Eukaryotic DNA topoisomerases I.
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Amino Acid Sequence↗
Biomedical subjects
Y Pommier
Publications and source records attributed to Y Pommier.
Topoisomerase I-related parameters and camptothecin activity in the colon carcinoma cell lines from the National Cancer Institute anticancer screen.
Camptothecin (CPT) derivatives are a new family of anticancer agents which are selective inhibitors of DNA topoisomerase I (top1) and have entered clinical trials with promising results. The cellular determinants for CPT activity were studied in the seven cell lines of the National Cancer Institute anticancer screen. These cell lines exhibit natural differences in sensitivity to CPT and can be divided into three groups, according to their increasing resistance: colo205, SW620, HCT116 < HT29, HCC2998 < HCT15, and KM12. The differential sensitivity range was approximately 17-fold between KM12 and colo205 cells. CPT uptake varied only by less than a factor of three among the cell lines. Top1 mRNA, measured by Northern blotting analysis, and top1 protein levels, measured by Western blotting, varied by 2-fold or less among the cell lines and were correlated neither with the CPT cytotoxicity nor the levels of cleavable complexes measured by alkaline elution in the various cell lines. An overall log-linear correlation was observed between CPT-induced top1-cleavable complexes and growth inhibition, indicating the importance of cleavable complex formation rather than top1 levels for cell killing in this panel of cell lines. Also, some cell lines displayed marked growth inhibition differences with minimal differences in cleavable complexes and S-phase fraction, suggesting that parameters downstream from the cleavable complexes are also critical for CPT cytotoxicity.
Camptothecin↗
Azatoxin derivatives with potent and selective action on topoisomerase II.
Azatoxin was rationally designed as a DNA topoisomerase II (top2) inhibitor [Leteurtre et al., Cancer Res 52: 4478-4483, 1992] and was also found to inhibit tubulin polymerization. Its cytotoxicity is due to action on tubulin at lower concentrations and on top2 at higher concentrations. At intermediate concentrations, the combination of the two mechanisms appears antagonistic [Solary et al., Biochem Pharmacol 45: 2449-2456, 1993]. The aim of this study was to design azatoxin derivatives that would act only on tubulin or on top2. Selective targeting of top2 or tubulin was tested using top2-mediated DNA cleavage assays, and tubulin polymerization and tubulin proteolysis assays, as well as COMPARE analyses of cytotoxicity assays in the National Cancer Institute in vitro Drug Screening Program. Selective inhibitors of top2 and tubulin polymerization have been obtained. Top2 inhibition, abolished by methylation at position 4', was enhanced by the addition of a bulky group at position 11. Bulky substitution at position 11 determined different patterns of top2 cleavage sites and suppressed the action on tubulin. Selective inhibition of tubulin was obtained with 4'-methylazatoxin that was found to bind to the colchicine site. These results are consistent with those obtained in the podophyllotoxin family to which azatoxin is structurally related. Some azatoxin derivatives are under consideration for further preclinical development.
Antineoplastic Agents↗
Inhibition of human immunodeficiency virus type-1 integrase by curcumin.
Curcumin (diferuloylmethane) is the yellow pigment in turmeric (Curcuma longa L.) that is widely used as a spice, food coloring (curry) and preservative. Curcumin exhibits a variety of pharmacological effects including antitumor, anti-inflammatory, and anti-infectious activities and is currently in clinical trials for AIDS patients. The effects of curcumin have been determined on purified human immunodeficiency virus type 1 (HIV-1) integrase. Curcumin has an inhibitory concentration50 (IC50) for strand transfer of 40 microM. Inhibition of an integrase deletion mutant containing only amino acids 50-212 suggests that curcumin interacts with the integrase catalytic core. Two structural analogs, methyl cinnamate and chlorogenic acid, were inactive. Energy minimization studies suggest that the anti-integrase activity of curcumin could be due to an intramolecular stacking of two phenyl rings that brings the hydroxyl groups into close proximity. The present data suggest that HIV-1 integrase inhibition may contribute to the antiviral activity of curcumin. These observations suggest new strategies for antiviral drug development that could be based upon curcumin as a lead compound for the development of inhibitors of HIV-1 integrase.
Antiviral Agents↗
Three-dimensional quantitative structure-activity relationship (QSAR) of HIV integrase inhibitors: a comparative molecular field analysis (CoMFA) study.
We present the results from a comparative molecular field analysis (CoMFA) of a set of flavone analogs that inhibit HIV-1 integrase-mediated cleavage (3'-processing step) and integration (strand transfer step) in vitro. The results indicate a strong correlation between the inhibitory activity of these flavones and the steric and electrostatic fields around them. CoMFA quantitative structure-activity relationship models with considerable predictive ability (cross-validated r2 as high as 0.8) were obtained.
Chemical Phenomena↗
Mutation at the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell line resistant to camptothecin.
We developed previously a resistant cell line, CEM/C2, from the human leukemia cell line CCRF-CEM by stepwise selection in camptothecin. This cell line is 974-fold more resistant to camptothecin than parental cells. Resistance is only partially explained by 2-fold reductions in topoisomerase I protein and mRNA levels. We further investigated biochemical and molecular features of topoisomerase I in the resistant cell line. Sequence analyses of the top1 cDNA from CEM/C2 identified mutations corresponding to two amino acid substitutions, Met370Thr and Asn722Ser. Asn722Ser is next to the catalytic Tyr723 in a region highly conserved among type I eukaryotic DNA topoisomerases. Recombinant top1 with the corresponding substitution was found to be catalytically active and resistant to camptothecin. These results indicate that camptothecin resistance of CEM/C2 is due to the mutation Asn722Ser and strongly suggest that the asparagine immediately flanking the catalytic tyrosine is important for the camptothecin action.
Amino Acid Sequence↗
Cosalane analogues with enhanced potencies as inhibitors of HIV-1 protease and integrase.
Several new analogues of the novel anti-HIV agent cosalane have been synthesized and evaluated as inhibitors of HIV-1 integrase and protease, HIV-1 replication, HIV-1 and HIV-2 cytopathicity, HIV-1- and HIV-2-mediated syncytium formation, and cytopathicity of a variety of human pathogenic viruses. The congeners displayed enhanced potencies relative to cosalane itself as inhibitors of HIV-1 integrase and protease. The two most potent analogues against HIV-1 integrase displayed IC50 values of 2.2 microM, while the three most potent compounds against HIV-1 protease had IC50 values in the 0.35-0.39 microM range. In addition to its activity against HIV-1 and HIV-2 cytopathicity, cosalane inhibited the cytopathic effects of herpes simplex virus-1, herpes simplex virus-2, and human cytomegalovirus at concentrations that were well below the cytotoxic concentrations. Potentially useful antiviral activities were also revealed for some of the new cosalane congeners against influenza virus, Junin virus, and Tacaribe virus.
Amino Acid Sequence↗
Unscheduled activation of cyclin B1/Cdc2 kinase in human promyelocytic leukemia cell line HL60 cells undergoing apoptosis induced by DNA damage.
We have studied changes in cyclin A- and B1-dependent kinases during apoptosis induced in human promyelocytic leukemia (HL60) cells treated with the topoisomerase I inhibitor camptothecin. We found that cyclin B1/Cdc2 kinase activity transiently increases within 30 min after camptothecin treatment. This increase is followed by a rapid inactivation of the cyclin B1/Cdc2 kinase that is associated with Cdc2 tyrosine phosphorylation without any change in Cdc2 or cyclin B1 protein levels. The DNA polymerase inhibitor aphidicolin abrogates camptothecin-induced changes in cyclin B1/Cdc2 kinase activity, indicating that DNA replication-induced DNA damage is essential for both Cdc2 alterations and apoptosis activation. Apoptosis and the initial cyclin B1/Cdc2 kinase activation were amplified using synchronized S-phase cells, and cyclin A/cdk2 kinase did not change under these conditions. The same transient activation and subsequent inactivation of cyclin B1/Cdc2 kinase were observed after DNA damage by etoposide or bis-(2-chloroethyl)methylamine hydrochloride. These observations suggest that DNA damage promotes the transient and unscheduled stimulation of cyclin B1/Cdc2 kinase activity in HL60 cells prior to apoptosis.
Aphidicolin↗
DNA recombinase activity of eukaryotic DNA topoisomerase I; effects of camptothecin and other inhibitors.
DNA oligonucleotides containing a strong topoisomerase I cleavage site were used to study the DNA cleavage and strand transferase activities of calf thymus topoisomerase I (top1) in the absence and presence of camptothecin. A partially single-stranded oligonucleotide with only two nucleotides on the 3' side of the cleavage site (positions +1 and +2) was cleaved at the same position as the corresponding duplex oligonucleotide. However, cleavage in the absence of camptothecin was more pronounced than in the duplex oligonucleotide and was only partially reversible in the presence of 0.5 M NaCl, consistent with release of the dinucleotide 3' to the top1 break. Another reaction took place generating a larger DNA fragment which resulted from religation (strand transfer) of the 5'-hydroxyl terminus of the non-scissile DNA strand to the 3' end of the top1-linked oligonucleotide after loss of the +1 and +2 nucleotides. Top1 religation activity appeared efficient since only the last 5' base of the single-stranded DNA acceptor was complementary to the 3' tail of the donor DNA. Religation was not detectable with a double-stranded DNA acceptor, which is consistent with the persistence of top1-induced DNA double-strand breaks in camptothecin-treated cells. Camptothecin and other top1 inhibitors enhanced cleavage in both the partially single-stranded and the duplex oligonucleotides, indicating that they did not inhibit the induction of top1-mediated DNA cleavage but primarily blocked the religation step of the enzyme catalytic cycle. The top1 DNA strand transferase activity was reversibly inhibited by camptothecin and several derivatives, as well as saintopin. These results are discussed in terms of camptothecin-induced DNA recombinations.
Animals↗
Inhibition of human immunodeficiency virus type 1 integrase by a hydrophobic cation: the phenanthroline-cuprous complex.
The human immunodeficiency virus type 1 integrase (HIV-1 integrase) is required for integration of a double-stranded DNA copy of the viral RNA genome into a host chromosome and for HIV replication. We have examined the effects of 2:1 1,10-phenanthroline-cuprous complexes on purified HIV-1 integrase. Although the uncomplexed phenanthrolines are not active below 100 microM, four of the cuprous complexes (neocuproine, 4-phenyl neocuproine, 2,3,4,7,8,9-hexamethyl phenanthroline, and 2,3,4,7,8-pentamethyl phenanthroline) have a 50% inhibitory concentration (IC50) for integration ranging between 1 and 10 microM. Disintegration is also inhibited by these phenanthroline-cuprous complexes at slightly higher concentrations (between 10 and 40 microM). Dialysis experiments showed that the inhibition is reversible and kinetic analyses revealed that the mode of inhibition by these cuprous complexes appears to be noncompetitive with respect to the substrate DNA. Consistent with these findings, binding assays demonstrate that, although these complexes can inhibit binding to DNA at high concentrations, they do not inhibit binding of integrase to the DNA substrate at their IC50 values. Because these complexes do not bind to B-DNA below 50 microM, inhibition via binding to a specific region on the enzyme was examined. Using deletion mutants of integrase, it was determined that neither the amino-terminal (zinc finger) nor the carboxy-terminal (DNA-binding) integrase domain is required for inhibition by the phenanthroline-cuprous complexes. Therefore, inhibition via binding to the enzyme catalytic core or to the interface between the enzyme and a noncanonical DNA structure generated during the enzymatic reaction is the probable mechanism. These results suggest the utility of neocuproine-cuprous complexes in developing inhibitors of HIV-1 integrase as well as probes for drug-binding sites and enzymatic reaction mechanism.
Base Sequence↗
T30177, an oligonucleotide stabilized by an intramolecular guanosine octet, is a potent inhibitor of laboratory strains and clinical isolates of human immunodeficiency virus type 1.
T30177, an oligonucleotide composed of only deoxyguanosine and thymidine, is 17 nucleotides in length and contains single phosphorothioate internucleoside linkages at its 5' and 3' ends for stability. This oligonucleotide does not share significant primary sequence homology with or possess any complementary (antisense) sequence motifs to the human immunodeficiency virus type 1 (HIV-1) genome. T30177 inhibited replication of multiple laboratory strains of HIV-1 in human T-cell lines, peripheral blood lymphocytes, and macrophages. T30177 was also found to be capable of inhibiting multiple clinical isolates of HIV-1 and preventing the cytopathic effect of HIV-1 in primary CD4+ T lymphocytes. In assays with human peripheral blood lymphocytes there was no observable toxicity associated with T30177 at the highest concentration tested (100 microM), while the median inhibitory concentration was determined to be in the range of 0.1 to 1.0 microM for the clinical isolates tested, resulting in a high therapeutic index for this drug. In temporal studies, the kinetics of addition of T30177 to infected cell cultures indicated that, like the known viral adsorption blocking agents dextran sulfate and Chicago sky blue, T30177 needed to be added to cells during or very soon after viral infection. However, analysis of nucleic acids extracted at 12 h postinfection from cells treated with T30177 at the time of virus infection established the presence of unintegrated viral cDNA, including circular proviral DNA, in the treated cells. In vitro analysis of viral enzymes revealed that T30177 was a potent inhibitor of HIV-1 integrase, reducing enzymatic activity by 50% at concentrations in the range of 0.050 to 0.09 microM. T30177 was also able to inhibit viral reverse transcriptase activity; however, the 50% inhibitory value obtained was in the range of 1 to 10 microM, depending on the template used in the enzymatic assay. No observable inhibition of viral protease was detected at the highest concentration of T30177 used (10 microM). In experiments in which T30177 was removed from infected cell cultures at 4 days post-HIV-1 infection, total suppression of virus production was observed for more than 27 days. PCR analysis of DNA extracted from cells treated in this fashion was unable to detect the presence of viral DNA 11 days after removal of the drug from the infected cell cultures. The ability of T30177 to inhibit both laboratory and clinical isolates of HIV-1 and the experimental data which suggest that T30177 represents a novel class of integrase inhibitors indicate that this compound is a viable candidate for evaluation as a therapeutic agent against HIV-1 in humans.
Antiviral Agents↗
Eukaryotic DNA topoisomerases mediated DNA cleavage induced by a new inhibitor: NSC 665517.
A compound with a novel structure, NSC 665517, was tested in the National Cancer Institute Preclinical Drug Discovery Screen. With the COMPARE algorithm, the pattern of differential cytotoxicity for NSC 665517 most closely resembled those of known topoisomerase II (top2) inhibitors. In vitro data showed that NSC 665517 induced DNA cleavage in the presence of top2 and topoisomerase I (top1) (at a higher concentration). The minimum concentration required to induce top2 cleavage was 0.5 microM. A substantial decrease in top2-induced cleavage by NSC 665517 was seen when the reaction mixtures were shifted to elevated temperature (55 degrees), suggesting that top2-induced cleavage occurs through the mechanism of stabilizing the reversible enzyme/DNA complex and inhibiting religation. The DNA cleavage pattern induced by NSC 665517 with top2 was different than that of other known top2 inhibitors, including etoposide, mitoxantrone, anthracyclines, amsacrine, and ellipticine. top2 cleavage sites induced by NSC 665517 showed strong preference for G located 3' to the top2-mediated DNA cleavage (position +1). NSC 665517 produced limited DNA unwinding at high drug concentration. DNA damage analyzed in KB cells by alkaline elution showed that NSC 665517 induced strand break. Data from the cytotoxicity in KB-V1 overexpressing P-glycoprotein and COMPARE analysis with rhodamine efflux assay indicated that NSC 665517 is a substrate of P-glycoprotein. These results strongly suggest that NSC 665517 is a novel topoisomerase-targeted drug. Preclinical evaluation of NSC 665517 as an antitumor agent is under way.
Antineoplastic Agents↗
Altered topoisomerase I expression in two subclones of human CEM leukemia selected for resistance to camptothecin.
Two camptothecin-resistant variants of the CEM human leukemia cell line were developed by stepwise selection in camptothecin (CPT) in vitro. The two lines, named CEM/C1 and CEM/C2, were found to be approximately 31- and 970-fold less sensitive to CPT, respectively, than the CEM parental line and variably cross-resistant to the CPT analogs 9-amino-CPT, 10,11-methylenedioxy-CPT, and topotecan. Levels of DNA-protein complex formation resulting from cell exposure to CPT were found to be progressively reduced in the CPT-resistant cells, despite equivalent CPT accumulation in the drug-sensitive and -resistant cells. Nuclear extracts (1.0 M NaCl) prepared from the CEM/C1 and CEM/C2 lines contained 1.5- to 2-fold less DNA topoisomerase I catalytic activity per microgram of protein than did extracts from the drug-sensitive CEM line, in association with altered sensitivity of the enzyme in the CEM/C1 and CEM/C2 extracts to the inhibitory activity of CPT. Only minor differences were noted in the CPT IC50s for the topoisomerase I activity in extracts from the two CPT-resistant cell lines, however, despite the marked differences in cellular sensitivity to CPT. There were notable differences in the level of CPT-induced cleavage of DNA oligonucleotides by topoisomerase I in nuclear extracts from CEM cells compared with the drug-resistant cell extracts, with very little oligonucleotide cleavage induced by enzyme in either drug-resistant cell type, despite the use of very high (100 microM) CPT concentrations. The alterations in topoisomerase I catalytic activity were associated with reduced cellular levels of both immunoreactive topoisomerase I protein (representing 59 +/- 19% [CEM/C1] and 49 +/- 12% [CEM/C2] of that in CEM, respectively) and mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Acute Disease↗
The role of cell cycle regulation and apoptosis triggering in determining the sensitivity of leukemic cells to topoisomerase I and II inhibitors.
Topoisomerase (topo) inhibitors induce enzyme-linked DNA breaks. Resulting DNA damage can lead to cell cycle arrest and/or cell death by apoptosis. The sensitivity of five human leukemic cell lines to topo I (camptothecin or CPT) and topo II (etoposide or VP-16) inhibitors varied widely (100-fold for CPT and 30-fold for VP-16). Three cell lines were more sensitive (BV173, HL60, U937) and two cell lines were resistant (K562, KCL22) to both drugs. None of these cell lines were selected for drug resistance and overexpressed mdr1 gene. Their sensitivity was not related to their doubling time nor to cell cycle repartition. The initial DNA damage (cleavable complexes) induced by topo I and II inhibitors was measured as DNA-protein crosslinks (DPC) using alkaline elution. Neither DPC level induced by 30-min treatment with CPT or VP-16 nor the levels of topo 1, topo II alpha and topo II beta mRNA were related to sensitivity. Electron microscopy and DNA fragmentation measured by filter elution and agarose gel electrophoresis demonstrated that apoptosis was induced by both drugs in the five cell lines. The kinetics of DNA fragmentation was related to cell sensitivity. At drug concentrations higher than IC50, DNA fragmentation increased very rapidly in the three sensitive, compared with the two resistant, cell lines. Continuous exposure to both drugs induced cell cycle arrest in either G2 or S phase that was related both to cell sensitivity and drug concentration. Comparison between cell lines indicated that the ability of cells to arrest cell cycle in G2 or S phase was related to their drug sensitivity and increased with cell resistance. In a given cell line, cell cycle progression was observed to be progressively inhibited by increasing drug concentrations. Treatment of synchronized cells demonstrated that highly cytotoxic drug concentration induced a complete inhibition of cell cycle progression. Altogether, these data suggest that the ability of leukemic cell lines to regulate cell cycle progression and to trigger apoptosis is more indicative of their sensitivity to topoisomerase poisons than cleavable complexes induced by these drugs.
Apoptosis↗
DNA topoisomerases I & II cleavage sites in the type 1 human immunodeficiency virus (HIV-1) DNA promoter region.
Topoisomerase sites were mapped in the 5'-long terminal repeat of HIV-1 DNA by agarose and sequencing gel electrophoresis. Topoisomerase II sites were observed in the absence and presence of teniposide and amsacrine in the transcription initiation region and the TATA box, consistent with a possible role of topoisomerase II in transcription. The NF-kB and Sp1 regions were poorly cleaved. Topoisomerase I sites were relatively unfrequent even in the presence of camptothecin. They were absent in the core promoter and were concentrated in the TAR and the upstream region near the junction with the host DNA.
Amsacrine↗
Saintopin, a dual inhibitor of DNA topoisomerases I and II, as a probe for drug-enzyme interactions.
Stabilization of the topoisomerase-cleavable complexes is the common initial event leading to the cytotoxicity of topoisomerase I and II (top1 and top2) inhibitors. Using saintopin (STP), a poison of both topoisomerases, we studied top1- and top2-cleavable complexes (Yamashita, Y., Kawada, S.-Z., Fujii, N., and Nakano, H. (1991) Biochemistry 30, 5838-5845). top1 and top2 sites induced in the presence of STP showed the same preferences for the base located 3' to the topoisomerase-induced DNA break (position +1): preference for G and not C. A camptothecin-resistant top1 with a mutation (Asn722-->Ser) next to the catalytic tyrosine (Tyr723) was cross-resistant to STP, suggesting that both STP and camptothecin interact with the protein near the catalytic tyrosine. These results are consistent with a dual interaction of the drug with the enzyme and the DNA and provide further evidence for the "drug-stacking" model. This model proposes that topoisomerase inhibitors bind, possibly through hydrogen bonding and/or stacking, with one of the bases flanking the DNA termini (guanine at position +1 in the case of STP) and within the enzyme catalytic pocket, most likely by stacking with the catalytic tyrosine.
Base Sequence↗
Methylphosphonodiester substitution near the conserved CA dinucleotide in the HIV LTR alters both extent of 3'-processing and choice of nucleophile by HIV-1 integrase.
We present evidence suggesting that the 3'-processing activity of HIV-1 integrase is dramatically affected by electrostatic and/or steric perturbations 3' to the conserved CA dinucleotide. When the phosphodiester bond 3' to the scissile phosphodiester is replaced by a methylphosphonodiester linkage, 3'-processing decreases by two orders of magnitude. This block of cleavage can be somewhat overcome by increasing the pH of the reaction. Labeling of the substrates at the 3'-end revealed blockage of water and glycerol, but stimulation of the viral DNA 3'-hydroxyl, acting as the nucleophile with the methylphosphonodiester substrate. Interestingly, a circular trinucleotide was formed using the phosphodiester and methylphosphonodiester substrates when the terminal nucleotide was 3'-deoxyadenosine but not 2'-deoxyadenosine. Mutagenesis of the enzyme active site has previously been shown to alter the choice of nucleophile in the 3'-processing reaction. Taken together, the results in this study suggest that 'mutagenesis' of the DNA backbone can also alter the choice of nucleophile.
Base Sequence↗
Streptonigrin-induced topoisomerase II sites exhibit base preferences in the middle of the enzyme stagger.
The non DNA intercalator streptonigrin was shown to inhibit topoisomerase II by stabilizing cleavable complexes (Yamashita et al, Cancer Res. 1990, 50, 5841). Streptonigrin-induced topoisomerase II cleavage sites were mapped in the c-myc proto-oncogene DNA. Streptonigrin induced a unique cleavage pattern. Its cleavage sites were less frequent than those induced by other topoisomerase II inhibitors. Strongly preferred bases were found in the middle of topoisomerase II DNA stagger, with thymine at position +2 and adenine at position +3, position +1 being the nucleotide covalently linked to topoisomerase II. Preference for bases not immediately flanking the cleavage sites has not been reported previously and indicates that a mechanism other than "drug stacking" within the DNA break is taking place with streptonigrin to stabilize cleavable complexes. An alternative model taking into account the unusual DNA binding properties of streptonigrin is proposed.
Base Sequence↗