To construct the pharmacophore model of the poly (ADP-ribose) polymerase-1 inhibitor and to investigate the possible inhibitory mechanisms, ten pharmacophore models of PARP-1 inhibitor were established from the training set of thirty-eight PARP-1 inhibitors with conformer analysis and pharmacophore mapping by using the Catalyst software. Based on the mechanism of action and the known structure-activity relationship of PARP-1 inhibitor, an optimal pharmacophore model including two hydrogen-bonding acceptors and two aromatic hydrophobic core was confirmed. The reliability of the optimal pharmacophore model is preferably with RMS = 0.46, Correl = 0.91, Weight = 2.06, and Config = 15.97. This pharmacophore model not only provided some information about the interaction between enzyme and compound, but also showed excellent forecast ability and contributes to design the PARP-1 inhibitors with undiscovered structure.
Computer-Aided Design ; Drug Design ; Enzyme Inhibitors ; chemistry ; pharmacology ; Models, Molecular ; Molecular Structure ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerase Inhibitors ; Poly(ADP-ribose) Polymerases ; chemistry ; Protein Conformation

The study aimed to investigate the effect of inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) activity on tau phosphorylation in HEK293/tau441 cells and its mechanism. HEK293/tau441 cells were treated with 3-aminobenzamide (3-AB), a PARP-1 inhibitor, at different doses (0.5, 1, 2, 4 mmol/L). After 24 h, the cell morphology was observed under phase contrast microscope, tau phosphorylation level in different sites (tau-1, tau-5, Thr231) and the activity of glycogen synthase kinase 3 (GSK-3) were detected by Western blotting. The results showed: (1) 3-AB at different doses failed to change the morphology of cells; (2) The 3-AB-induced decrease in activity of PARP-1 resulted in increase of unphosphorylation level in tau-1(Ser195/198/199/202) sites; (3) The phosphorylation of tau was decreased in Thr231 site, while the total tau was slightly changed after 3-AB treatment; (4) With the increased phosphorylation of GSK-3 at Ser9 site, the activity of GSK-3 was decreased after 3-AB treatment. The results suggest that the inhibition of PARP-1 by 3-AB could decrease tau phosphorylation in HEK293/tau441 cells probably through decreasing GSK-3 activity.
Benzamides ; pharmacology ; Depression, Chemical ; Glycogen Synthase Kinase 3 ; metabolism ; HEK293 Cells ; Humans ; Phosphorylation ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerase Inhibitors ; Poly(ADP-ribose) Polymerases ; metabolism ; tau Proteins ; metabolism

PARP [poly(ADP-ribose)polymerase] represents a novel potential target in cancer therapy. It is involved in a DNA repair process by catalyzing the transfer of ADP-ribose units from NAD to a number of its substrate proteins. In this work, a series of novel azaindole derivatives was designed and synthesized. Moreover, 16 target molecules were screened and 8 compounds displayed inhibitory activity against PARP-1. It has been demonstrated that these azaindoles bearing cycloamine substituents at 2-position were active to both PARP-1 and PARP-2.
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Aza Compounds ; chemical synthesis ; chemistry ; pharmacology ; Indoles ; chemical synthesis ; chemistry ; pharmacology ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism

PARP1 is an important part of DNA repair machinery. In recent years, PARP1 as novel anti-cancer therapeutic target has been broadly explored. In this study, we expressed hPARP1 enzyme in the baculovirus system and tested its activity. We inserted hPARP1 gene into the pFastBac1, a baculovirus transfer vector and then transformed it into DH10Bac containing a shuttle vector of Bacmid. After co-transfecting the recombinant plasmid into Sf9 insect cells, the expressed hPARP1 was purified by 3-aminobezamide affinity chromatography. The expression of hPARPI was visualized by SDS-PAGE and Western blotting; the activity of expressed and purified hPARP1 was confirmed by the reaction of consumption of NAD+ by hPARP1 in vitro. After the purification by 3-aminobezamide affinity column, 3.2 mg protein was obtained and its specific activity was 1.988 nmol/(min x microg).
Animals ; Baculoviridae ; genetics ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Genetic Vectors ; Humans ; Insecta ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; biosynthesis ; Recombinant Proteins ; Sf9 Cells ; Transfection

OBJECTIVETo investigate the DNA methylation changes induced by hydroquinone (HQ) in human bronchial epithelial cells and to explore the role of poly (ADP-ribose) polymerase-l (PARP-l) in this process.

METHODSHuman bronchial epithelial 16HBE cells and PARP-l-deficient 16HBE cells (16HBE-shPARP-l cells) were exposed to HQ (10, 20, 40, 60, and 80 µmol/L) for 48h, while control cells were treated with an equal volume of PBS solution. The changes in genomic DNA methylation were investigated by high-performance capillary electrophoresis, and the expression levels of PARP-l and DNA methyltransferase 1 (DNMT1) were measured.

RESULTSThe percentages of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP-l cells were 4.89%±0.07% and 9.53%±0.51%, respectively; after treatment with 5-aza-2'-deoxycytidine for 72 h, mCpG% decreased to 3.07±0.12% and 6.34%±0.3%, respectively. The one-way analysis of variance revealed significant differences in mCpG% between the cells exposed to different concentrations of HQ in both 16HBE and 16HBE-shPARP-l groups (F = 61.25, P < 0.01; F = 60.36, P < 0.01). For 16HBE cells treated with HQ (10, 20, 40, 60, and 80 µmol/L), the mRNA expression levels of PARP-1 were 145.0%, 159.0%, 169.0%, 215.0%, and 236.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all); for 16HBE-shPARP-l cells treated with HQ (10, 20, 40, 60, and 80 µmol/L), the mRNA expression levels of PARP-l were 170.0%, 223.0%, 264.0%, 327.0%, and 320.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all). When the dose of HQ reached 20, 40, 60, and 80 µmol/L, the mRNA expression levels of DNMT1 in 16HBE group were 114.0%, 126.0%, 136.0%, and 162.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all); when the dose of HQ reached 10, 20, 40, 60, and 80 µmol/L, the mRNA expression levels of DNMT1 in the 16HBE-shPARP-l group were 141.0%, 165.2%, 186.9%, 202.1%, and 217.3%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all).

CONCLUSIONHQ can induce hypomethylation in 16HBE cells, and PARP-1 can regulate DNA methylation in 16HBE cells by influencing the expression and activity of DNMT1.

Cells, Cultured ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Damage ; DNA Methylation ; Epithelial Cells ; metabolism ; Humans ; Hydroquinones ; toxicity ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism

OBJECTIVETo investigate DNA methylation variation in human cells induces by B(a)P, and to explore the role of PARP1 during this process.

METHODSThe changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B(a)P (1.0, 2.0, 5.0, 10.0, 15.0, 30.0 µmol/L) were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP 1 and DNMT 1 were monitored dynamically.

RESULTSThe percentage of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP1 cells were separately (4.04 ± 0.08)% and (9.69 ± 0.50)%. After being treated by 5-DAC for 72 hours, mCpG% decreased to (3.15 ± 0.14)% and (6.07 ± 0.54)%. After both being exposed to B(a)P for 72 hours, the mCpG% in 16HBE group (ascending rank) were separately (5.10 ± 0.13), (4.25 ± 0.10), (3.91 ± 0.10), (4.23 ± 0.27), (3.70 ± 0.15), (3.08 ± 0.07); while the figures in 16HBE-shPARP1 group (ascending rank) were respectively (10.63 ± 0.60), (13.08 ± 0.68), (9.75 ± 0.55), (7.32 ± 0.67), (6.90 ± 0.49) and (6.27 ± 0.21). The difference of the results was statistically significant (F values were 61.67 and 60.91, P < 0.01). For 16HBE group, expression of PARP 1 and DNMT 1 were 141.0%, 158.0%, 167.0%, 239.0%, 149.0%, 82.9% and 108.0%, 117.0%, 125.0%, 162.0%, 275.0%, 233.0% comparing with the control group, whose difference also has statistical significance (t values were 11.45, 17.32, 32.24, 33.44, 20.21 and 9.87, P < 0.01). For 16HBE-shPARP1 group, expression of PARP 1 and DNMT 1 were 169.0%, 217.0%, 259.0%, 323.0%, 321.0%, 256.0% and 86.0%, 135.0%, 151.0%, 180.0%, 229.0%, 186.0% comparing with the control group, with statistical significance (t values were 9.06, 15.92, 22.68, 26.23, 37.19 and 21.15, P < 0.01). When the dose of B(a)P reached 5.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE group (ascending rank) were 125.0%, 162.0%, 275.0%, 233.0% times of it in control group, with statistical significance (t values were 12.74, 24.92, 55.11, 59.07, P < 0.01); while the dose of B(a)P reached 2.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE-shPARP1 group were 135.0%, 151.0%, 180.0%, 229.0%, 186.0% of the results in control group, and the differences were statistically significant (t values were 23.82, 40.17, 32.69, 74.85, 46.76, P < 0.01).

CONCLUSIONThe hypomethylation of 16HBE cells induced by B(a)P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.

Benzo(a)pyrene ; adverse effects ; Cell Line ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; metabolism ; DNA Damage ; DNA Methylation ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; genetics ; metabolism

OBJECTIVETo study hPARP1 genetic polymorphism in southern Chinese Han and Miao populations.

METHODSBlood samples from 187 and 210 southern healthy Han and Miao populations were collected. The mutations of exons 12,13,16 and 17 of hPARP1 gene were investigated by PCR-single-strand conformation polymorphism(SSCP).

RESULTSFragments of 253 bp,313 bp,175 bp,362 bp within exons 12,13,16 and 17 respectively of hPARP1 gene were amplified by multiple PCR. An SSCP variant in exons 12,13,16 and 17 of PARP1 gene in 187 healthy Han and 210 healthy Miao individuals was identified. Seven single-base substitutions compared with the sequence of PARP1 gene were identified: a T to C transition in exon 12 (Phe548Ser), a G to T transition in exon 13 (Ala683Ser), a G to T transition in exon 16 (Asp798Tyr), and a A to G transition in exon 17 (His808Arg).

CONCLUSIONThere were polymorphism sites in exons 12,13,16,17 of hPARP1 gene in southern Chinese Han and Miao populations; these results may be useful for the establishment of PARP1 genotyping, and these newly described PARP1 alleles would be advantageous indicators for population studies.

Adult ; Alleles ; China ; Exons ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; genetics ; Polymorphism, Single-Stranded Conformational

Erythropoietin (EPO) is the major means of treating anemia of chronic disease (ACD) through stimulating hematopoiesis, inhibiting hepcidin and decreasing proinflammatory factors. Recently, it has been found that monocytes are another source of hepcidin. EPO can reduce the hepcidin stimulated by IL-6 in monocytes, it is assumed that EPO can reduce hepcidin indirectly by reducing IL-6. However, the specific mechanism of EPO inhibiting the proinflammatory cytokines in monocytes is unclear now. This study was purposed to investigate the effect of EPO on monocyte proinflammatory factors and its molecular mechanism. IL-6 mRNA and TNF-α mRNA were detected by real time PCR, level of signaling molecule PARP-1 protein was detected by Western blot. THP-1 monocytes were stimulated by 1 µg/ml lipopolysaccharide (LPS) to observe the impact of EPO at different concentrations (0.5, 1, 2, 5, 10 U/ml) for different time (0, 3, 6, 12, 24 hours) on the expression of IL-6 mRNA, TNF-α mRNA and PARP-1 protein. 1 µg/ml or 5 µg/ml EPO receptor (EPOR) antibody and/or 3-aminobenzamide (3-AB, PARP-1 inhibitor) were added to observe the antagonistic effect on EPO and the impact on PARP-1. The results showed that LPS could stimulate the THP-1 cells. EPO could decrease the levels of IL-6 and TNF-α stimulated by LPS in a dose- and time-dependent manners. The most significant decrease in IL-6 mRNA expression was observed in 2 U/ml EPO for 6 hours. And down-regulation of TNF-α mRNA expression was pronounced at 10 U/ml EPO for 3 hours. IL-6 mRNA expression could be stimulated by LPS, PARP-1 protein was induced at the same time. EPO inhibited the expression of IL-6 mRNA, while PARP-1 protein also decreased. Down-regulation of IL-6 mRNA and PARP-1 protein level was pronounced at 2 U/ml EPO for 6 hours. 3AB is a direct inhibitor of PARP-1. Similar to 3AB, EPO receptor antibody could antagonize the decline of IL-6 induced by EPO. It is concluded that EPO can inhibit the expression of IL-6 and TNF-α in monocytes, and the inhibition of IL-6 expression may be associated with decrease of PARP level.
Anemia ; metabolism ; Cell Line ; Erythropoietin ; pharmacology ; Humans ; Interleukin-6 ; metabolism ; Monocytes ; drug effects ; metabolism ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate whether or not adaptive response of hPARP-1 protein normal and deficient cells is induced by low dose of hydroquinone (HQ), and to analyze the relationship between the adaptive response and micronuclei formation, and cell cycle alteration in human embryo lung fibroblasts (HLF), so as to elucidate the mechanism of adaptive response.

METHODSHLF, HLFC and HLFP cells pretreated with low concentration were retreated by high concentration of HQ. Cellular viability, the rate of micronuclei and abnormal nuclei, cell cycle and DNA strand break were determined.

RESULTSThe tolerance to 80.0 micromol/L concentration of HQ was enhanced when HLF, HLFC and HLFP cells were pretreated with HQ from 0.001 - 0.050 micromol/L. There were varying degrees of micronuclei and abnormal nuclei in three cells pretreated with low concentration of HQ and then retreated with high concentration of HQ; the cell numbers of G1, G2, S phase in cell cycle were obviously different. When compared with only high attack dose, the micronuclei rate and abnormal nuclei rate of HLF, HLFC and HLFP decreased by pretreatment with HQ at high concentration (P < 0.05), meanwhile increased by pretreatment with HQ at low concentration (P < 0.05). HLF, HLFC and HLFP showed blockage in G2 phase when pretreated with HQ at 0 approximately 0.05 micromol/L, but HLFP showed blockage in G1 phase, and in S phase at 1.0 and 2.0 micromol/L.

CONCLUSIONThe level of adaptive response of hPARP-1 protein deficient cells was lower than normal cell, suggesting that hPARP-1 protein may play an important role in the adaptive response of cells, which may be related with the regulation of cell cycle.

Cell Cycle ; Cell Nucleus ; Cell Survival ; Cells, Cultured ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Hydroquinones ; toxicity ; Lung ; cytology ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism

OBJECTIVETo study the effect of silicon dioxide nanoparticles on the expression and promoter region CpG islands methylation of (Poly [ADP-ribose] polymerase 1, PARP-1) gene in human HaCaT Cell.

METHODSHaCaT Cells were treated with nm-SiO₂at 0, 2.5, 5 and 10 µg/mL and micro-SiO₂at 10 µg/ml for 24 h and DAC treatment was given at 10 µg/ml group for 48 h. Real-time PCR and western blot assay was used to detect the expression of PARP-1 mRNA and protein. BSP (Bisulfite Pyrosequence, BSP) assay was used to detect the promoter region CpG islands methylation status of PARP-1 gene.

RESULTSAfter exposure to nano-SiO₂particles, compared to CTRL group, the mRNA and protein expression of PARP-1 in micro-SiO₂and 2.5 µg/ml group unchanged, but he mRNA and protein expression of PARP-1 in 5, 10 µg/ml as well as DAC group was down-regulated and there are statistical significance between CTRL group and 5, 10 µg/ml as well as DAC group and the PARP-1 promoter region CpG islands showed methylation.

CONCLUSIONnano-SiO₂can down-regulate PARP-1 expression in HaCaT Cell and this is associated with the change in the methylation of PARP-1 gene promoter region CpG islands induced by nano-SiO₂particles.

Cell Line, Tumor ; CpG Islands ; DNA Methylation ; Humans ; Nanoparticles ; adverse effects ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Silicon Dioxide ; adverse effects