100 compounds. A more rigorous scoring method was used to rescreen the 3100 compounds against both ASV IN and HIV-1 IN. Twenty-two of those compounds were selected for inhibition assays against HIV-1 IN. Thirteen of the 22 showed inhibitory activity against HIV-1 IN at concentrations less than 200 microM and three of them showed antiviral activities in HIV-1 infected CEM cells with effective concentrations (EC50) ranging from 0.8 to 200 microM. Analysis of the computer-generated binding modes of the active compounds to HIV-1 IN showed that simultaneous interaction with the Y3 site and the catalytic site is possible. In addition, interactions between the active compounds and the flexible loop involved in the binding of DNA by IN are indicated to occur. The structural details and the unique binding motif between the HIV-1 IN and its inhibitors identified in the present work may contribute to the future development of IN inhibitors.

Avian Sarcoma Viruses↗
PubMed2000

A gene expression database for the molecular pharmacology of cancer.

We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.

Antineoplastic Agents↗