共查询到20条相似文献,搜索用时 46 毫秒
1.
Zhang YW Regairaz M Seiler JA Agama KK Doroshow JH Pommier Y 《Nucleic acids research》2011,39(9):3607-3620
Poly(ADP-Ribose) (PAR) polymerase (PARP) inhibitors represent a promising class of novel anticancer agents. The present study explores the molecular rationale for combining veliparib (ABT-888) with camptothecin (CPT) and its clinical derivatives, topotecan and irinotecan. ABT-888 inhibited PAR induction by CPT and increased CPT-induced cell killing and histone γH2AX. Increased DNA breaks by ABT-888 were not associated with a corresponding increase of topoisomerase I cleavage complexes and were further increased by inactivation of tyrosyl-DNA phosphodiesterase 1. SiRNA knockdown for the endonuclease XPF-ERCC1 reduced the ABT-888-induced γH2AX response in non-replicating and replicating cells but enhanced the antiproliferative effect of ABT-888 in CPT-treated cells. Our findings indicate the involvement of XPF-ERCC1 in inducing γH2AX response and repairing topoisomerase I-induced DNA damage as an alternative pathway from PARP and tyrosyl-DNA phosphodiesterase 1. 相似文献
2.
Liu X Shi Y Guan R Donawho C Luo Y Palma J Zhu GD Johnson EF Rodriguez LE Ghoreishi-Haack N Jarvis K Hradil VP Colon-Lopez M Cox BF Klinghofer V Penning T Rosenberg SH Frost D Giranda VL Luo Y 《Molecular cancer research : MCR》2008,6(10):1621-1629
Poly(ADP-ribose) polymerase (PARP) senses DNA breaks and facilitates DNA repair via the polyADP-ribosylation of various DNA binding and repair proteins. We explored the mechanism of potentiation of temozolomide cytotoxicity by the PARP inhibitor ABT-888. We showed that cells treated with temozolomide need to be exposed to ABT-888 for at least 17 to 24 hours to achieve maximal cytotoxicity. The extent of cytotoxicity correlates with the level of double-stranded DNA breaks as indicated by gammaH2AX levels. In synchronized cells, damaging DNA with temozolomide in the presence of ABT-888 during the S phase generated high levels of double-stranded breaks, presumably because the single-stranded DNA breaks resulting from the cleavage of the methylated nucleotides were converted into double-stranded breaks through DNA replication. As a result, treatment of temozolomide and ABT-888 during the S phase leads to higher levels of cytotoxicity. ABT-888 inhibits poly(ADP-ribose) formation in vivo and enhances tumor growth inhibition by temozolomide in multiple models. ABT-888 is well tolerated in animal models. ABT-888 is currently in clinical trials in combination with temozolomide. 相似文献
3.
《DNA Repair》2014
Anti-cancer topoisomerase I (Top1) inhibitors (camptothecin and its derivatives irinotecan and topotecan, and indenoisoquinolines) induce lethal DNA lesions by stabilizing Top1-DNA cleavage complex (Top1cc). These lesions are repaired by parallel repair pathways including the tyrosyl-DNA phosphodiesterase 1 (TDP1)-related pathway and homologous recombination. As TDP1-deficient cells in vertebrates are hypersensitive to Top1 inhibitors, small molecules inhibiting TDP1 should augment the cytotoxicity of Top1 inhibitors. We developed a cell-based high-throughput screening assay for the discovery of inhibitors for human TDP1 using a TDP1-deficient chicken DT40 cell line (TDP1−/−) complemented with human TDP1 (hTDP1). Any compounds showing a synergistic effect with the Top1 inhibitor camptothecin (CPT) in hTDP1 cells should either be a TDP1-related pathway inhibitor or an inhibitor of alternate repair pathways for Top1cc. We screened the 400,000-compound Small Molecule Library Repository (SMLR, NIH Molecular Libraries) against hTDP1 cells in the absence or presence of CPT. After confirmation in a secondary screen using both hTDP1 and TDP1−/− cells in the absence or presence of CPT, five compounds were confirmed as potential TDP1 pathway inhibitors. All five compounds showed synergistic effect with CPT in hTDP1 cells, but not in TDP1−/− cells, indicating that the compounds inhibited a TDP1-related repair pathway. Yet, in vitro gel-based assay revealed that the five compounds did not inhibit TDP1 catalytic activity directly. We tested the compounds for their ability to inhibit poly(ADP-ribose)polymerase (PARP) because PARP inhibitors are known to potentiate the cytotoxicity of CPT by inhibiting the recruitment of TDP1 to Top1cc. Accordingly, we found that the five compounds inhibit catalytic activity of PARP by ELISA and Western blotting. We identified the most potent compound (Cpd1) that offers characteristic close to veliparib, a leading clinical PARP inhibitor. Cpd1 may represent a new scaffold for the development of PARP inhibitors. 相似文献
4.
Xuesong Liu Joann Palma Yan Shi Paul A Ellis Milagros Colon-Lopez David LeBlond David J. Frost Vincent L. Giranda 《Analytical biochemistry》2008,381(2):240-247
Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. The DNA repair capacity of the tumor represents a common mechanism used by cancer cells to survive DNA-damaging therapy. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that is activated by DNA damage and has critical roles in DNA repair. Inhibition of PARP potentiates the activity of DNA-damaging agents such as temozolomide, topoisomerase inhibitors and radiation in both in vitro and in vivo preclinical models. Recently, several PARP inhibitors have entered clinical trials either as single agents or in combination with DNA-damaging chemotherapy. Because PARP inhibitors are not cytotoxic, a biomarker assay is useful to guide the selection of an optimal biological dose. We set out to develop an assay that enables us to detect 50% PAR reduction in human tumors with 80% power in a single-plate assay while assuring no more than a 10% false-positive rate. We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) to measure PARP activity that meets the above-mentioned criterion. This robust assay is able to detect PAR levels of 30-2000 pg/ml in both tumor and peripheral blood monocyte samples. In a B16F10 mouse syngeneic tumor model, PARP inhibitor ABT-888 potentiates the effect of temozolomide in suppressing tumor growth, and PARP activity is greatly reduced by ABT-888 at efficacious doses. In summary, the ELISA assay described here is suitable for biomarker studies in clinical trials of PARP inhibitors. 相似文献
5.
Benu Brata Das Shar-yin N. Huang Junko Murai Ishita Rehman Jean-Christophe Amé Souvik Sengupta Subhendu K. Das Papiya Majumdar Hongliang Zhang Denis Biard Hemanta K. Majumder Valérie Schreiber Yves Pommier 《Nucleic acids research》2014,42(7):4435-4449
Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3′-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1–PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1. 相似文献
6.
Patel AG Flatten KS Schneider PA Dai NT McDonald JS Poirier GG Kaufmann SH 《The Journal of biological chemistry》2012,287(6):4198-4210
Poly(ADP-ribose) polymerase-1 (PARP1) plays critical roles in the regulation of DNA repair. Accordingly, small molecule inhibitors of PARP are being developed as agents that could modulate the activity of genotoxic chemotherapy, such as topoisomerase I poisons. In this study we evaluated the ability of the PARP inhibitor veliparib to enhance the cytotoxicity of the topoisomerase I poisons topotecan and camptothecin (CPT). Veliparib increased the cell cycle and cytotoxic effects of topotecan in multiple cell line models. Importantly, this sensitization occurred at veliparib concentrations far below those required to substantially inhibit poly(ADP-ribose) polymer synthesis and at least an order of magnitude lower than those involved in selective killing of homologous recombination-deficient cells. Further studies demonstrated that veliparib enhanced the effects of CPT in wild-type mouse embryonic fibroblasts (MEFs) but not Parp1(-/-) MEFs, confirming that PARP1 is the critical target for this sensitization. Importantly, parental and Parp1(-/-) MEFs had indistinguishable CPT sensitivities, ruling out models in which PARP1 catalytic activity plays a role in protecting cells from topoisomerase I poisons. To the contrary, cells were sensitized to CPT in a veliparib-independent manner upon transfection with PARP1 E988K, which lacks catalytic activity, or the isolated PARP1 DNA binding domain. These results are consistent with a model in which small molecule inhibitors convert PARP1 into a protein that potentiates the effects of topoisomerase I poisons by binding to damaged DNA and preventing its normal repair. 相似文献
7.
Ji J Kinders RJ Zhang Y Rubinstein L Kummar S Parchment RE Tomaszewski JE Doroshow JH 《PloS one》2011,6(10):e26152
Background
Poly(ADP-ribose) polymerase (PARP)facilitates DNA repair and PARP inhibitors may potentiate the effect of DNA-damaging chemotherapeutic agents in patients with cancer. Collection of peripheral blood mononuclear cells (PBMCs)as a surrogate tissue to monitor PARP inhibitor pharmacodynamic effects has several advantages over tumor biopsy collection, including minimally invasive sample collection and the ability to collect multiple samples for longitudinal assessment of drug effect.Methodology/Principal Findings
Using our previously validated immunoassay for measuring poly(ADP-ribose) (PAR), a product of PARP, in tumor biopsies, we validated a method to quantify PAR levels in PBMCs to monitor the pharmacodynamic effects of the PARP inhibitor ABT-888 in clinical trials. The inter-individual variation in PAR levels was large. No significant difference (P = 0.67) was measured between median baseline PAR levels in 144 healthy volunteers (131.7 pg/1×107 PBMCs [interquartile range, 79.5–241.6]) and 49 patients with cancer (149.2 pg/1×107 PBMCs [interquartile range, 83.2–249.3]). In addition, PAR levels monitored in healthy volunteers over 3 weeks had considerable intra- and inter-individual variation (range, 44–1073 pg PAR/1×107 PBMCs). As a pharmacodynamic model, we quantified changes in PAR levels in human PBMCs treated ex vivo with clinically relevant concentrations of ABT-888. Of 40 healthy volunteer PBMC samples treated with ABT-888, 47.5% had greater than 50% PAR reduction compared to vehicle-treated controls. Considerable inter-sample heterogeneity in PAR levels was measured, and several ABT-888–insensitive samples were identified.Conclusions/Significance
Our results emphasize the importance of using a validated method to measure PAR levels, and support further investigation into the role of PARP in PBMCs. To this end, the PAR immunoassay has been validated for use with PBMCs and incorporated into clinical trials to assess PBMCs as a potential pharmacodynamic surrogate for tumor biopsies in clinical trials of PARP inhibitors. 相似文献8.
9.
Richard Tuli Andrew J. Surmak Juvenal Reyes Michael Armour Amy Hacker-Prietz John Wong Theodore L. DeWeese Joseph M. Herman 《Translational oncology》2014,7(3):439-445
OBJECTIVESTo determine whether poly (ADP-ribose) polymerase-1/2 (PARP-1/2) inhibition enhances radiation-induced cytotoxicity of pancreatic adenocarcinoma in vitro and in vivo, and the mechanism by which this occurs.MethodsPancreatic carcinoma cells were treated with ABT-888, radiation, or both. In vitro cell viability, apoptosis, and PARP activity were measured. Orthotopic xenografts were generated in athymic mice and treated with ABT-888 (25 mg/kg), radiation (5 Gy), both, or no treatment. Mice were monitored with bioluminescence imaging.RESULTSIn vitro, treatment with ABT-888 and radiation led to higher rates of cell death after 8 days (P < .01). Co-treatment with 5 Gy and 1, 10 or 100 μmol/l of ABT-888 led to dose enhancement factors of 1.29, 1.41 and 2.36, respectively. Caspase activity was not significantly increased when treated with ABT-888 (10 μmol/l) alone (1.28-fold, P = .08), but became significant when radiation was added (2.03-fold, P < .01). PARP activity increased post-radiation and was abrogated following co-treatment with ABT-888. In vivo, treatment with ABT-888, radiation or both led to tumor growth inhibition (TGI) of 8, 30 and 39 days, and survival at 60 days of 0%, 0% and 40%, respectively.CONCLUSIONSABT-888 with radiation significantly enhanced tumor response in vitro and in vivo. ABT-888 inhibited PAR protein polymerization resulting in dose-dependent feedback up-regulation of PARP and p-ATM suggesting increased DNA damage. This translated into enhancement in TGI and survival with radiation in vivo. In vitro PAR levels correlated with levels of tumor apoptosis suggesting potential as a predictive biomarker. These data are being used to support a Phase I study in locally advanced pancreatic cancer. 相似文献
10.
Plo I Liao ZY Barceló JM Kohlhagen G Caldecott KW Weinfeld M Pommier Y 《DNA Repair》2003,2(10):1087-1100
DNA topoisomerase I (Top1) is converted into a cellular poison by camptothecin (CPT) and various endogenous and exogenous DNA lesions. In this study, we used X-ray repair complementation group 1 (XRCC1)-deficient and XRCC1-complemented EM9 cells to investigate the mechanism by which XRCC1 affects the cellular responses to Top1 cleavage complexes induced by CPT. XRCC1 complementation enhanced survival to CPT-induced DNA lesions produced independently of DNA replication. CPT-induced comparable levels of Top1 cleavage complexes (single-strand break (SSB) and DNA-protein cross-links (DPC)) in both XRCC1-deficient and XRCC1-complemented cells. However, XRCC1-complemented cells repaired Top1-induced DNA breaks faster than XRCC1-deficient cells, and exhibited enhanced tyrosyl DNA phosphodiesterase (Tdp1) and polynucleotide kinase phosphatase (PNKP) activities. XRCC1 immunoprecipitates contained Tdp1 polypeptide, and both Tdp1 and PNKP activities, indicating a functional connection between the XRCC1 single-strand break repair pathway and the repair of Top1 covalent complexes by Tdp1 and PNKP. 相似文献
11.
12.
Cetuximab augments cytotoxicity with poly (adp-ribose) polymerase inhibition in head and neck cancer
Nowsheen S Bonner JA Lobuglio AF Trummell H Whitley AC Dobelbower MC Yang ES 《PloS one》2011,6(8):e24148
Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of head and neck cancers and confers increased resistance and inferior survival rates. Despite targeted agents against EGFR, such as cetuximab (C225), almost half of treated patients fail this therapy, necessitating novel therapeutic strategies. Poly (ADP-Ribose) polymerase (PARP) inhibitors (PARPi) have gained recent attention due to their unique selectivity in killing tumors with defective DNA repair. In this study, we demonstrate that C225 enhances cytotoxicity with the PARPi ABT-888 in UM-SCC1, UM-SCC6, and FaDu head and neck cancer cells. The mechanism of increased susceptibility to C225 and PARPi involves C225-mediated reduction of non-homologous end-joining (NHEJ)- and homologous recombination (HR)-mediated DNA double strand break (DSB) repair, the subsequent persistence of DNA damage, and activation of the intrinsic apoptotic pathway. By generating a DSB repair deficiency, C225 can render head and neck tumor cells susceptible to PARP inhibition. The combination of C225 and the PARPi ABT-888 can thus be an innovative treatment strategy to potentially improve outcomes in head and neck cancer patients. Furthermore, this strategy may also be feasible for other EGFR overexpressing tumors, including lung and brain cancers. 相似文献
13.
Fiorani P Reid RJ Schepis A Jacquiau HR Guo H Thimmaiah P Benedetti P Bjornsti MA 《The Journal of biological chemistry》2004,279(20):21271-21281
DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT). During S-phase, collisions with replication forks convert these complexes into cytotoxic DNA lesions that trigger cell cycle arrest and cell death. To investigate cellular responses to CPT-induced DNA damage, a yeast genetic screen identified conditional tah mutants with enhanced sensitivity to self-poisoning DNA topoisomerase I mutant (Top1T722Ap), which mimics the action of CPT. Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered. A nonsense mutation in DOA4 eliminated the catalytic residues of the Doa4p deubiquitinating enzyme, yet retained the rhodanase domain. At 36 degrees C, this doa4-10 mutant exhibited increased sensitivity to CPT, osmotic stress, and hydroxyurea, and a reversible petite phenotype. However, the accumulation of pre-vacuolar class E vesicles that was observed in doa4Delta cells was not detected in the doa4-10 mutant. Mutations in SLA1 or SLA2, which alter actin cytoskeleton architecture, induced a conditional synthetic lethal phenotype in combination with doa4-10 in the absence of DNA damage. Here actin cytoskeleton defects coincided with the enhanced fragility of large-budded cells. In contrast, the enhanced sensitivity of doa4-10 mutant cells to Top1T722Ap was unrelated to alterations in endocytosis and was selectively suppressed by increased dosage of the ribonucleotide reductase inhibitor Sml1p. Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons. These findings indicate a functional link between ubiquitin-mediated proteolysis and cellular resistance to CPT-induced DNA damage. 相似文献
14.
Colley WC van der Merwe M Vance JR Burgin AB Bjornsti MA 《The Journal of biological chemistry》2004,279(52):54069-54078
Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding. 相似文献
15.
Diana Nguyen Maria Zajac-Kaye Larry Rubinstein Donna Voeller Joseph E Tomaszewski Shivaani Kummar Alice P Chen Yves Pommier James H Doroshow Sherry X Yang 《Cell cycle (Georgetown, Tex.)》2011,10(23):4074-4082
Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G1/S checkpoint, ATM and p53 signaling pathways in p53-wild-type cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G1/S checkpoint pathway in p53 wild-type lines and not in p53-mutant cells. These responses are coupled with G2/G1 checkpoint effectors p21CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G2 cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wild-type and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G2 arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wild-type and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.Key words: DNA damaging agent, G2 arrest, microarray, PARP inhibition, p53, topotecan, veliparib (ABT-888) 相似文献
16.
Parise RA Shawaqfeh M Egorin MJ Beumer JH 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2008,872(1-2):141-147
ABT-888, a poly(ADP-ribose) polymerase (PARP) -inhibitor in clinical trials, potentiates DNA-damaging agents. We developed and validated, according to FDA guidelines, an LC-MS assay for sensitive, accurate and precise quantitation of ABT-888 and its metabolite M8 in 0.2mL human plasma. After ethyl acetate extraction, separation is achieved with a hydro-Synergi column and a 0.1% formic acid in acetonitrile/water-gradient. Detection uses electrospray, positive-mode ionization mass spectrometry. Between 10 (LOQ) and 1000ng/mL, accuracy was 95.5-98.5% for ABT-888 and 91.4-100.9% for M8, and precision was 0.1-4.9% for ABT-888 and 0-13.7% for M8. The assay is being applied to samples generated in several clinical trials. 相似文献
17.
《DNA Repair》2016
PARP inhibitors in combination with other agents are in clinical trial against cancer, but its effect on cancer stem cells (CSCs) is limited. CSCs are responsible for drug resistance, metastasis and cancer relapse due to high DNA repair capacity. Here, we present preclinical effects of Quinacrine (QC) with ABT-888, a PARP inhibitor, on highly metastatic breast cancer stem cells (mBCSCs). An increased level of Adenomatous polyposis coli (APC) was noted after treatment with ABT-888 in QC pre-treated mBCSCs cells. Increased APC physically interacts with PARP-1 and inhibits PARylation causing the non assembly of base excision repair (BER) multiprotein complex, resulting in an irreparable DNA damage and subsequent apoptosis. Knockdown of APC in mBCSCs inhibited DNA damage, increased BER and PARylation, reduces apoptosis while the over-expression of APC in BT20 (APC low expressing) cells reversed the effect. Thus, combination of QC and ABT-888 decreased mBCSCs growth by activating APC and inhibiting BER within the cells. 相似文献
18.
Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which is activated in response to genotoxic insults by binding damaged DNA and attaching polymers of ADP-ribose to nuclear proteins at the expense of its substrate NAD+. In persons affected with ataxia telangiectasia (A-T), associated mutations in the ataxia telangiectasia mutated gene render cells unable to cope with the genotoxic stresses from ionizing radiation and oxidative damage, thus resulting in a higher concentration of unrepaired DNA damage and the activation of PARP in an uncontrolled manner. In primary A-T fibroblasts, we observed a 58-96% increase in PARP activity and a concomitant loss of cellular NAD+ and ATP content. PARP protein by Western blot analysis increased only slightly in these cells, supporting the observation that the steady state levels of DNA damage is higher in A-T cells than in normals. When treated with PARP inhibitors 3-aminobenzamide or 1,5-dihydroisoquinoline, cellular growth rates reached those observed in normal fibroblast cultures. The improvement of cellular growth and NAD+ levels in A-T cells with PARP inhibition suggests that the cellular metabolic status of A-T cells is compromised and the inhibition of PARP may relieve some of the drain on cellular pyridine nucleotides and ATP. Thus, therapy utilizing PARP inhibitors may provide a benefit for individuals affected with A-T. 相似文献
19.
Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear protein in most of the eukaryotic tissues. When activated by DNA damage, PARP synthesizes poly(ADP-ribose) from NAD. Conventional radioactive PARP enzyme assay requires the separation of the polymer product from the NAD substrate, a rate-limiting step that hampers large-scale chemical library screening to identify novel small-molecule PARP inhibitors. By using biotinylated NAD, we have developed a scintillation proximity assay (SPA) for PARP. We demonstrated that PARP can incorporate the biotinylated ADP-ribose units into the radioactive poly(ADP-ribose) polymer, which can directly bind and excite the streptavidin-conjugated scintillation beads. PARP-SPA can be readily adapted to a 96-well format for automatic high-throughput screening for PARP inhibitors. 相似文献
20.
Ren-Kuo Lin Chia-Wen Ho Leroy F. Liu Yi Lisa Lyu 《The Journal of biological chemistry》2013,288(10):7182-7192
Camptothecin (CPT), a topoisomerase (Top) I-targeting drug that stabilizes Top1-DNA covalent adducts, can induce S-phase-specific cytotoxicity due to the arrest of progressing replication forks. However, CPT-induced non-S-phase cytotoxicity is less well characterized. In this study, we have identified topoisomerase IIβ (Top2β) as a specific determinant for CPT sensitivity, but not for many other cytotoxic agents, in non-S-phase cells. First, quiescent mouse embryonic fibroblasts (MEFs) lacking Top2β were shown to be hypersensitive to CPT with prominent induction of apoptosis. Second, ICRF-187, a Top2 catalytic inhibitor known to deplete Top2β, specifically sensitized MEFs to CPT. To explore the molecular basis for CPT hypersensitivity in Top2β-deficient cells, we found that upon CPT exposure, the RNA polymerase II large subunit (RNAP LS) became progressively depleted, followed by recovery to nearly the original level in wild-type MEFs, whereas RNAP LS remained depleted without recovery in Top2β-deficient cells. Concomitant with the reduction of the RNAP LS level, the p53 protein level was greatly induced. Interestingly, RNAP LS depletion has been well documented to lead to p53-dependent apoptosis. Altogether, our findings support a model in which Top2β deficiency promotes CPT-induced apoptosis in quiescent non-S-phase cells, possibly due to RNAP LS depletion and p53 accumulation. 相似文献