Showing papers by "Yves Pommier published in 2007"
TL;DR: Results show that replication forks moving from one origin, as well as from neighboring origins, tend to exhibit the same velocity, although the plasticity of the replication program allows for their adaptation to variable interorigin distances.
Abstract: The spatial organization of replicons into clusters is believed to be of critical importance for genome duplication in higher eukaryotes, but its functional organization still remains to be fully clarified. The coordinated activation of origins is insufficient on its own to account for a timely completion of genome duplication when interorigin distances vary significantly and fork velocities are constant. Mechanisms coordinating origin distribution with fork progression are still poorly elucidated, because of technical difficulties of visualizing the process. Taking advantage of a single molecule approach, we delineated and compared the DNA replication kinetics at the genome level in human normal primary and malignant cells. Our results show that replication forks moving from one origin, as well as from neighboring origins, tend to exhibit the same velocity, although the plasticity of the replication program allows for their adaptation to variable interorigin distances. We also found that forks that emanated from closely spaced origins tended to move slower than those associated with long replicons. Taken together, our results indicate a functional role for origin clustering in the dynamic regulation of genome duplication.
208 citations
TL;DR: It is demonstrated that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers.
Abstract: To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication-mediated DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. After a short dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in late-S-phase, compared to early-S-phase, cells and was due to both an inhibition of initiation and elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers. DNA replication was actively inhibited by checkpoint activation since 7-hydroxystaurosporine (UCN-01), the specific Chk1 inhibitor CHIR-124, or transfection with small interfering RNA targeting Chk1 restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced camptothecin-induced DNA damage at replication sites where histone gamma-H2AX colocalized with replication foci. Together, our study demonstrates that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation.
196 citations
TL;DR: In this article, two indenoisoquinoline derivatives (NSC 725776 and NSC 724998) have been selected for therapeutic development, which are potent Top1 inhibitors and induce Top1 cleavage at unique genomic positions compared with CPT.
Abstract: Camptothecin (CPT) derivatives are effective anticancer drugs, especially against solid tumors. As CPTs are chemically unstable and have clinical limitations, we have synthesized indenoisoquinolines as novel topoisomerase I (Top1) inhibitors. We presently report two indenoisoquinoline derivatives, NSC 725776 and NSC 724998, which have been selected for therapeutic development. Both are potent Top1 inhibitors and induce Top1 cleavage at unique genomic positions compared with CPT. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with NSC 725776 and NSC 724998 at nanomolar concentrations. Those drug-induced protein-linked DNA breaks persisted longer after drug removal than those produced by CPT. Studies in human cells in culture show that NSC 725776 and NSC 724998 exert antiproliferative activity at submicromolar concentrations. Furthermore, NSC 725776 and NSC 724998 show cross-resistance in cells deficient or silenced for Top1, which is consistent with their selective Top1 targeting. Similar to other known Top1 inhibitors, NSC 725776-treated and NSC 724998-treated cells show an arrest of cell cycle progression in both S and G(2)-M and a dependence on functional p53 for their cytotoxicity. Dose-dependent gamma-H2AX foci formation was readily observed in cells treated with NSC 725776 and NSC 724998. These gamma-H2AX foci were detectable at pharmacologically relevant doses for up to 24 h and thus could be used as biomarkers for clinical trials (phase 0).
116 citations
Journal Article•
TL;DR: Indenoisoquinoline derivatives are synthesized as novel topoisomerase I (Top1) inhibitors and induce Top1 cleavage at unique genomic positions compared with CPT, and show cross-resistance in cells deficient or silenced for Top1, which is consistent with their selective Top1 targeting.
Abstract: C219 Camptothecin (CPT) derivatives are effective anticancer drugs, especially against solid tumors. As camptothecins are chemically unstable and have clinical limitations, we have synthesized indenoisoquinolines as novel topoisomerase I (Top1) inhibitors. We presently report two indenoisoquinoline derivatives NSC 725776 and NSC 724998 that have been selected for therapeutic development. Both are potent Top1 inhibitors and induce Top1 cleavage at unique genomic positions compared to camptothecin. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with NSC 725776 and NSC 724998 at nanomolar concentrations. Those drug-induced protein-linked DNA breaks persisted longer after drug removal than those produced by CPT, topotecan or SN-38, the active metabolite of irinotecan. Studies in human cells in culture show that NSC 725776 and NSC 724998 exert antiproliferative activity at nanomolar concentrations. Furthermore, NSC 725776 and NSC 724998 demonstrate cross-resistance in cells deficient or silenced for Top1, which is consistent with their selective Top1 targeting. Whereas topotecan and SN-38 exhibit resistance in cells overexpressing ABCG2 (MXR/BCRP), NSC 724998 (and to a lesser extent NSC 725776) showed full activity in ABCG2-overexpressing cells, indicating that the new indenoisoquinolines can overcome resistance to camptothecins. Similar to other known Top1 inhibitors, NSC 725776 and NSC 724998 treated cells show an arrest of cell cycle progression in both S and G2/M and a dependence on functional p53 for their cytotoxicity. Dose dependent histone gamma-H2AX foci formation was readily observed in cells treated with NSC 725776 and NSC 724998. These gamma-H2AX foci were detectable at pharmacologically relevant doses for up to 24 hours and thus could be used as biomarkers for clinical trials (phase 0-I).
107 citations
TL;DR: Furamidine represents the most potent Tdp1 inhibitor reported to date and is effective both with single- and double-stranded substrates but is slightly stronger with the duplex DNA.
Abstract: By enzymatically hydrolyzing the terminal phosphodiester bond at the 3'-ends of DNA breaks, tyrosyl-DNA phosphodiesterase (Tdp1) repairs topoisomerase-DNA covalent complexes and processes the DNA ends for DNA repair. To identify novel Tdp1 inhibitors, we developed a high-throughput assay that uses electrochemiluminescent (ECL) substrates. Subsequent to screening of 1981 compounds from the 'diversity set' of the NCI-Developmental Therapeutics Program, here we report that furamidine inhibits Tdp1 at low micromolar concentrations. Inhibition of Tdp1 by furamidine is effective both with single- and double-stranded substrates but is slightly stronger with the duplex DNA. Surface plasmon resonance studies show that furamidine binds both single- and double-stranded DNA, though more weakly with the single-stranded substrate DNA. Thus, the inhibition of Tdp1 activity could in part be due to the binding of furamidine to DNA. However, the inhibition of Tdp1 by furamidine is independent of the substrate DNA sequence. The kinetics of Tdp1 inhibition by furamidine was influenced by the drug to enzyme ratio and duration of the reaction. Comparison with related dications shows that furamidine inhibits Tdp1 more effectively than berenil, while pentamidine was inactive. Thus, furamidine represents the most potent Tdp1 inhibitor reported to date.
106 citations
TL;DR: It is shown that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability, and apoptosis, and the pleiotropic nature of human Top1 activities is shown.
Abstract: The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1 small interfering RNA (siRNA) cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those clones, Top1 is reduced approximately 5-fold and Top2alpha compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone gamma-H2AX foci associated with replication defects. siTop1 cells also show rDNA and nucleolar alterations and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) expression was reduced in siTop1 cells and in cells with transient Top1 down-regulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacologic profiling showed L-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, indenoisoquinoline, aphidicolin, hydroxyurea, and staurosporine and hypersensitivity to etoposide and actinomycin D show that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability, and apoptosis. Overall, our studies show the pleiotropic nature of human Top1 activities. In addition to its classic DNA nicking-closing functions, Top1 plays critical nonclassic roles in genomic stability, gene-specific transcription, and response to various anticancer agents. The reported cell lines and approaches described in this article provide new tools to perform detailed functional analyses related to Top1 function.
99 citations
TL;DR: It is reported that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX, ATM, and Chk2 colocalizing in nuclear foci.
Abstract: The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.
89 citations
TL;DR: In this paper, two series of indenoisoquinoline topoisomerase I inhibitors have been prepared to investigate optimal substituents on the indenone ring at the 9 position.
Abstract: Two series of indenoisoquinoline topoisomerase I inhibitors have been prepared to investigate optimal substituents on the indenone ring at the 9-position. The more exhaustive series was prepared using a nitrated isoquinoline ring that has been previously demonstrated to enhance biological activity. After preliminary biological evaluation, a more focused series of inhibitors was prepared utilizing a 2,3-dimethoxy-substituted isoquinoline ring. The results of the two series indicate the existence of superior functional groups such as methoxy, fluorine, and cyano for the indenoisoquinoline 9-position. Interestingly, these functional groups coincide with established structure−activity relationships for the 11-position of camptothecin.
88 citations
TL;DR: These studies suggest AF inhibits the growth of breast cancer cells in part, by inducing ROS production, oxidative DNA damage and apoptosis and has the potential to treat hormone‐independent breast cancer.
Abstract: Aminoflavone (5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methylchromen-4-one; AF; NSC 686288), a novel anticancer candidate agent, is undergoing clinical evaluation. AF induces DNA-protein cross-links (DPCs), Gamma-H2AX phosphorylation, aryl hydrocarbon receptor (AhR) signaling, apoptosis and its own metabolism via cytochrome P4501A1 and 1A2 (CYP1A1/1A2) activation in sensitive estrogen receptor positive (ER+) MCF7 breast cancer cells. Estrogen receptor negative (ER-) breast cancer is typically more aggressive with a poorer prognosis. In this investigation, we evaluated the ability of AF to induce reactive oxygen species (ROS) formation, oxidative DNA damage and apoptosis in ER- MDA-MB-468 breast cancer cells. The antioxidant, N-acetyl-L-cysteine (NAC), attenuated the cytotoxic effects of AF in MDA-MB-468 cells; an effect is also observed in ER+ T47D breast cancer cells. Nonmalignant MCF10A breast epithelial cells were resistant to the cytotoxic effects of AF. AF increased intracellular ROS, an effect blocked by NAC and the CYP1A1/1A2 inhibitor, alpha-Naphthoflavone (alpha-NF). AF induced oxidative DNA damage as evidenced by increased 8-oxo-7,8-dihydroguanine (8-oxodG) levels and DPC formation in these cells. AF caused S-phase arrest corresponding to an increase in p21((waf1/cip1)) protein expression. AF induced caspase 3, 8 and 9 activation, caspase-dependent apoptotic body formation and poly [ADP-ribose] polymerase (PARP) cleavage. Pretreatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone inhibited apoptosis and partially inhibited ROS formation and oxidative DNA damage. Pretreatment with NAC attenuated AF-induced apoptotic body formation and caspase 3 activation. These studies suggest AF inhibits the growth of breast cancer cells in part, by inducing ROS production, oxidative DNA damage and apoptosis and has the potential to treat hormone-independent breast cancer.
80 citations
TL;DR: Batracylin is a dual Top1 and Top2 inhibitor and gamma-H2AX could be considered a biomarker in the ongoing clinical trials.
Abstract: Batracylin (8-aminoisoindolo [1,2-b]quinazolin-10(12H)-one; NSC320846) is an investigational clinical anticancer agent. Previous animal studies showed activity against solid tumors and Adriamycin-resistant leukemia. We initially sought to test the proposed Top2-mediated DNA cleavage activity of batracylin and identify potential biomarkers for activity. COMPARE analysis in the NCI-60 cell lines showed batracylin activity to be most closely related to the class of Top2 inhibitors. The 50% growth inhibition (GI50) value for batracylin in HT29 colon carcinoma cells was 10 micromol/L. DNA-protein cross-links, consistent with Top2 targeting, were measured by alkaline elution. DNA single-strand breaks were also detected and found to be protein associated. However, only a weak induction of DNA double-strand breaks was observed. Because batracylin induced almost exclusively DNA single-strand breaks, we tested batracylin as a Top1 inhibitor. Batracylin exhibited both Top1- and Top2alpha/beta-mediated DNA cleavage in vitro and in cells. The phosphorylation of histone (gamma-H2AX) was tested to measure the extent of DNA damage. Kinetics of gamma-H2AX "foci" showed early activation with low micromol/L concentrations, thus presenting a useful early biomarker of DNA damage. The half-life of gamma-H2AX signal reversal after drug removal was consistent with reversal of DNA-protein cross-links. The persistence of the DNA-protein complexes induced by batracylin was markedly longer than by etoposide or camptothecin. The phosphorylated DNA damage-responsive kinase, ataxia telangiectasia mutated, was also found activated at sites of gamma-H2AX. The cell cycle checkpoint kinase, Chk2, was only weakly phosphorylated. Thus, batracylin is a dual Top1 and Top2 inhibitor and gamma-H2AX could be considered a biomarker in the ongoing clinical trials.
72 citations
TL;DR: These new findings demonstrate that replication fork velocity (fork progression) is coordinated with inter-origin distance and that it can be actively slowed down by Chk1-dependent mechanisms in response to DNA damage.
Abstract: The regulation of DNA replication initiation is well documented, for both unperturbed and damaged cells. The regulation of elongation, or fork velocity, however, has only recently been revealed with the advent of new techniques allowing us to view DNA replication at the single cell and single DNA molecule levels. Normally in S phase, the progression of replication forks and their stability are regulated by the ATR-Claspin-Chk1 pathway. We recently showed that replication fork velocity varies across the human genome in normal and cancer cells, but that the velocity of a given fork is positively correlated with the distance between origins on the same DNA fiber. (19) Accordingly, in DNA replication-deficient Bloom's syndrome cells, reduced fork velocity is associated with an increased density of replication origins. (21) Replication elongation is also regulated in response to DNA damage. In human colon carcinoma cells treated with the topoisomerase I inhibitor camptothecin, DNA replication is inhibited both at the level of initiation and at the level of elongation through a Chk1-dependent checkpoint mechanism. (10) Together, these new findings demonstrate that replication fork velocity (fork progression) is coordinated with inter-origin distance and that it can be actively slowed down by Chk1-dependent mechanisms in response to DNA damage. Thus, we propose that the intra-S phase checkpoint consist of at least three elements: (1) stabilization of damaged replication forks; (2) suppression of firing of late origins; and (3) arrests of normal ongoing forks to prevent further DNA lesions by replication of a damaged DNA template.
TL;DR: In vitro data show the specific inhibition of Chk2 kinase activity by NSC 109555, a novel chemotype for the development of potent and selective inhibitors of ChK2, which may ultimately be useful in combination with current DNA-damaging agents used in the clinic.
Abstract: Chk2 is a protein kinase involved in the ATM-dependent checkpoint pathway (http://discover.nci.nih.gov/mim). This pathway is activated by genomic instability and DNA damage and results in either cell cycle arrest, to allow DNA repair to occur, or cell death (apoptosis). Chk2 is activated by ATM-mediated phosphorylation and autophosphorylation and in turn phosphorylates its downstream targets (Cdc25A, Cdc25C, BRCA1, p53, Hdmx, E2F1, PP2A, and PML). Inhibition of Chk2 has been proposed to sensitize p53-deficient cells as well as protect normal tissue after exposure to DNA-damaging agents. We have developed a drug-screening program for specific Chk2 inhibitors using a fluorescence polarization assay, immobilized metal ion affinity-based fluorescence polarization (IMAP). This assay detects the degree of phosphorylation of a fluorescently linked substrate by Chk2. From a screen of over 100,000 compounds from the NCI Developmental Therapeutics Program, we identified a bis-guanylhydrazone [4,4'-diacetyldiphenylureabis(guanylhydrazone); NSC 109555] as a lead compound. In vitro data show the specific inhibition of Chk2 kinase activity by NSC 109555 using in vitro kinase assays and kinase-profiling experiments. NSC 109555 was shown to be a competitive inhibitor of Chk2 with respect to ATP, which was supported by docking of NSC 109555 into the ATP binding pocket of the Chk2 catalytic domain. The potency of NSC 109555 was comparable with that of other known Chk2 inhibitors, such as debromohymenialdisine and 2-arylbenzimidazole. These data define a novel chemotype for the development of potent and selective inhibitors of Chk2. This class of drugs may ultimately be useful in combination with current DNA-damaging agents used in the clinic.
TL;DR: A series of simplified indenoisoquinoline analogues possessing a linker spacing of 0-12 carbon atoms between the lactam nitrogen and the terminal amino group have been prepared, determining that 2-4-atom lengths are optimal for topoisomerase I inhibition and cytotoxicity.
Abstract: Indenoisoquinolines with lactam substituents such as ethylamino, propylamino, and butylamino have previously demonstrated potent biological activity, but an optimal length has never been established. In the present study, a series of simplified indenoisoquinoline analogues possessing a linker spacing of 0-12 carbon atoms between the lactam nitrogen and the terminal amino group have been prepared, determining that 2-4-atom lengths are optimal for topoisomerase I inhibition and cytotoxicity. Using these lengths, analogues were prepared with the amino group and portions of the linker replaced by a pyridine ring. A three-carbon spacer within the pyridine series still demonstrated potent topoisomerase I inhibition.
TL;DR: This study provides a type of detailed analysis of methylation that can also be applied to other cancer-related genes, and found E-cad expression to be correlated with E- cad methylation at highly statistically significant levels.
Abstract: E-cadherin (E-cad) is a transmembrane adhesion glycoprotein, the expression of which is often reduced in invasive or metastatic tumors. To assess E-cad's distribution among different types of cancer cells, we used bisulfite-sequencing for detailed, base-by-base measurement of CpG methylation in E-cad's promoter region in the NCI-60 cell lines. The mean methylation levels of the cell lines were distributed bimodally, with values pushed toward either the high or low end of the methylation scale. The 38 epithelial cell lines showed substantially lower (28%) mean methylation levels compared with the nonepithelial cell lines (58%). The CpG site at -143 with respect to the transcriptional start was commonly methylated at intermediate levels, even in cell lines with low overall DNA methylation. We also profiled the NCI-60 cell lines using Affymetrix U133 microarrays and found E-cad expression to be correlated with E-cad methylation at highly statistically significant levels. Above a threshold of ∼20% to 30% mean methylation, the expression of E-cad was effectively silenced. Overall, this study provides a type of detailed analysis of methylation that can also be applied to other cancer-related genes. As has been shown in recent years, DNA methylation status can serve as a biomarker for use in choosing therapy. [Mol Cancer Ther 2007;6(2):391–403]
TL;DR: The chemical stability of the keto analogues and the ability of S39625 to produce high levels of persistent Top1-DNA cleavage complex and its potent antiproliferative activity against human cancer cell lines make S39 625 a promising new anticancer drug candidate.
Abstract: Camptothecin (CPT) analogues are powerful anticancer agents but are chemically unstable due to their α-hydroxylactone six-membered E-ring structure, which is essential for trapping topoisomerase I (Top1)-DNA cleavage complexes. To stabilize the E-ring, CPT keto analogues with a five-membered E-ring lacking the oxygen of the lactone ring (S38809 and S39625) have been synthesized. S39625 has been selected for advanced preclinical development based on its promising activity in tumor models. Here, we show that both keto analogues are active against purified Top1 and selective against Top1 in yeast and human cancer cells. The keto analogues show improved cytotoxicity toward colon, breast, and prostate cancer cells and leukemia cells compared with CPT. The drug-induced Top1-DNA cleavage complexes induced by the keto analogues show remarkable persistence both with purified Top1 and in cells following 1-h drug treatments. Moreover, we find that S39625 is not a substrate for either the ABCB1 (multidrug resistance-1/P-glycoprotein) or ABCG2 (mitoxantrone resistance/breast cancer resistance protein) drug efflux transporters, which sets S39625 apart from the clinically used CPT analogues topotecan or SN-38 (active metabolite of irinotecan). Finally, we show that nanomolar concentrations of S38809 or S39625 induce intense and persistent histone γ-H2AX. The chemical stability of the keto analogues and the ability of S39625 to produce high levels of persistent Top1-DNA cleavage complex and its potent antiproliferative activity against human cancer cell lines make S39625 a promising new anticancer drug candidate. Histone γ-H2AX could be used as a biomarker for the upcoming clinical trials of S39625. [Mol Cancer Ther 2007;6(12):3229–38]
TL;DR: This review summarizes the current knowledge of mitochondrial topoisomerases (type IA, IB and type II) in eukaryotes including budding and fission yeasts and protozoan parasites and includes new data showing alternative splice variants of human TOP1mt.
TL;DR: Two HIV-1 integrase-DNA cross-linking assays are used to probe the binding sites of integrase inhibitors from different chemical families and with different strand transfer selectivities, providing clues to the molecular interactions between integrase and the viral cDNA.
Abstract: HIV-1 integrase binds site-specifically to the ends of the viral cDNA. We used two HIV-1 integrase-DNA cross-linking assays to probe the binding sites of integrase inhibitors from different chemical families and with different strand transfer selectivities. The disulfide assay probes cross-linking between the integrase residue 148 and the 5'-terminal cytosine of the viral cDNA, and the Schiff base assay probes cross-linking between an integrase lysine residue and an abasic site placed at selected positions in the viral cDNA. Cross-linking interference by eight integrase inhibitors shows that the most potent cross-linking inhibitors are 3'-processing inhibitors, indicating that cross-linking assays probe the donor viral cDNA (donor binding site). In contrast, strand transfer-selective inhibitors provide weak cross-linking interference, consistent with their binding to a specific acceptor (cellular DNA) site. Docking and crystal structure studies illustrate specific integrase-inhibitor contacts that prevent cross-linking formation. Four inhibitors that prevented Schiff base cross-linking to the conserved 3'-terminal adenine position were examined for inhibition at various positions within the terminal 21 bases of the viral cDNA. Two of them selectively inhibited upper strand cross-linking, whereas the other two had a more global effect on integrase-DNA binding. These findings have implications for elucidating inhibitor binding sites and mechanisms of action. The cross-linking assays also provide clues to the molecular interactions between integrase and the viral cDNA.
TL;DR: The results suggest that the cellular responses to AF are determined at least in part by Mdm2 and p21CIP1/WAF1 protein levels, as well as by Akt activity, leading either to cell cycle arrest when Mdm 2 and p 21CIP 1/Waf1 are elevated, or to apoptosis when MDM2 andp21C IP1/ WAF1 are degraded by the proteasome and Akt insufficiently activated to protect against apoptosis
Abstract: Aminoflavone (AF, NSC 686,288) is beginning clinical trials. It induces replication-mediated histone H2AX phosphorylation, DNA-protein crosslinks and activates p53. Here, we studied p21(CIP1/WAF1) and Mdm2 responses to AF. Although p53 stabilization and phosphorylation at serine 15 increased with dose and time of exposure, Mdm2 and p21(CIP1/WAF1) protein levels displayed a biphasic response, as they accumulated at submicromolar doses and then decreased with increasing AF. As both Mdm2 and p21(CIP1/WAF1) mRNA levels increased with AF concentration without reduction at higher concentrations, we measured the half-lives of Mdm2 and p21(CIP1/WAF1) proteins. Mdm2 and p21(CIP1/WAF1) half-lives were shortened with increasing AF concentrations. Proteasomal degradation appears responsible for the decrease of both Mdm2 and p21(CIP1/WAF1), as MG-132 prevented their degradation and revealed AF-induced Mdm2 polyubiquitylation. AF also induced protein kinase B (Akt) activation, which was reduced with increasing AF concentrations. Suppression of Akt by small interfering RNA was associated with downregulation of Mdm2 and p21(CIP1/WAF1) and with enhanced apoptosis. These results suggest that the cellular responses to AF are determined at least in part by Mdm2 and p21(CIP1/WAF1) protein levels, as well as by Akt activity, leading either to cell cycle arrest when Mdm2 and p21(CIP1/WAF1) are elevated, or to apoptosis when Mdm2 and p21(CIP1/WAF1) are degraded by the proteasome and Akt insufficiently activated to protect against apoptosis.
TL;DR: The method described here provides the first direct stable-isotope labeling method to definitely detect phosphorylation sites within proteins, and could be readily confirmed using tandem MS.
Abstract: A novel stable-isotope labeling approach for identification of phosphopeptides that utilizes adenosine triphosphate, in which four oxygen-16 atoms attached to the terminal phosphate group are substituted with oxygen-18 [γ(18O4)-ATP], has been developed. The ability to use γ(18O4)-ATP to monitor phosphorylation modification within various proteins was conducted by performing in vitro kinase reactions in the presence of a 1:1 mixture of γ(18O4)-ATP and normal isotopic abundance ATP (ATP). After tryptic digestion, the peptides were analyzed using mass spectrometry (MS). Phosphorylated peptides are easily recognized within the MS spectrum owing to the presence of doublets separated by 6.01 Da; representing versions of the peptide modified by ATP and γ(18O4)-ATP. Standard peptides phosphorylated using γ(18O4)-ATP via in vitro kinase reactions showed no exchange loss of 18O with 16O. The identity of these doublets as phosphorylated peptides could be readily confirmed using tandem MS. The method described here p...
TL;DR: The data suggests that MM intensity from PM signal can be a major source of error analysis, leading to fewer potentially biologically important candidate genes.
Abstract: Motivation: For Affymetrix microarray platforms, gene expression is determined by computing the difference in signal intensities between perfect match (PM) and mismatch (MM) probesets. Although the use of PM is not controversial, MM probesets have been associated with variance and ultimately inaccurate gene expression calls. A principal focus of this study was to investigate the nature of the MM signal intensities and demonstrate its contribution to the experimental results.
Results: While most MM intensities were likely associated with random noise, a subset of ~20% (99 485) of the MM probes displayed relatively high signal intensities to the corresponding PM probes (MM > PM) in a non-random fashion; 13 440 of these probes demonstrated exceptionally high ‘outlier’ intensities. About 15 938 PM probes also demonstrated exceptionally high outlier intensities consistently across all hybridizations. About 92% of the MM > PM probes had either a dThymidine (dT) or a dCytidine (dC) at the 13th position of the probe sequence. MM and PM probes displaying extremely high outlier intensities contained high dC rich nucleotides, and low dA contents at other nucleotides positions along the 25mer probe sequence. Differentially expressed genes generated using Genechip Operating System (GCOS) or modified PM-only methods were also examined. Of those candidate genes identified in the PM-only method, 157 of them were designated by GCOS as absent across all datasets and many others contained probes with MM > PM signal intensities. Our data suggests that MM intensity from PM signal can be a major source of error analysis, leading to fewer potentially biologically important candidate genes.
Contact: wangyong@mail.nih.gov
Supplementary information: Supplementary data are available at Bioinformatics online.
TL;DR: Virtual screening methods including docking and filtering by predicted ADME/Tox properties yielded several microM level inhibitors of the strand transfer reaction catalyzed by wild-type HIV-1 integrase.
TL;DR: These isosteric compounds represent the first examples of beta-diketo phosphonic acids of structural, synthetic, and antiviral interest.
TL;DR: The results suggest the crucial role played by the N-terminal extension of the small subunit of DNA topoisomerase I in both relaxation activity and CPT sensitivity of LdTop1 is suggested.
TL;DR: Treatment with single doses of ABT-888, a novel potent PARP-1 inhibitor, reduced PAR levels in peripheral blood mononuclear cells (PBMCs) and tumor biopsies in an on-going Phase-0 trial at the NIH Clinical Center.
Abstract: 14024 Background: Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is a molecular sensor of DNA breaks that facilitates DNA repair and controls genomic stability. Treatment with single doses of ABT-888, a novel potent PARP-1 inhibitor, reduced PAR levels in peripheral blood mononuclear cells (PBMCs) and tumor biopsies in an on-going Phase-0 trial at the NIH Clinical Center. As a corollary to this study, we investigated whether ABT-888 can act as a sensitizer for radiation therapy and chemotherapy in human cancer cell culture, xenograft tumors and PBMCs to support future combination clinical trials. Methods: Inhibition of PARP-1 by ABT-888 was determined by a quantitative PAR chemiluminescence immunoassay validated for the Phase 0 trial. Since gamma-H2AX (?-H2AX) is a marker of DNA damage, we also developed and validated ?H2AX assays to monitor the effects of PARP-1 inhibition during treatment with Topo I inhibitors and radiation. Human monocytic leukemia (THP-1) and breast carcinoma (MCF-7) cell lines were tr...
Journal Article•
TL;DR: In this article, the authors conducted a single dose pilot toxicity study in Beagle dogs and found that the maximum tolerated dose level is 3 mg/kg for NSC-725776 and 7.75 mg/ kg for NC-724998 when dosed via a 15 min intravenous infusion and neither compound displays increased sensitivity in dogs.
Abstract: 1562 The activity of traditional DNA topoisomerase I (Top1) inhibitors, such as camptothecin (CPT) and its derivatives, have been limited due to their chemical instability, rapid cleavage complex reversibility after drug removal, drug resistance and side effects. Through the extensive ongoing work to develop new, novel Top1 inhibitors without these limitations, the indenoisoquinoline analogs, NSC-725776 and NSC-724998, have emerged as potential lead candidates. Both analogs are chemically stable, and exhibit increased resistance to reversal of effects after drug removal, relative to CPT and its derivatives. In order to determine whether NSC-725776 and NSC-724998 would produce similar adverse events as CPT and its derivatives, i.e., neutropenia and diarrhea, and show similar species sensitivity, we conducted a single dose pilot toxicity study in Beagle dogs. NSC-725776 and NSC-724998 were administered via intravenous injection (60 seconds) or infusion (15 minutes) to Beagle dogs at target dose levels of 1.5, 3 and 5 mg/kg or 3.75, 7.5, 10, and 25 mg/kg, respectively. Mortality was observed in dogs treated with NSC-725776 (5 mg/kg) and NSC-724998 (10 or 25 mg/kg). Prior to death, the animals exhibited excessive salivation, emesis, respiratory distress, cyanotic mucous membrane, lethargy, weak pulses, vocalization, mucoid feces and/or soft feces. At necropsy, gelatinous material in bronchial lymph nodes and clear fluid in the trachea and/or foam in the airways were noted in some of the dogs. For surviving animals treated with NSC-725776, a dose of 1.5 mg/kg via injection or 3 mg/kg via infusion produced respiratory distress and prostration, whereas, a dose of 1.5 mg/kg via infusion produced prostration and diarrhea/soft feces. Surviving animals dosed with NSC-724998 via infusion, exhibited signs of breathing difficulty (7.5 mg/kg) or mucoid/soft feces (3.75 mg/kg). Consistent with the signs of respiratory distress in dogs that died prematurely, was a concomitant decrease in serum carbon dioxide CO2 levels just prior to their death. Modest increases in serum total bilirubin concentration (TBILI) and alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK) activity were also noted, however because of the small magnitude of these changes, these clinical chemistry effects were probably not biologically relevant. Alterations in hematology parameters consistent with neutropenia, thrombocytopenia and hemoconcentration were noted in animals treated with NSC-725776 (5 mg/kg) or NSC-724998 (10 or 25 mg/kg). No necropsy findings were noted in dogs treated with NSC-725776 ≤ 1.5 mg/kg or NSC-724998 doses ≤10 mg/kg. In conclusion, the maximum tolerated dose level is 3 mg/kg for NSC-725776 and 7.5 mg/kg for NSC-724998 when dosed via a 15 min intravenous infusion and neither compound displays increased sensitivity in dogs. (Supported by NCI Contract N01-CM-42204).
Journal Article•
TL;DR: Results have indicated that MAP3K7 silencing preferentially sensitizes MDA-MB-231 cells to camptothecin and doxorubicin, as compared to the relatively normal mammary epithelial cell line MCF10A, which can be associated with long-term exposure to chemotherapeutic agents.
Abstract: PR-3 Specific molecular targets have yet to be identified for many cancers, necessitating treatment with non-specific, cytotoxic agents. In an effort to develop strategies that improve the efficacy of such agents, we have conducted a siRNA-based RNAi screen to identify genes that, when targeted by siRNA, improve compound activity. Screening was conducted using a set of siRNAs corresponding to over 400 apoptosis-related genes in the triple negative breast cancer cell line MDA-MB-231. Triple negative breast cancers lack the canonical treatment targets progesterone receptor, estrogen receptor, and amplified ErbB2. Screens were performed using the well-known anti-cancer agents camptothecin and doxorubicin. During the course of these studies, we identified the silencing of MAP3K7 as a significant potentiator of the activity of both compounds. These effects were reproduced in follow-up analyses and further corroborated using an additional siRNA targeting MAP3K7. MAP3K7 encodes TAK1 kinase, an enzyme that is central to the regulation of many processes associated with the growth of cancer cells (e.g. NFκB signaling). MAP3K7 silencing was not found to be toxic independent of compound addition. Moreover, results have indicated that MAP3K7 silencing preferentially sensitizes MDA-MB-231 cells to camptothecin and doxorubicin, as compared to the relatively normal mammary epithelial cell line MCF10A. Ongoing studies are directed towards evaluating the magnitude of this effect in additional breast cell lines and examining if enhanced compound activity can circumvent drug resistance, which can be associated with long-term exposure to chemotherapeutic agents.