OBJECTIVETo evaluate the effect of Zhuanggu Jianxi Decoction (, ZGJXD) on interleukin-1 β (IL-1 β)-induced degeneration of chondrocytes (CDs) as well as the activation of caveolin-p38 mitogen-activated protein kinase (MAPK) signal pathway, investigating the possible molecular mechanism that ZGJXD treats osteoarthritis.

METHODSSerum pharmacology was applied in the present study, where ZGJXD was orally administrated to New Zealand rabbits and then ZGJXD containing serum (ZGJXD-S) was collected for following in vitro experiments. CDs were isolated aseptically from New Zealand rabbits and then cultured in vitro. Upon IL-1 β stimulation, the degeneration of CDs was verified by inverted microscope, toluidine blue stain and type II collagen immunocytochemistry. After IL-1 β-stimulated CDs were intervened with blank control serum, ZGJXD-S, together with or without SB203580 (a specific inhibitor of p38 MAPK) for 48 h, caveolin-1 protein expression and the phosphorylation level of p38 were determined by Western blotting, and the mRNA expression of IL-1 β, tumor necrosis factor α (TNF-α), matrix metalloproteinase 3 (MMP-3) and MMP-13 were examined by real-time polymerase chain reaction.

RESULTSIL-1 β stimulation induced degeneration of CDs, increased caveolin-1 expression and p38 phosphorylation, up-regulated the mRNA level of IL-1 β, TNF-α, MMP-3 and MMP-13. However, the IL-1 β-induced activation of caveolin-p38 signaling and alteration in the expression of p38 downstream target genes were suppressed by ZGJXD-S and/or SB203580 in CDs.

CONCLUSIONZGJXD can prevent CDs degeneration via inhibition of caveolin-p38 MAPK signal pathway, which might be one of the mechanisms that ZGJXD treats osteoarthritis.

Animals ; Base Sequence ; Blotting, Western ; Caveolins ; metabolism ; Chondrocytes ; drug effects ; enzymology ; metabolism ; DNA Primers ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Profiling ; Interleukin-1beta ; physiology ; MAP Kinase Signaling System ; Male ; RNA, Messenger ; genetics ; Rabbits ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

The study aims at cloning the CDS fragment of erk2 gene cDNA in Inner Mongolia Cashmere Goat and analyzing its tissue-specific expression, erk2 gene cDNA was cloned by RT-PCR. The nucleotide sequence was analyzed by Blast and amino acid sequence was analyzed by online softwares SMART and Psite. The tissue-specific expression pattern of erk2 was analyzed by quantitative RT-PCR. The expression of erk2 in testis of goat was detected by Immunohistochemistry. The cloned erk2 gene cDNA (GenBank Accession No. JX569765) was 1 083 bp in length, including a complete ORF encoding 360 amino acids residues. The amino acid sequence shares 100% identity with the Bos Taurus ERK2 (Bos Taurus BC133588.1). Analysis by SMART suggests that the encoded protein contained a "TEY" structure and an S-TKc domain possessing serine/threonine kinase catalytic activity. Analysis with Psite indicates one cAMP-/cGMP-dependent protein kinase phosphorylation site, 3 protein kinase C phosphorylation sites, 5 casein kinase II phosphorylation sites, 2 protein kinases ATP-binding region signatures and one serine/threonine protein kinases active-site signature in this protein. Analysis by Psort (k-NN prediction) suggestes that this protein most probably is localized in cytoplasm. The results of quantitative RT-PCR show that the expression of erk2 mRNA was higher in heart, skin and breast, whereas lower in spleen and kidney. ERK2 protein was detected in testis by immunohistochemistry.
Amino Acid Sequence ; Animals ; China ; Cloning, Molecular ; DNA, Complementary ; genetics ; Goats ; genetics ; Male ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Molecular Sequence Data ; Real-Time Polymerase Chain Reaction ; Testis ; metabolism

Benzene is an established leukotoxin and leukemogen in humans. We have previously reported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to mediate the cellular response to DNA double strand break (DSB) caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026), as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phosphorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apoptosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.
Apoptosis ; drug effects ; physiology ; Benzene ; toxicity ; Catalysis ; Chromones ; pharmacology ; DNA Damage ; drug effects ; genetics ; DNA Repair ; drug effects ; physiology ; DNA-Activated Protein Kinase ; antagonists & inhibitors ; metabolism ; Humans ; K562 Cells ; Morpholines ; pharmacology ; Protein Subunits

PURPOSE: To investigate whether the G6721T polymorphism (rs.7003908) of the non-homologous end-joining DNA repair XRCC7 gene contributes to the development of exudative age-related macular degeneration (ARMD). METHODS: The present case-control study consisted of 111 patients with exudative ARMD and 112 sex frequency-matched healthy controls that were randomly selected from unrelated volunteers in the same clinic. Genotypes were determined by the Restriction Fragment Length Polymorphism (PCR-RFLP) based method. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for ARMD risk associated with polymorphism of XRCC7. In all analysis the GG genotype was considered to be the reference genotype. RESULTS: There was no significant association between genotypes of XRCC7 and susceptibility to ARMD. Considering the significant difference in age distribution between cases and controls, age was used as a covariate in further analysis. After ORs were adjusted for age, the same result was observed. In the next step we stratified our subjects into outdoor and indoor groups according to their job titles. The outdoor and indoor patients were occupationally exposed to sunlight and not exposed to sunlight, respectively. Our present study showed that among indoor subjects there was no association between XRCC7 polymorphism and susceptibility to ARMD. However, among outdoor subjects, the GT + TT genotypes compared to the GG genotype increased the risk of ARMD (OR, 3.13; 95% CI, 1.04-9.39; p = 0.042). CONCLUSIONS: Our study revealed that the T allele of the G6721T polymorphism of XRCC7 increased the risk of ARMD among outdoor subjects.
Aged ; DNA/*genetics ; DNA-Activated Protein Kinase/*genetics/metabolism ; *Environmental Exposure ; Exudates and Transudates ; Female ; Follow-Up Studies ; *Genetic Predisposition to Disease ; Genotype ; Humans ; Macular Degeneration/*genetics/metabolism ; Male ; Middle Aged ; Nuclear Proteins/*genetics/metabolism ; Polymerase Chain Reaction ; *Polymorphism, Genetic ; Retrospective Studies ; Risk Factors

DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.
Antigen-Antibody Complex ; metabolism ; Antigens, Nuclear ; genetics ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; Cellular Senescence ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair ; DNA-Activated Protein Kinase ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Fibroblasts ; cytology ; HeLa Cells ; Homologous Recombination ; genetics ; physiology ; Humans ; Ku Autoantigen ; Nicotinamide Phosphoribosyltransferase ; genetics ; metabolism ; physiology ; Nuclear Proteins ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; beta-Galactosidase ; metabolism

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin-damaging agents including pectenotoxin-2 (PTX-2) increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced-tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA-PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.
Actins/*metabolism ; Apoptosis ; Catalytic Domain ; Cell Cycle Proteins/genetics/*metabolism ; Cell Line ; Cell Line, Tumor ; DNA-Activated Protein Kinase/chemistry/genetics/*metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Furans/pharmacology ; *G1 Phase ; Gene Knockdown Techniques ; Humans ; Phosphorylation/drug effects ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; Pyrans/pharmacology ; Tumor Suppressor Protein p53/*metabolism ; Tumor Suppressor Proteins/genetics/*metabolism

This study was aimed to explore the expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in adult acute leukemia and its correlation with clinical characteristics, karyotype and prognosis. Indirect immunofluorescent cytometry was used to detect the expression of DNA-PKcs in bone marrow mononuclear cells of 105 patients with acute leukemia before chemotherapy and 41 of them after 2 cycles of chemotherapy. Cytogenetic data were obtained from 26 of them by R band karyotypic analysis. The results showed that the expression of DNA-PKcs was correlated with higher WBC count level in peripheral blood (p < 0.05), but was not obviously associated with median age, gender, percentage of bone marrow blasts, clinical classification, median hemoglobin level and median platelet count (p > 0.05). The middle and strong positive expression of DNA-Pkcs in non-remission group was significantly higher than that in remission group (p < 0.05). The positive rate of DNA-PKcs in abnormal chromosome group was significantly higher than that in chromosome normal group (p < 0.05). It is concluded that the DNA-PKcs expression level is closely related with the increased WBC count, and the expression of DNA-PKcs is correlated also with karyotype and clinical prognosis in adult acute leukemia.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Chromosome Aberrations ; DNA-Activated Protein Kinase ; genetics ; metabolism ; Female ; Humans ; Karyotype ; Leukemia ; diagnosis ; genetics ; metabolism ; Male ; Middle Aged ; Nuclear Proteins ; genetics ; metabolism ; Prognosis ; Young Adult

OBJECTIVETo study the expression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) in non-small cell lung cancer (NSCLC), and the interaction between hnRNP A2/B1 protein and mRNA of DNA repair enzymes O(6)-methylguanine DNA-methyltransferase (MGMT), 8-oxoguanine DNA glycosylase (OGG1), redox factor 1(Ref-1), DNA-dependent protein kinase (including DNA-PKcs and ku).

METHODSThe expression and distribution of hnRNP A2/B1 were detected by immunohistochemistry and Western blot on 50 NSCLC samples from patients who underwent resection in Zhongshan Hospital. The hnRNP A2/B1 mRNA expression was tested by real-time PCR. Co-immunoprecipitation (co-IP) combined RT-PCR was used to investigate whether hnRNP A2/B1 could be bound with the mRNA of the above mentioned 5 DNA repair enzymes in human lung cancer cell line (HTB-182). Then immunohistochemistry and real-time PCR were used to detect the expression of MGMT in the same group of patients.

RESULTSHnRNP A2/B1 protein and mRNA expressions were increased in the NSCLC tissues than that in the corresponding normal lung tissues. HnRNP A2/B1 was expressed predominantly in the nuclei of tumor cells. The positive rate and immunohistochemistry score of hnRNP A2/B1 in tumor tissue were significantly higher than that in normal tissue (P < 0.01). In stage III-IV NSCLC, hnRNP A2/B1 expression was higher than that in stage I-II. There was no significant differences of hnRNP A2/B1 expression among patients of different age, sex, histological type, and smoking history. The results of co-IP combined RT-PCR suggested that hnRNP A2/B1 is bound with MGMT mRNA, and MGMT expression is decreased in tumor tissue of NSCLC.

CONCLUSIONSThe results of this study show that hnRNP A2/B1 protein and mRNA are highly expressed in NSCLC, and hnRNP A2/B1 is bound with MGMT mRNA, which indicate that it might be one of the mechanisms of hnRNP A2/B1 participating in the pathogenesis of NSCLC.

Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; genetics ; DNA-Activated Protein Kinase ; metabolism ; Guanine ; analogs & derivatives ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B ; genetics ; metabolism ; Humans ; Immunohistochemistry ; Immunoprecipitation ; Lung ; chemistry ; Lung Neoplasms ; genetics ; RNA, Messenger ; analysis ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the roles of DNA dependent protein kinase (DNA-PK)in silica-induced cell cycle changes and expressions of CyclinE and CDK2 in human embryo lung fibroblasts (HELF).

METHODSThe expressions of Ku80 and DNA-PKcs proteins were inhibited by siRNA plasmids, respectively. Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression levels of CyclinE and CDK2 after cells were exposed to 200 microg/ml silica for 0, 3, 6, 12, 24 h.

RESULTSThe proportion of G1 phases in negative control cells decreased from 83.53% +/- 2.24% to 69.11% +/- 3.12% after exposure to silica; the proportion of G1 phases in H-Ku80 and H-PKcs cells exposed to silica decreased from 85.16% +/- 3.73% to 59.92% +/- 3.31% and from 75.06% +/- 2.23% to 58.32% +/- 1.35%, respectively (P < 0.05). The exposure to silica resulted in the increasing protein expression levels of CyclinE and CDK2 in negative control cells, and the expression levels of CyclinE were obviously suppressed in H-Ku80 and H-PKcs as compared with control cells. However, the expression level of CDK2 protein did not change significantly.

CONCLUSIONDNA-PK might play a role in silica-induced alternations of cell cycle and regulate silica-induced overexpression of CyclinE in human embryo lung fibroblasts.

Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; DNA-Activated Protein Kinase ; genetics ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Lung ; cytology ; Nuclear Proteins ; genetics ; metabolism ; Oncogene Proteins ; metabolism ; Silicon Dioxide ; pharmacology

Sequential activation of the JNK pathway components, including Rac1/Cdc42, MLKs (mixed-lineage kinases), MKK4/7 and JNKs, plays a required role in many cell death paradigms. Those components are organized by a scaffold protein, POSH (Plenty of SH3's), to ensure the effective activation of the JNK pathway and cell death upon apoptotic stimuli. We have shown recently that the expression of POSH and MLK family proteins are regulated through protein stability. By generating a variety of mutants, we provide evidence here that the Nterminal half of POSH is accountable for its stability regulation and its over-expression-induced cell death. In addition, POSH's ability to induce apoptosis is correlated with its stability as well as its MLK binding ability. MLK family's stability, like that of POSH, requires activation of JNKs. However, we were surprised to find out that the widely used dominant negative (d/n) form of c-Jun could down-regulate MLK's stability, indicating that peptide from d/n c-Jun can be potentially developed into a therapeutical drug.
Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Apoptosis ; physiology ; Base Sequence ; Cell Line ; DNA Primers ; genetics ; Humans ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; genetics ; metabolism ; MAP Kinase Kinase Kinases ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; PC12 Cells ; Peptide Fragments ; genetics ; metabolism ; Protein Stability ; Rats ; Recombinant Proteins ; genetics ; metabolism ; Signal Transduction ; physiology ; Transfection ; Ubiquitin-Protein Ligases ; genetics ; metabolism