Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nucleoside analogue cordycepin (3'-deoxyadenosine), when protected against ADA deamination, is specifically cytotoxic for TdT-positive leukemia cells. Cordycepin-treated, ADA-inhibited, TdT-positive cells undergo the classic changes associated with drug-induced apoptosis: reduction in cell volume, chromatin clumping, membrane blebbing, and 180-bp multimer DNA laddering on agarose gels. In common with the apoptosis seen in normal TdT-positive thymocytes, following exposure to various agents, apoptosis induced by cordycepin in TdT-positive leukemia cells was associated with increased protein kinase A (PK-A) activity. Unlike thymocyte apoptosis however, no elevation in cAMP levels was seen preceding the rise in PK-A activity. Ex vivo we show that cordycepin monophosphate can activate PK-A as efficiently as cAMP. On this basis we speculate that cordycepin monophosphate in TdT-positive cells may be able to activate PK-A in place of cAMP, and that PK-A may phosphorylate TdT, augmenting its activity as an endonuclease. In cell-free experiments, the activity of recombinant TdT as an endonuclease digesting supercoiled plasmid DNA into linear fragments was dramatically increased following phosphorylation of TdT by PK-A. A role for TdT as an apoptotic endonuclease in TdT-positive leukemia cells following cordycepin exposure is now the subject of on-going work.
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PMID:Induction of apoptosis by cordycepin in ADA-inhibited TdT-positive leukemia cells. 866 37

Previously we reported that phorbol ester, a protein kinase C (PKC) activator, exhibits a unique pattern of potentiation of nitric oxide (NO)-related apoptosis in HL-60 human promyelocytic leukemia cells. Here we show that elevation of intracellular cAMP could protect HL-60 cells from NO- or NO plus PMA-induced DNA damage. Exposure of cells to sodium nitroprusside (SNP; 0.5 to 4 mM), a NO-generating agent, induced apoptotic cell death as monitored by morphological means, gel electrophoresis, and in situ TdT-apoptosis assay. However, concomitant incubation of the cells with DB-cAMP markedly inhibited SNP-induced apoptotic cell death in a dose-dependent manner. Similar results were obtained with other commonly used cAMP analogs such as CPT-cAMP and 8-C1-cAMP and the intracellular cAMP-elevating agent such as forskolin. In contrast, pretreatment of HL-60 cells with H89 or KT5720, which are known to inhibit cAMP-dependent protein kinase (PKA), abolished the protective effect of cAMP analogs and forskolin on SNP-induced apoptosis. Synergism between SNP and phorbol ester to induce apoptosis was also inhibited by prior treatment of HL-60 cells with DB-cAMP or forskolin. The effect of DB-cAMP in maintaining cell viability was not associated with the onset of G0/G1 cell cycle arrest. In addition, neither dimethyl sulfoxide nor retinoic acid (which produce granulocyte differentiation) could produce cAMP effect. Under the same conditions, DB-cAMP also inhibited NO- or NO plus phorbol ester-induced apoptosis in another transformed cell line, U-937 cells. Taken together, these findings suggest that exposure of HL-60 cells to cAMP analogs renders them more resistant to NO-induced DNA damage and further suggest the existence of specific down-modulatory mechanisms related to NO-induced apoptotic DNA fragmentation.
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PMID:Cyclic adenosine monophosphate inhibits nitric oxide-induced apoptosis in human leukemic HL-60 cells. 957 15

Two membrane-permeable and RNase-resistant antisense poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotides (poly-DNP-RNAs) have been synthesized as inhibitors of human breast cancer, with nucleotide sequences complementary to the genes of RIalpha subunit of protein kinase A (RIalpha/PKA) and erbB-2, respectively. Both compounds inhibit the proliferation of SK-Br-3 breast cancer cells in culture above the concentration of 10 microg/ml, but have no effect on nontumorigenic MCF-10A breast cells. These antisense inhibitors also block the cell colony formation in methylcellulose medium, whereas the control poly-DNP-RNA with either random or sense sequence has no effect. RT-PCR data show that the antisense inhibition decreases the concentration of the mRNA. TdT-mediated dUTP nick-end labeling (TUNEL) fluorescence assay indicates that the targeted antisense inhibition by poly-DNP-RNAs leads to apoptosis of SK-Br-3 cells but does not affect nontumorigenic MCF-10A cells. The control poly-DNP-RNAs with random or sense nucleotide sequence are completely inactive.
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PMID:Specific inhibition of breast cancer cells by antisense poly-DNP-oligoribonucleotides and targeted apoptosis. 1010 Jul 55

beta(1)-Adrenoceptor autoantibodies are present in about 30% of patients suffering from dilated cardiomyopathy. The apoptotic effects mediated by beta(1)-adrenoceptor antibodies remain to be studied. Monoclonal antibodies were raised against a synthetic peptide corresponding to the second extracellular loop of the human beta(1)-adrenoceptor in balb/C mouse, and were characterized by enzyme immunoassay. Purified immunoglobulin G from nonimmunized animals (controls) did not influence the rate of apoptosis. beta(1)-Adrenoceptor antibodies caused a dose-related increase in apoptotic cells: annexin test (dilution 1:2: 21+/-1.1% apoptotic cells vs. 4+/-0.4% apoptotic cells in controls; p<0.01); TdT-mediated dUTP nick end labeling (TUNEL) test (dilution 1:2: 26+/-2% apoptotic cells vs. 10+/-2% apoptotic cells in controls; p<0.01). The effect of the beta(1)-adrenoceptor antibodies was blocked by the antigenic peptide and by the antagonist metoprolol (10 micromol/l). The apoptotic effect induced by isoproterenol was attenuated by the beta(1)-adrenoceptor antibody. After pre-incubation of cardiomyocytes with the protein kinase A inhibitor Rp-Adenosine-3',5'-cyclic monophosphothioate triethylamine (RpcAMPS), beta(1)-adrenoceptor antibody was not capable of inducing an increase of the rate of apoptosis. beta(1)-Adrenoceptor antibodies induced apoptosis in adult rat cardiomyocytes via the protein kinase A cascade.
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PMID:Beta1-adrenoceptor antibodies induce apoptosis in adult isolated cardiomyocytes. 1267 35

Kainic acid induces excitotoxicity and nerve cell degeneration in vulnerable regions of rat brain, most markedly in hippocampus and amygdala. Part of the cell death following kainic acid is apoptotic as shown by caspase 3 activation and chromatin condensation. Here we have studied the regulation of pro- and anti-apoptotic proteins belonging to the Bcl-2 family in rat hippocampus and amygdala by kainic acid in relationship to ensuing neuronal death. The pro-apoptotic protein Bax was up-regulated in hippocampus 6 h after kainic acid administration. The increase in Bax was followed by the appearance of TdT-mediated dUTP nick end labelling-positive cells which were prominent at 24 h. Immunohistochemistry for active Bax revealed a punctuated labelling of neurons in the CA3 and hilar regions of hippocampus as well as in amygdala. Double staining for NeuN, a marker for nerve cells, and TdT-mediated dUTP nick end labelling showed that mainly neurons undergo degeneration after kainic acid treatment. In contrast to Bax, the pro-apoptotic BH3-only Bcl-2 proteins Bim and Harakiri/DP5 were down-regulated by kainic acid. This was also observed for the anti-apoptotic proteins Bcl-x and Bcl-w. Immunoreactive Bcl-2 was up-regulated in hippocampus after kainic acid together with an increase in the phosphorylation of serine-87 in Bcl-2, suggesting a post-transcriptional modification of the protein. This was confirmed using immunoprecipitation of total Bcl-2 from hippocampus and amygdala which revealed an increase in serine-87 phospho-Bcl-2 after kainic acid. Inhibition of the c-jun N-terminal protein kinase pathway reduced both serine-87 phosphorylation and cell death after kainic acid. This indicates an important role of Bcl-2 phosphorylation in controlling neuronal death after kainic acid. In contrast to the situation in trophic factor-deprived neurons, no up-regulation of Bim or Harakiri/DP5 proteins occurred after kainic acid, suggesting alternative pathways for regulation of cell death in excitotoxicity. The results indicate that not only the relative levels of Bcl-2 family proteins but also conformation changes and post-translational modifications contribute to neuronal death following kainic acid.
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PMID:Increase in Bcl-2 phosphorylation and reduced levels of BH3-only Bcl-2 family proteins in kainic acid-mediated neuronal death in the rat brain. 1295 12

CGRP is a well-known neuropeptide that has various protective effects on cardiovascular system. Our previous studies have shown that CGRP inhibits vascular smooth muscle cell (VSMC) proliferation in vitro. The present study aimed to explore the role of the CGRP in neointimal formation after balloon injury in the rat aortic wall and the underlying mechanism. Gene transfer of CGRP was performed with the use of intramuscular electroporation in a balloon-injured rat aorta model. Apoptosis in VSMCs was determined by electrophoresis assessment of DNA fragmentation and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay. Overexpression of the CGRP gene significantly inhibited the neointimal formation after balloon injury compared with the mock transfer, as assessed by the intima-to-media ratio 14 days after balloon injury (29.2 +/- 3.7% vs. 52.7 +/- 5.4%; n = 9-12, P < 0.05). In addition, CGRP gene expression increased the number of apoptotic cells in the neointima in vivo 14 days after balloon injury. Similarly, the addition of bioactive CGRP and the nitric oxide donor induced similar apoptosis in cultured VSMCs. The antagonist of the CGRP(1) receptor and inhibitors of cAMP-PKA and nitric oxide blocked CGRP-mediated apoptosis. Furthermore, CGRP gene transfer increased inducible nitric oxide synthase and p53 but decreased PCNA and Bcl-2 protein levels in balloon-injured rat aorta. Our data demonstrated that CGRP potently inhibited neointimal thickening in the rat aorta, at least in part through its distinct effects on apoptosis and proliferation of VSMCs both in vivo and in vitro. Therefore, delivery of the CGRP gene may have therapeutic implications in limiting vascular restenosis.
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PMID:Intramuscular gene transfer of CGRP inhibits neointimal hyperplasia after balloon injury in the rat abdominal aorta. 1537 Dec 65

To explore the molecular mechanism of c-jun antisense gene transfection in alleviating injury of cardiomyocytes treated with burn serum and hypoxia, burn serum was collected from Wistar rats inflicted with 30% third-degree burn of the total body surface area. The cardiomyocytes of neonatal Wistar rats were cultured and then treated with burn serum and hypoxia (a gas mixture containing 1% O2). The constructed c-jun antisense gene recombinant was transfected into the cardiomyocytes of neonatal Wistar rats. TdT-mediated d-utp nick end labeling (TUNEL) was adopted to examine cardiomyocyte apoptosis. Morphological changes of cardiomyocytes were observed under an optic-microscope and an electron-microscope. Expression of troponin T and beta-tubulin protein, c-jun protein, protein kinase Ca (PKCa), and c-jun N-terminal kinase (JNK) were assayed with Western blot in the transfected and non-transfected groups. The morphology of cardiomyocytes in the non-transfected group changed explicitly, but the change was not so obvious in the transfected cardiomyocytes. The expression of beta-tubulin and troponin increased significantly in the transfected group as compared with the non-transfected group. In the non-transfected group, numbers of apoptotic cardiomyocytes were significantly higher than in the transfected group. The c-jun protein, PKCa, and JNK were significantly expressed in the non-transfected group, and they reached a maximum at the 24th hour after cardiomyocytes were treated with burn serum and hypoxia. In the transfected group, however, expressions of c-jun protein, PKCa, and JNK decreased significantly compared with the non-transfected group. The c-jun antisense gene recombinant transfection alleviates injury to cardiomyocytes treated with burn serum and hypoxia, probably through low expression of PKCa and JNK.
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PMID:Molecular mechanism of c-jun antisense gene transfection in alleviating injury of cardiomyocytes treated with burn serum and hypoxia. 1557 45

The aim of our in-vitro experiments was to examine, whether leptin can directly control functions of avian ovarian cells and to outline potential intracellular mediators of its effects. Granulosa cells or fragments of ovarian follicular wall were cultured with leptin (0, 1, 10 or 100 ng/mL medium). The expression of peptides involved in apoptosis (TdT, bax, its binding protein, bcl-2, ASK-1 and p53), cell cycle-related peptides (PCNA and cyclin B1), release of hormones (progesterone, testosterone, estradiol, arginine-vasotocin), as well as the expression of protein kinases (PKA, MAPK/ERK1,2 and CDK/p34) in the ovarian cells were examined by using immunocytochemistry, TUNEL, SDS-PAGE-Western immunoblotting, EIA and RIA. It was found that leptin inhibited expression of all markers of cytoplasmic apoptosis (bax, ASK-1 and p53), stimulated expression of anti-apoptotic peptide bcl-2, but did not affect nuclear DNA fragmentation (TdT). Furthermore, leptin inhibited expression of PCNA (marker of S-phase of mitosis), but not of cyclin B1 (marker of G phase of cell cycle). Moreover, it promoted release of progesterone and estradiol, suppressed release of testosterone, but did not affect arginine-vasotocin. Finally, leptin inhibited expression of MAPK/ERK1,2 and CDK/p34 and stimulated expression of PKA. The present observations demonstrate that leptin can directly control basic chicken ovarian functions - inhibit cytoplasmic apoptosis and proliferation (S-phase, but not G-phases of mitosis), regulate secretory activity (release of steroids, but not nonapeptide hormone) and expression of MAPK, PKA and CDC2, which might be potential intracellular mediators of leptin action.
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PMID:Leptin directly controls proliferation, apoptosis and secretory activity of cultured chicken ovarian cells. 1760 68

Treatment of exponentially growing MCF-7 human breast carcinoma cells with tamoxifen (TAM) inhibits cell growth in a dose-dependent manner. However, the molecular basis for the drug's activity and its relationship to the cell cycle have not yet been clearly established. In this study, we analyzed cell cycle-related proteins used for immunoblotting and flow cytometry in TAM-treated MCF-7 cells. In addition, the ratio of apoptosis in the cell was analyzed using labeling of DNA strand breaks (TdT assay). In flow-cytometric DNA distribution analysis, the S-phase fraction showed a marked decrease and a concomitant increase in G1- and G2-phase cells accompanying the inhibitory effect of TAM; these changes were time- and dose-dependent. Immunoblotting revealed that the levels of p53 and p21(WAF1/CIP1) in TAM-treated cells increased in a time- and dose-dependent manner, whereas those of p27(KIP1) and p16 slightly increased or remained unchanged. Furthermore, cyclin D3 and B showed sharp decreases, in contrast with p53 and p21(WAF1/CIP1) DNA-apoptosis dual analysis using flow cytometry revealed that the TAM-treated samples contained apoptotic cells, the majority of which were arrested in G1 or G2 and showed suppression of Bcl-2 protein. These results suggest that the tumorigenic effect of TAM on MCF-7 cells arises through antitumor effects that are due to the expression of cyclin-dependent kinase inhibitors, especially p21(WAF1/CIP1) and these are regulated by the decrease of wild-type p53. The proposed mechanism is similar to that underlying the cytotoxic effects of other agents and ionizing irradiation that cause DNA damage.
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PMID:G1 arrest and expression of cyclin-dependent kinase inhibitors in tamoxifen-treated MCF-7 human breast cancer cells. 1839 72

We had earlier demonstrated that chronic exposure (30 days) to micro-molar concentration (0.50 microM) of arsenic induced head kidney macrophage (HKM) death in Clarias batrachus. The purpose of the present study is to characterize the nature of HKM death induced by arsenic and elucidate the signal transduction pathways involved in the process. Arsenic-induced HKM death was apoptotic in nature as evident from DNA gel, Annexin V-propidium iodide, Hoechst 33342 staining and TdT-mediated dUTP nick end labeling (TUNEL) assays. Inhibitor studies and immunoblot analyses further demonstrated that arsenic-induced HKM apoptosis involved activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, a well-characterized caspase-3 substrate. Preincubation with antioxidants N-acetyl-cysteine or dimethyl sulfoxide significantly lowered reactive oxygen species (ROS) levels in arsenic-treated HKM and prevented caspase activation, malondialdehyde formation and HKM apoptosis. Arsenic induced membrane translocation of the NADPH oxidase subunit p47(phox). Preincubation with apocynin and diphenyleneiodonium chloride, both selective inhibitors of NADPH oxidases, prevented p47(phox) translocation, ROS production and HKM death. Exposure of HKM to arsenic induced the activation of mitogen-activated protein kinase family (MAPK) proteins including c-Jun NH(2)-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (p38). Preincubation of HKM with p38 inhibitor SB203580 and JNK inhibitor SP600125 protected the HKM against arsenic-induced apoptosis. We conclude that exposure to micro-molar concentration of arsenic induces ROS generation through the activation of NADPH oxidases, which in turn causes caspase-3 mediated HKM apoptosis. In addition, the study also indicates a role of p38-JNK pathway in arsenic-induced HKM apoptosis in C. batrachus.
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PMID:Low concentration of arsenic could induce caspase-3 mediated head kidney macrophage apoptosis with JNK-p38 activation in Clarias batrachus. 1978 97


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