OBJECTIVETo construct antisense c-Jun N-terminal kinase 1 (JNK1) eukaryotic fluorescent expressing vector and JNK1-/- human embryo lung fibroblasts cell line.

METHODSTrizol reagent was used to extract total RNA in HELF. The proper primers of JNK1 were chosen and synthesized. RT-PCR and gene recombinant techniques were used to construct the fragment of JNK1. After purification, the PCR products were cut, and JNK1 were inserted reversely into eukaryotic fluorescent expressing vector pEGFP-C1. Enzyme-cutting and DNA auto-sequencing were used to prove the successful construction of JNK1 eukaryotic expressing vector. Then plasmids were extracted and transfected into HELF cells and screen by G418 24 h later. Monoclone was chosen and cultured. Fluorescent imaging and Western blot were used to identify the JNK1-/- HELF cell line.

RESULTSSequence analysis of pEGFP-C1-as JNK1 plasmids was same as expected. The expression level of JNK1 was inhibited markedly.

CONCLUSIONConstruction of antisense JNK1 eukaryotic fluorescent expressing vectors and JNK1-/- HELF cell line is successful.

Cell Line ; DNA, Antisense ; genetics ; Fibroblasts ; metabolism ; Genetic Vectors ; Humans ; Mitogen-Activated Protein Kinase 8 ; genetics ; metabolism ; Transfection

Bone Marrow Cells ; metabolism ; DNA-Activated Protein Kinase ; genetics ; metabolism ; Humans ; Leukemia ; genetics ; Nuclear Proteins ; genetics ; metabolism ; PTEN Phosphohydrolase ; genetics ; metabolism ; RNA, Messenger ; Telomerase ; genetics ; metabolism

OBJECTIVETo study the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in silica-induced DNA double-strand break repair in human embryo lung fibroblasts (HELF).

METHODSTwo stable transfectants, HELF transfected with DNA-PKcs siRNA (HELF-PKcs) and with negative control siRNA (HELF-NC), were established. HELF cells were treated with 0, 25, 50, 100, 200, 300 and 400 microg/ml silica for 12 h and with 200 microg/ml silica for different times (0, 1, 2, 6, 12 and 24 h). HELF-PKcs and HELF-NC were treated with 200 microg/ml silica for 0, 12 and 24 h. The expression levels of DNA-PKcs and phosphor-H2AX (H2AX) were determined by Western blot. DNA double strand breaks were measured by neutral comet assay.

RESULTSAfter treatment with different doses of silica for 12 h, the levels of H2AX and the percentages of tail DNA increased in concentration-dependent manner. After treatment with 200 microg/ml silica for different times, the levels of H2AX increased in a time-dependent manner. The percentages of tail DNA increased significantly at 6 h, and reaching maximum at 12 h and then decreasing at 24 h. The expression level of DNA-PKcs was suppressed in HELF-PKcs. After treatment with silica at 12 h, the level of H2AX was lower in HELF-PKcs than in HELF-NC, and the percentages of tail DNA increased obviously in both HELF-PKcs and HELF-NC compared with non-treated cells, but no significant difference was found in the percentages of tail DNA between them. The percentages of tail DNA decreased markedly in silica-treated HELF-NC and was significantly lower than in HELF-PKcs at 24 h (P < 0.05).

CONCLUSIONSilica can induce DNA double strand breaks in human embryo lung fibroblasts. DNA-PKcs might play a major role in silica-induced DNA double strand break repair. Silica-induced histone H2AX phosphorylation was dependent on DNA-PKcs.

Cell Line ; DNA Breaks, Double-Stranded ; drug effects ; DNA Repair ; DNA-Activated Protein Kinase ; genetics ; metabolism ; Fibroblasts ; drug effects ; physiology ; Histones ; metabolism ; Humans ; Phosphorylation ; Silicon Dioxide ; pharmacology ; Transfection

OBJECTIVETo investigate the effects of protein tyrosine phosphatase-SHP2 and dual-specificity MAPK phosphatase-MKP5 on the activation of MAPKs and cell invasion induced by P2Y purinergic receptor in human prostate cancer cell lines with different metastatic potentials.

METHODSThe wide type (-wt) SHP2, mutant type (-cs) SHP2 and wide type (-wt) MKP5 cDNA expression vectors were constructed and stably transfected into 1E8 cells (highly metastatic) and/or 2B4 cells (non-metastatic). The tyrosine phosphorylation of SHP2 was examined by immunoprecipitation. The activation of ERK1/2 and p38 induced by P2Y receptor agonist ATP was analyzed by Western blot with phospho-specific antibodies against the dually phosphorylated, active forms of ERK1/2 and p38. The in-vitro invasive ability through Matrigel was measured by boyden-chamber assay.

RESULTSATP induced significant SHP2 phosphorylation, which was stronger and lasted longer in 1E8 than in 2B4. SHP2-wt enhanced the ERK1/2 activation induced by ATP in 2B4 cells, while SHP2-cs delayed and decreased this effect in 1E8 cells. Both SHP2-wt and SHP2-cs had no obvious influence on p38 activation. ATP stimulated cell invasion of both 1E8 and 2B4, while transfection of SHP2-wt into 2B4 cells further increased the invasive-stimulating ability of ATP (18.7% increase compared with ATP treatment alone). Transfection of SHP2-cs into 1E8 cells, however, antagonized the invasive-stimulating ability of ATP (40.9% decrease compared with ATP treated group). Up-regulation of MKP5-wt inhibited phosphorylation of p38 by ATP and reduced cell invasion stimulated by ATP (22.4% and 28.7% decrease compared with ATP treated group of 1E8 and 2B4, respectively).

CONCLUSIONSBoth SHP2 and MKP5 play some roles in P2Y receptor-mediated activation of MEK/ERK, p38 signaling pathways and prostate cancer invasion. SHP2 positively regulates ERK activation and prostate cancer invasion, whereas MKP5 inhibits the invasion by suppressing p38 activation.

Adenosine Triphosphate ; pharmacology ; Cell Line, Tumor ; DNA, Complementary ; genetics ; Dual-Specificity Phosphatases ; Genetic Vectors ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase Phosphatases ; Neoplasm Invasiveness ; Phosphorylation ; Prostatic Neoplasms ; metabolism ; pathology ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; Protein Tyrosine Phosphatases ; genetics ; metabolism ; Receptors, Purinergic P2 ; physiology ; Signal Transduction ; Transfection ; p38 Mitogen-Activated Protein Kinases ; metabolism

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

In eukaryotic cells, DNA damage triggers activation of checkpoint signaling pathways that coordinate cell cycle arrest and repair of damaged DNA. These DNA damage responses serve to maintain genome stability and prevent accumulation of genetic mutations and development of cancer. The p38 MAPK was previously implicated in cellular responses to several types of DNA damage. However, the role of each of the four p38 isoforms and the mechanism for their involvement in DNA damage responses remained poorly understood. In this study, we demonstrate that p38γ, but not the other p38 isoforms, contributes to the survival of UV-treated cells. Deletion of p38γ sensitizes cells to UV exposure, accompanied by prolonged S phase cell cycle arrest and increased rate of apoptosis. Further investigation reveal that p38γ is essential for the optimal activation of the checkpoint signaling caused by UV, and for the efficient repair of UV-induced DNA damage. These findings have established a novel role of p38γ in UV-induced DNA damage responses, and suggested that p38γ contributes to the ability of cells to cope with UV exposure by regulating the checkpoint signaling pathways and the repair of damaged DNA.
Animals ; Apoptosis ; Cell Cycle Proteins ; metabolism ; Cells, Cultured ; DNA Damage ; DNA Repair ; Enzyme Activation ; Fibroblasts ; metabolism ; radiation effects ; Gene Deletion ; Histones ; metabolism ; Mice ; Mitogen-Activated Protein Kinase 12 ; genetics ; metabolism ; Phosphorylation ; S Phase ; Tumor Suppressor Protein p53 ; metabolism ; Ultraviolet Rays

OBJECTIVETo study the role of extracellular signal-regulated kinase (ERK) in cerebral ischemia and the mechanism of protective effects of U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene) on ischemic brain.

METHODSMice underwent left middle cerebral artery occlusion (MCAO) by introducing a suture in the lumen. U0126 was injected intravenously through the internal jugular vein. The immuno-activity of phosphorylated ERK1/2 (pERK1/2), phosphorylated mitogen activated protein kinase kinase (pMEK), and phosphorylated Elk-1 (pElk-1) was assessed by Western blot analysis and immunohistochemistry. Interleukin (IL)-1beta mRNA level was measured by ribonuclease protection assay.

RESULTSPhosphorylated ERK1/2 in 2 hours MCAO mice was down-regulated after intravenous injection of U0126. The inhibition was dose dependent and treatment time related. pMEK and pElk-1 were also reduced in a similar fashion after U0126 treatment. IL-1beta mRNA increased after 1 and 2 hours of MCAO. After injection of U0126, it was down-regulated during 1 to 4 hours after MCAO.

CONCLUSIONIntravenous administration of the MEK inhibitor U0126 inhibits pMEK, pERK1/2, and pElk-1 up-regulation induced by cerebral ischemia. The protective effect of U0126 against ischemic injury is probably resulted from the reduction of IL-1beta mRNA via the inhibition of ERK pathway.

Animals ; Butadienes ; pharmacology ; DNA-Binding Proteins ; metabolism ; Enzyme Inhibitors ; pharmacology ; Infarction, Middle Cerebral Artery ; metabolism ; Interleukin-1 ; biosynthesis ; genetics ; Male ; Mice ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Phosphorylation ; Proto-Oncogene Proteins ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Transcription Factors ; metabolism ; ets-Domain Protein Elk-1

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

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