Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 35
Filter
1.
Biochem Med (Zagreb) ; 21(3): 291-6, 2011.
Article in English | MEDLINE | ID: mdl-22420243

ABSTRACT

INTRODUCTION: We hypothesized that patients with stable angina have increased plasma levels of mRNA from genes responsible for atherosclerotic plaque development and destabilisation, i.e. from death-associated protein kinase (DAPK1) and monocyte chemotactic protein-1 (CCL2). MATERIALS AND METHODS: Nucleic acids were isolated from plasma of patients with stabile angina and healthy subjects as controls. mRNAs were transcribed to cDNAs, quantified by real-time PCR and standardized to the amount of a reference gene. Reagents for PCR quantification are declared to be mRNA specific, but in our test conditions DNA was found to interfere in both assays. RESULTSs: Patients had 5.1-times higher plasma level of DAPK1 nucleic acids (mRNA and DNA) than controls (P < 0.001) and the highest levels were associated with the presence of diabetes. However, plasma levels of CCL2 tended to be lower than in controls, and in statin-treated patients the decrement reached significance (-66.3%; P = 0.041). CONCLUSION: The estimated levels are explicable in terms of current knowledge. Further studies with specific assays for mRNA PCR quantification are reasonable to access whether this approach offers non-invasive in vive assessment and monitoring of gene expression profile in atherosclerotic vascular beds.


Subject(s)
Angina, Stable/blood , Apoptosis Regulatory Proteins/genetics , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Chemokine CCL2/genetics , Nucleic Acids/blood , Adult , Aged , Aged, 80 and over , Angina, Stable/genetics , Apoptosis Regulatory Proteins/blood , Calcium-Calmodulin-Dependent Protein Kinases/blood , Case-Control Studies , Chemokine CCL2/blood , Death-Associated Protein Kinases , Female , Humans , Male , Middle Aged , Nucleic Acids/analysis , RNA, Messenger/analysis , RNA, Messenger/blood , Up-Regulation
2.
J Surg Oncol ; 100(5): 414-7, 2009 Oct 01.
Article in English | MEDLINE | ID: mdl-19653236

ABSTRACT

BACKGROUND AND OBJECTIVES: To further improve the screening, diagnosis and therapy of patients with non-small cell lung cancer (NSCLC) additional diagnostic tools are desperately warranted. Aim of this study was to investigate the potential of the DNA methylation of DAPK, MGMT, and GSTPI in serum of patients with NSCLC as a prognostic molecular marker in this disease. METHODS: Seventy-six patients with NSCLC were included in this study. The analysis of DNA methylation in serum of patients was performed on pre-operative samples. Following DNA isolation and bisulfite-treatment, DNA methylation was analyzed by quantitative-methylation-specific real-time PCR with beta-actin as the internal reference gene. RESULTS: DNA methylation was detectable with following frequencies: DAPK 68.4%, MGMT 7.9%, GSTPI 0%. There were no associations between DNA methylation status and histology, tumor stage, grading or gender detectable. With a mean follow-up of 19.7 months the median survival was 26.3 months. There were no associations between the status of DNA methylation in patient's serum and prognosis detectable. CONCLUSION: The analysis of DNA methylation in serum of patients with NSCLC by quantitative-methylation-specific real-time PCR is technically feasible. Although our results suggest quantification of DNA methylation in serum not of prognostic significance in this disease, further studies are warranted to determine the future potential of this molecular approach.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Carcinoma, Non-Small-Cell Lung/mortality , DNA Methylation , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Glutathione S-Transferase pi/genetics , Lung Neoplasms/mortality , Tumor Suppressor Proteins/genetics , Aged , Apoptosis Regulatory Proteins/blood , Biomarkers, Tumor , Calcium-Calmodulin-Dependent Protein Kinases/blood , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , DNA Modification Methylases/blood , DNA Repair Enzymes/blood , Death-Associated Protein Kinases , Female , Glutathione S-Transferase pi/blood , Humans , Lung Neoplasms/blood , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Middle Aged , Polymerase Chain Reaction , Prognosis , Tumor Suppressor Proteins/blood
3.
J Cancer Res Clin Oncol ; 135(9): 1231-7, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19259700

ABSTRACT

BACKGROUND: Death-associated protein kinase (DAPK) and adenomatous polyposis coli gene (APC) have been recently shown to be associated with outcome in patients with esophageal carcinoma, especially adenocarcinoma. We wanted to validate the correlation of these two markers with outcome by detecting methylated DNA sequences in peripheral blood. METHODS: Circulating cell-free DNA extracted from blood plasma of 59 patients with esophageal cancer was analyzed before and after surgical resection by quantitative real-time methylation-specific RT-PCR (TaqMan) assays. RESULTS: Thirty-six of 59 patients (61.0%) with esophageal cancer had detectable levels of methylated DAPK or APC promoter DNA and preoperative detection was significantly associated with an unfavorable prognosis as revealed by multivariate Cox proportional hazards regression analysis [Exp(b) = 4.578; P = 0.01]. The combination of both markers significantly increased sensitivity and specificity for discriminating between short (<2.5 years) and long survivors (P = 0.04, ROC curve analysis). Postoperative APC detection was significantly different if residual tumor was apparent (P = 0.03). CONCLUSIONS: Preoperative measurement of methylated DAPK and APC promoter DNA in peripheral blood may contribute to better estimate postoperative survival chances of patients with esophageal carcinoma, especially adenocarcinoma. The postoperative detection of methylated APC in peripheral blood might provide crucial information on apparent residual tumor and might be used as a "molecular" R0-Marker in addition to the pathologic examination.


Subject(s)
Adenomatous Polyposis Coli/genetics , Apoptosis Regulatory Proteins/genetics , Calcium-Calmodulin-Dependent Protein Kinases/genetics , DNA Methylation/genetics , DNA, Neoplasm/blood , Esophageal Neoplasms/genetics , Neoplasm, Residual/genetics , Promoter Regions, Genetic/genetics , Apoptosis Regulatory Proteins/blood , Apoptosis Regulatory Proteins/metabolism , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Calcium-Calmodulin-Dependent Protein Kinases/blood , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA, Neoplasm/genetics , Death-Associated Protein Kinases , Esophageal Neoplasms/blood , Esophageal Neoplasms/surgery , Humans , Multivariate Analysis , Neoplasm, Residual/blood , Neoplasm, Residual/surgery , Prognosis , ROC Curve , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Survival Rate , Treatment Outcome
4.
Neoplasma ; 54(2): 123-6, 2007.
Article in English | MEDLINE | ID: mdl-17319784

ABSTRACT

The DAPK1 gene works as a regulator of apoptosis and is frequently inactivated in cancer by aberrant promoter hypermethylation. Loss of DAPK1 expression is associated with a selective advantage for tumor cells to resist apoptotic stimuli, allowing them to separate from the original tumor; from this point of view, DAPK1 could be considered a tumor metastases inhibitor gene. To verify the participation of DAPK1 silencing in cerebral invasion, we analyzed its promoter methylation status in a series of 28 samples from cerebral metastases using MSP and sequencing of the MSP-product. We have found hypermethylation in 53.6% (15/28) metastatic tumor samples as well as in 27.8% (5/18) of its peripheral blood samples. Our data suggest an important role of DAPK1 for silencing through promoter CpG island hypermethylation in the development of brain metastases from solid tumors. The detection of aberrant hypermethylation on DAPK1 promoter from peripheral blood samples has potential clinical implications as a tumor prognosis marker.


Subject(s)
Apoptosis Regulatory Proteins/blood , Apoptosis Regulatory Proteins/genetics , Brain Neoplasms/genetics , Calcium-Calmodulin-Dependent Protein Kinases/blood , Calcium-Calmodulin-Dependent Protein Kinases/genetics , DNA Methylation , Promoter Regions, Genetic , Antimetabolites, Antineoplastic/pharmacology , Azacitidine/pharmacology , Biomarkers, Tumor/metabolism , Brain Neoplasms/blood , Brain Neoplasms/secondary , CpG Islands , DNA, Neoplasm/genetics , Death-Associated Protein Kinases , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans
5.
Oncol Rep ; 10(4): 1031-3, 2003.
Article in English | MEDLINE | ID: mdl-12792765

ABSTRACT

Death-associated protein (DAP) kinase is a gene that participates in apoptosis induced by interferon gamma. It appears to play a role in lung cancer metastasis in animal models, suggesting that DAP-kinase may function as a metastasis suppressor by inducing apoptosis. Expression silencing through CpG island methylation of DAP-kinase has been frequently found in connection with adverse survival, as cells lacking DAP-kinase appear to be more invasive and more metastatic in lung cancer. The purpose of this study was to analyze the promoter methylation status of DAP-kinase gene in brain metastases of solid tumors. Methylation-specific PCR was performed on ten brain metastasis samples derived from malignant melanoma (three cases), lung cancer (two), breast carcinoma (two), ovarian carcinoma (two) and colon carcinoma (one case), and in corresponding peripheral blood DNA samples. Two normal brain tissue samples were also analyzed, no promoter hypermethylation was observed in either case. DAP-kinase hypermethylated alleles were identified in nine metastases (90%), and in peripheral blood lymphocytes DNA from four cases. Our data suggest that silencing of DAP-kinase through promoter hypermethylation is a common event in the multistep process of tumor metastasis, including brain involvement.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/secondary , Calcium-Calmodulin-Dependent Protein Kinases/genetics , DNA Methylation , Promoter Regions, Genetic , Adult , Apoptosis Regulatory Proteins , Brain Neoplasms/enzymology , Breast Neoplasms/blood , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Calcium-Calmodulin-Dependent Protein Kinases/blood , Colonic Neoplasms/blood , Colonic Neoplasms/enzymology , Colonic Neoplasms/genetics , DNA, Neoplasm/genetics , Death-Associated Protein Kinases , Female , Humans , Lung Neoplasms/blood , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Male , Melanoma/blood , Melanoma/enzymology , Melanoma/genetics , Middle Aged , Ovarian Neoplasms/blood , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/genetics , Polymerase Chain Reaction , Sulfites
6.
Cancer Lett ; 194(1): 99-105, 2003 May 08.
Article in English | MEDLINE | ID: mdl-12706863

ABSTRACT

Death-associated protein (DAP)-kinase is frequently inactivated by promoter methylation in human cancers. To understand the involvement of the DAP-kinase gene in colorectal cancer (CRC), we investigated the methylation of the DAP-kinasegene in primary CRC to define the frequency of this epigenetic aberration and the clinicopathological significance. For this reason, methylation-specific polymerase chain reaction (MSP) was used to detect DAP-kinase gene methylation in DNA from 122 cases of CRC and 18 paired serum samples. Methylation of the DAP-kinase gene was found in 67 of 122 (55%) cases of primary CRC. Study of the serum DNA from 14 patients exhibiting methylated DAP-kinase gene revealed aberrant methylation in three patients (21%). False positives were not obtained in any of the patients who did not exhibit methylation. No association was found between the promoter methylation of the DAP-kinase gene in primary CRC and gender, localization, tumor differentiation, invasion depth, regional lymph node involvement, or tumor stage. In conclusion, methylation of the DAP-kinase gene is common in CRC. The detection of the methylation of the DAP-kinase gene has a potential clinical application as a diagnostic tumor marker for CRC.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , DNA Methylation , Promoter Regions, Genetic , Apoptosis Regulatory Proteins , Blotting, Western , Calcium-Calmodulin-Dependent Protein Kinases/blood , Colorectal Neoplasms/blood , DNA/metabolism , Death-Associated Protein Kinases , Female , Humans , Male , Polymerase Chain Reaction , Sulfites/pharmacology
7.
Cancer Lett ; 193(2): 207-16, 2003 Apr 25.
Article in English | MEDLINE | ID: mdl-12706879

ABSTRACT

Gene methylation and K-ras mutations were examined in tumor and paired serum DNA of 50 resected non-small-cell lung cancer patients. RASSF1A, death associated protein kinase and target of methylation-induced silencing were methylated in 17/50 (34%), 23/50 (45%) and 18/50 (35%) tumors, respectively, and in 17/50 (34%), 20/50 (40%) and 17/50 (34%) sera, respectively. Methylation in tumor and serum were closely correlated (P=0.001), but no correlation was found with survival. Twelve K-ras mutations (cysteine) were found in serum and nine mutations were found in tumor (five cysteine, one alanine, one aspartic, one arginine, and one valine). K-ras mutations in serum correlated significantly with survival (P=0.01).


Subject(s)
Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/genetics , DNA Methylation , Genes, ras/genetics , Lung Neoplasms/blood , Lung Neoplasms/genetics , Mutation , Aged , Aged, 80 and over , Apoptosis Regulatory Proteins , Calcium-Calmodulin-Dependent Protein Kinases/blood , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Codon , DNA/metabolism , DNA Mutational Analysis , Death-Associated Protein Kinases , Female , Humans , Male , Middle Aged , Polymerase Chain Reaction , Prognosis , Time Factors
8.
Int J Cardiol ; 83(2): 151-8, 2002 May.
Article in English | MEDLINE | ID: mdl-12007687

ABSTRACT

BACKGROUND: Plasma level of B-type natriuretic peptide is a sensitive marker of left ventricular dysfunction and the level is markedly elevated in patients with hypertrophic obstructive cardiomyopathy. Percutaneous transluminal septal myocardial ablation, a catheter-based treatment of hypertrophic obstructive cardiomyopathy, has been widely used as a new therapeutic option for the disease. This study was designed to evaluate clinical implications of natriuretic peptides after the new treatment. METHODS: Seven consecutive patients with hypertrophic obstructive cardiomyopathy unresponsive to usual medical treatments (age: 57.9+/-22.0 years) were enrolled in the study. Serial changes in atrial and B-type natriuretic peptide in plasma were examined after percutaneous transluminal septal myocardial ablation. RESULTS: Atrial and B-type natriuretic peptides levels (pg/ml, mean+/-S.D.) at baseline were higher in hypertrophic obstructive cardiomyopathy than in control (80.0+/-43.0 vs. 12.8+/-5.2, P<0.0001; 858.0+/-458.4 vs. 12.4+/-7.0, P<0.0001; respectively). Left ventricular outflow-tract pressure gradient (mmHg) immediately decreased from 115.3+/-23.3 to 30.6+/-12.4 (P<0.0001) after the treatment and concomitantly B-type natriuretic peptide level decreased (858.0+/-458.4 to 264.1+/-137.7, P=0.0084). The level re-increased and peaked at the 2nd day (634.4+/-429.6) and gradually decreased again until 4 weeks. Reduction rate of left ventricular outflow-tract pressure gradient between before and 4 weeks after percutaneous transluminal septal myocardial ablation positively correlated with that of B-type natriuretic peptide (r(2)=0.817, P=0.0053). Changes in atrial natriuretic peptide were not significant in contrast to those of B-type natriuretic peptide. CONCLUSIONS: Plasma B-type natriuretic peptide level could be useful to predict the effects of percutaneous transluminal septal myocardial ablation in patients with hypertrophic obstructive cardiomyopathy.


Subject(s)
Atrial Natriuretic Factor/blood , Calcium-Calmodulin-Dependent Protein Kinases/blood , Cardiomyopathy, Hypertrophic/blood , Cardiomyopathy, Hypertrophic/surgery , Catheter Ablation/methods , Creatine Kinase/blood , Isoenzymes/blood , Adolescent , Aged , Analysis of Variance , Biomarkers/analysis , Creatine Kinase, MB Form , Female , Humans , Linear Models , Male , Middle Aged , Postoperative Period , Probability , Prognosis , Prospective Studies , Sensitivity and Specificity
9.
Clin Cancer Res ; 8(2): 433-7, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11839660

ABSTRACT

PURPOSE: Death-associated protein (DAP)-kinase gene is frequently inactivated by promoter hypermethylation in cancer. The aim of this study was to evaluate the promoter methylation status of the DAP-kinase gene in nasopharyngeal carcinoma (NPC). EXPERIMENTAL DESIGN: The methylation status was evaluated by methylation-specific PCR (MSP). Thirty-two NPC biopsy specimens, plasma and buffy coat of 12 patients, 5 NPC cell lines, 3 normal nasopharyngeal biopsy tissues, and 2 normal nasopharyngeal epithelial primary cultures were included in this study. RESULTS: There was no promoter hypermethylation in all 3 normal nasopharyngeal tissues and 2 normal nasopharyngeal primary cultures. Hypermethylation was found in 24 (75%) NPC primary tumor biopsies and 4 (80%) NPC cell lines. Of the 24 patients with hypermethylation of DAP-kinase promoter in the primary tumors, 12 patients had their plasma and buffy coat DNA available for MSP study. Hypermethylated DAP-kinase promoter was detectable in 5 patients in the plasma but not in the buffy coat, 2 patients in the buffy coat but not in the plasma, and 1 patient in both plasma and buffy coat. Four patients had no detectable hypermethylated DAP-kinase promoter in both plasma and buffy coat. Hypermethylation of DAP-kinase promoter was found in both early- and late-stage NPC. CONCLUSIONS: Our results show that hypermethylation of the DAP-kinase promoter is a common early event in NPC. The high frequency of identification of hypermethylated DAP-kinase promoter in plasma and buffy coat of NPC patients illustrates its potential clinical application as tumor marker for the diagnosis and monitoring of treatment result.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/blood , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Carcinoma/enzymology , DNA Methylation , Nasopharyngeal Neoplasms/enzymology , Promoter Regions, Genetic , Apoptosis Regulatory Proteins , Biopsy , Calcium-Calmodulin-Dependent Protein Kinases/biosynthesis , Carcinoma/blood , Death-Associated Protein Kinases , Female , Humans , Male , Nasopharyngeal Neoplasms/blood , Sulfites/metabolism
10.
Blood ; 96(9): 3215-23, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11050006

ABSTRACT

Human granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction-based strategy was used to identify novel granulocyte-specific kinases. A novel protein kinase complementary DNA with an open reading frame of 357 amino acids was identified with homology to calcium-calmodulin-dependent kinase I (CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of CKLiK messenger RNA (mRNA) expression in hematopoietic cells demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during neutrophilic differentiation of CD34(+) stem cells. CKLiK kinase activity was dependent on Ca(++) and calmodulin as analyzed by in vitro phosphorylation of cyclic adenosine monophosphate responsive element modulator (CREM). Furthermore, CKLiK- transfected cells treated with ionomycin demonstrated an induction of CRE- binding protein (CREB) transcriptional activity compared to control cells. Additionally, CaMK-kinasealpha enhanced CKLiK activity. In vivo activation of CKLiK was shown by addition of interleukin (IL)-8 to a myeloid cell line stably expressing CKLiK. Furthermore inducible activation of CKLiK was sufficient to induce extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase activity. These data identify a novel Ca(++)/calmodulin-dependent PMN- specific kinase that may play a role in Ca(++)-mediated regulation of human granulocyte functions.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/blood , Granulocytes/enzymology , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Calcium-Calmodulin-Dependent Protein Kinase Type 1 , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Chloramphenicol O-Acetyltransferase/genetics , Chlorocebus aethiops , Cloning, Molecular , Hematopoietic Stem Cells/enzymology , Humans , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Transfection
11.
Cancer Res ; 59(16): 3980-4, 1999 Aug 15.
Article in English | MEDLINE | ID: mdl-10463595

ABSTRACT

Components of cell signaling pathways provide important targets for anticancer drugs. Protein kinase C (PKC) is a serine/threonine-specific kinase that regulates cell growth and differentiation. It is also implicated in tumor promotion. The staurosporine analogue CGP41251 is a PKC inhibitor, and it is currently in a Phase I clinical trial for treatment of advanced cancer. However, it is difficult to define its biological activity. We have used two approaches to measure the in vivo biological response to CGP41251: (a) sequential whole blood samples were taken from 27 patients before and during treatment and incubated with mitogen (PHA), and cytokine [tumor necrosis factor (TNF)-alpha and interleukin (IL)-6] release was measured ex vivo; and (b) peripheral blood lymphocytes were isolated from seven of these patients, and the levels of extracellular signal-regulated kinase 2 were measured by Western blotting. Response to PHA was significantly lowered during treatment (P < 0.001 for TNF-alpha production; P < 0.03 for IL-6). This was most evident at 7 and 28 days after the start of treatment in patients receiving higher doses (150-300 mg/day; P = 0.002 and P = 0.02, respectively, for TNF-alpha and P = 0.001 and P = 0.003, respectively, for IL-6 release). Whole blood cytokine production returned to pretreatment levels after drug administration ceased. The levels of extracellular signal-regulated kinase 2 were reduced by 50-97% during treatment in all seven patients tested. These results show for the first time that a PKC inhibitor can block in vivo signaling pathways in cancer patients. The assays we describe complement toxicity studies in selecting relevant doses for Phase II trial of novel agents, particularly when biological activity occurs at doses below those that cause obvious side effects.


Subject(s)
Antineoplastic Agents/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/blood , Cytokines/blood , Enzyme Inhibitors/pharmacology , Neoplasms/blood , Neoplasms/drug therapy , Staurosporine/analogs & derivatives , Adult , Aged , Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Humans , Lymphocytes/metabolism , Middle Aged , Mitogen-Activated Protein Kinase 1 , Protein Kinase C/antagonists & inhibitors , Staurosporine/pharmacology , Staurosporine/therapeutic use
12.
J Biol Chem ; 274(31): 21847-52, 1999 Jul 30.
Article in English | MEDLINE | ID: mdl-10419502

ABSTRACT

We have measured the activation of the small GTPase Ral in human neutrophils after stimulation with fMet-Leu-Phe (fMLP), platelet activating factor (PAF), and granulocyte macrophage-colony stimulating factor and compared it with the activation of two other small GTPases, Ras and Rap1. We found that fMLP and PAF, but not granulocyte macrophage-colony stimulating factor, induce Ral activation. All three stimuli induce the activation of both Ras and Rap1. Utilizing specific inhibitors we demonstrate that fMLP-induced Ral activation is mediated by pertussis toxin-sensitive G-proteins and partially by Src-like kinases, whereas fMLP-induced Ras activation is independent of Src-like kinases. PAF-induced Ral activation is mediated by pertussis toxin-insensitive proteins, Src-like kinases and phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase is not involved in PAF-induced Ras activation. The calcium ionophore ionomycin activates Ral, but calcium depletion partially inhibits fMLP- and PAF-induced Ral activation, whereas Ras activation was not affected. In addition, 12-O-tetradecanoylphorbol-13-acetate-induced activation of Ral is completely abolished by inhibitors of protein kinase C, whereas 12-O-tetradecanoylphorbol-13-acetate-induced Ras activation is largely insensitive. We conclude that in neutrophils Ral activation is mediated by multiple pathways, and that fMLP and PAF induce Ral activation differently.


Subject(s)
GTP-Binding Proteins/blood , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/physiology , Platelet Activating Factor/pharmacology , Signal Transduction/physiology , Calcium/blood , Calcium-Calmodulin-Dependent Protein Kinases/blood , Enzyme Activation , GTP Phosphohydrolases/blood , GTPase-Activating Proteins , Humans , In Vitro Techniques , Kinetics , Mitogen-Activated Protein Kinase 1 , Neutrophils/drug effects , Phosphorylation , Protein Kinase C/blood , Proteins/metabolism , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , ral GTP-Binding Proteins , rap GTP-Binding Proteins , ras GTPase-Activating Proteins
13.
J Biol Chem ; 274(31): 22072-80, 1999 Jul 30.
Article in English | MEDLINE | ID: mdl-10419535

ABSTRACT

Neutrophil-mediated organ damage is a common feature of many disease states. We previously demonstrated that resuscitation with hypertonic salt solutions prevented the endotoxin-induced leukosequestration and consequent lung injury, and this effect was partially attributed to an altered surface expression of adhesion molecules, CD11b and L-selectin. In this study we investigated the mechanisms whereby osmotic stress evokes L-selectin shedding. The metalloprotease inhibitor RO 31-9790 prevented the osmotic down-regulation of L-selectin, indicating that this process was catalyzed by the same "sheddase" responsible for L-selectin cleavage induced by diverse inflammatory stimuli. The trigger for hypertonic shedding was cell shrinkage and not increased osmolarity, ionic strength, or intracellular pH. Volume reduction caused robust tyrosine phosphorylation and its inhibition by genistein and erbstatin abrogated shedding. Shrinkage stimulated tyrosine kinases Hck, Syk, and Pyk2, but prevention of their activation by the Src-family inhibitor PP1 failed to affect the L-selectin response. Hypertonicity elicited the Src family-independent activation of p38, and the inhibition of this kinase by SB203580 strongly reduced shedding. p38 was also essential for the N-formyl-methionyl-leucyl-phenylalanine- and lipopolysaccharide-induced shedding but not the phorbol ester-induced shedding. Thus, cell volume regulates L-selectin surface expression in a p38-mediated, metalloprotease-dependent manner. Moreover, p38 has a central role in shedding induced by many inflammatory mediators.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/blood , Hydroxamic Acids , L-Selectin/blood , Mitogen-Activated Protein Kinases , Neutrophils/cytology , Neutrophils/physiology , Cell Size/drug effects , Cytosol/physiology , Enzyme Inhibitors/pharmacology , Genistein/pharmacology , Humans , Hydrogen-Ion Concentration , Hypertonic Solutions , Imidazoles/pharmacology , In Vitro Techniques , L-Selectin/genetics , Macrophage-1 Antigen/blood , Metalloendopeptidases/antagonists & inhibitors , Neutrophils/drug effects , Phosphotyrosine/blood , Protease Inhibitors/pharmacology , Pyridines/pharmacology , p38 Mitogen-Activated Protein Kinases
14.
Biochem Biophys Res Commun ; 261(1): 58-63, 1999 Jul 22.
Article in English | MEDLINE | ID: mdl-10405323

ABSTRACT

Thrombopoietin (TPO) plays a crucial role in megakaryocyte differentiation and platelet production. c-Mpl, a receptor for TPO, is also expressed in terminally differentiated platelets. We investigated the effects of TPO on activation of p38 mitogen-activated protein kinase in human platelets. Thrombin, a thrombin receptor agonist peptide, a thromboxane A(2) analogue, collagen, crosslinking the glycoprotein VI, ADP, and epinephrine, but not phorbol 12, 13-dibutyrate activated p38. TPO did not activate p38 by itself, whereas TPO pretreatment potentiated the agonist-induced activation of p38. TPO did not promote phosphorylation of Hsp27 and cytosolic phospholipase A(2) by itself, but enhanced thrombin-induced phosphorylation of them. The specific p38 inhibitor SB203580 strongly inhibited such phosphorylation. Thus, TPO possesses the priming effect on p38 activation in human platelets and could affect platelet functions through the p38 pathway.


Subject(s)
Blood Platelets/enzymology , Calcium-Calmodulin-Dependent Protein Kinases/blood , Heat-Shock Proteins , Mitogen-Activated Protein Kinases , Receptors, Thrombin/agonists , Thrombopoietin/pharmacology , Blood Platelets/drug effects , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Drug Synergism , Enzyme Activation/drug effects , HSP27 Heat-Shock Proteins , Humans , Imidazoles/pharmacology , Molecular Chaperones , Neoplasm Proteins/blood , Phospholipases A/blood , Phosphorylation/drug effects , Phosphotyrosine/blood , Pyridines/pharmacology , Receptors, Thrombin/physiology , Thrombin/agonists , Thrombin/antagonists & inhibitors , Thrombin/pharmacology , p38 Mitogen-Activated Protein Kinases
15.
Free Radic Biol Med ; 26(7-8): 1010-8, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10232846

ABSTRACT

We have investigated the inhibitory effect of 2-hydroxymethyl-1-naphthol diacetate (TAC) on the respiratory burst of rat neutrophils and the underlying mechanism of action was also assessed in this study. TAC caused concentration-related inhibition of the formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2*-) generation (IC50 10.2+/-2.3 and 14.1+/-2.4 microM, respectively) and O2 consumption (IC50 9.6+/-2.9 and 13.3+/-2.7 microM, respectively) of neutrophils. TAC did not scavenge the generated O2*- during dihydroxyfumaric acid autoxidation. TAC inhibited both the transient elevation of [Ca2+]i in the presence or absence of [Ca2+]o (IC50 75.9+/-8.9 and 84.7+/-7.9 microM, respectively) and the generation of inositol trisphosphate (IP3) (IC50 72.0+/-9.7 microM) in response to fMLP. Cytosolic phospholipase C (PLC) activity was also reduced by TAC at a same range of concentrations. The PMA-induced PKC-beta associated to membrane was attenuated by TAC (about 80% inhibition at 30 microM). Upon exposure to fMLP, the cellular cyclic AMP level was decreased in neutrophils pretreated with TAC. TAC attenuated fMLP-induced phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 (IC50 17.4+/-1.7 microM), but not p38. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP was inhibited by TAC in a concentration-dependent manner (IC50 25.4+/-2.4 and 25.9+/-1.4 microM, respectively). TAC had no effect on the O2*- generation of PMA-stimulated and arachidonic acid (AA)-stimulated NADPH oxidase preparations. However, TAC caused concentration-related decrease of the membrane associated p47phoX in PMA-stimulated neutrophils (about 80% inhibition at 30 microM). We conclude that inhibition by TAC of the neutrophil respiratory burst is probably attributable to the blockade of the p42/44 MAPK and phospholipase D (PLD) pathways, the membrane translocation of PKC, and to the failure in assembly of a functional NADPH oxidase complex. Blockade of the PLC pathway by TAC probably plays a minor role.


Subject(s)
Naphthols/pharmacology , Neutrophils/physiology , Respiratory Burst/drug effects , Superoxides/blood , Animals , Calcium/blood , Calcium-Calmodulin-Dependent Protein Kinases/blood , Cell Membrane/enzymology , Cyclic AMP/blood , Cytochalasin B/analogs & derivatives , Cytochalasin B/pharmacology , In Vitro Techniques , Kinetics , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , NADPH Dehydrogenase/blood , NADPH Oxidases/blood , Neutrophils/drug effects , Oxygen Consumption/drug effects , Phospholipase D/blood , Phosphoproteins/blood , Protein Kinase C/blood , Rats , Tetradecanoylphorbol Acetate/pharmacology
16.
Ann Cardiol Angeiol (Paris) ; 48(4): 253-7, 1999 Apr.
Article in French | MEDLINE | ID: mdl-12555365

ABSTRACT

Plasma myosin heavy chain assay, which can be easily performed during the acute phase of myocardial infarction, is a recent method allowing quantitative assessment of the extent of infarction. However, to our knowledge, its prognostic value has not been studied in contrast with serum myosin light chain assay. We monitored the state of health of 40 patients (including 37 men with a mean age of 56 years) for two years after a first myocardial infarction, thrombolized during the acute phase. Their survival (mortality) and the development of "cardiac events" (MI, angina, sudden death, etc.) were evaluated at 2 years. The results observed at 2 years were correlated with the initial plasma myosin assay results and other direct and indirect methods of assessment of the extent of infarction, performed during the acute phase of myocardial infarction (cardiac enzymes, contrast angiography). The main result of this study is the demonstration that an unusual plasma myosin release kinetic (complex appearance) is predictive for the medium-term development of heart failure (p = 0.04) and/or destabilization of coronary insufficiency (p = 0.02). These results need to be emphasized, as with only 5 serum myosin assays performed over a 10-day period, it seems possible to identify a group of patients at high risk of medium-term complications, who possess a complex release kinetic during the acute phase of myocardial infarction and a value for area under the curve greater than 10.470 microliters U/L (cut-off value, p = 0.043).


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/blood , Myocardial Infarction/blood , Myocardial Infarction/drug therapy , Thrombolytic Therapy , Acute Disease , Coronary Angiography , Creatine Kinase/blood , Female , Heart Failure/etiology , Humans , Male , Middle Aged , Multivariate Analysis , Myocardial Infarction/complications , Myocardial Infarction/diagnosis , Myocardial Infarction/mortality , Predictive Value of Tests , Prognosis , Proportional Hazards Models , Protozoan Proteins , Risk Factors , Survival Analysis , Time Factors , Treatment Outcome
17.
Proc Natl Acad Sci U S A ; 95(24): 14540-5, 1998 Nov 24.
Article in English | MEDLINE | ID: mdl-9826736

ABSTRACT

The anti-inflammatory effects of high-dose salicylates are well recognized, incompletely understood and unlikely due entirely to cyclooxygenase (COX) inhibition. We have previously reported a role for activation of the kinase Erk in CD11b/CD18 integrin-dependent adhesiveness of human neutrophils, a critical step in inflammation. We now report the effects of salicylates on neutrophil Erk and adhesion. Exposure of neutrophils to aspirin or sodium salicylate (poor COX inhibitor) inhibited Erk activity and adhesiveness of formylmethionyl-leucyl-phenylalanine- and arachidonic acid-stimulated neutrophils, consistent with anti-inflammation but not COX inhibition (IC50s = 1-8 mM). In contrast, indomethacin blocked neither Erk nor adhesion. Inhibition of Mek (proximal activator of Erk) also blocked stimulation of Erk and adhesion by formylmethionyl-leucyl-phenylalanineand arachidonic acid. Salicylate inhibition of Erk was independent of protein kinase A activation and generation of extracellular adenosine. These data are consistent with a role for Erk in stimulated neutrophil adhesion, and suggest that anti-inflammatory effects of salicylates may be mediated via inhibition of Erk signaling required for integrin-mediated responses.


Subject(s)
Aspirin/analogs & derivatives , Aspirin/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/blood , Mitogen-Activated Protein Kinases , Neutrophils/physiology , Sodium Salicylate/pharmacology , Acetaminophen/pharmacology , Antigens, CD/physiology , Arachidonic Acid/pharmacology , CD18 Antigens/physiology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Aggregation/drug effects , Cell Aggregation/physiology , Cell Survival/drug effects , Cyclooxygenase Inhibitors/pharmacology , Humans , In Vitro Techniques , Macrophage-1 Antigen/physiology , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/cytology , Neutrophils/drug effects , Tetradecanoylphorbol Acetate/pharmacology
18.
J Leukoc Biol ; 64(4): 537-45, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9766635

ABSTRACT

The signal transduction pathways activated by tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) that lead to priming of polymorphonuclear leukocytes (PMNs) are unknown. The hypotheses that these cytokines stimulate multiple mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and p38 MAPK, and that these MAPKs participate in priming of human PMNs were examined. TNF-alpha stimulated a dose-dependent increase in ERK and p38 MAPK activities that was maximal at 10 min. JNKs were not stimulated by TNF-alpha or GM-CSF. GM-CSF stimulated ERK activity comparable to that of TNF-alpha, but GM-CSF was a less potent stimulus of p38 MAPK activity. The tyrosine kinase inhibitor, genistein, inhibited ERK and p38 MAPK stimulation by both cytokines. The phosphatidylinositol 3-kinase inhibitor, wortmannin, attenuated stimulation of ERKs and p38 MAPK by GM-CSF, but not TNF-alpha. GM-CSF, but not TNF-alpha, stimulated wortmannin-sensitive activation of Raf-1. TNF-alpha and GM-CSF priming of superoxide release stimulated by N-formyl-methionyl-leucyl-phenylalanine was significantly attenuated by the MEK inhibitor, PD098059, and the p38 MAPK inhibitor, SB203580. Incubation with both MAPK inhibitors produced an additive effect. Our data suggest that TNF-alpha and GM-CSF activate ERKs and p38 MAPK by different signal transduction pathways. Both ERK and p38 MAPK cascades contribute to the ability of TNF-alpha and GM-CSF to prime the respiratory burst response in human PMNs.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/blood , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Neutrophils/physiology , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/pharmacology , Amino Acid Sequence , Enzyme Activation , Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Humans , Intracellular Signaling Peptides and Proteins , JNK Mitogen-Activated Protein Kinases , Kinetics , MAP Kinase Kinase 1 , Molecular Sequence Data , Neutrophils/drug effects , Neutrophils/enzymology , Protein Serine-Threonine Kinases/blood , Protein Serine-Threonine Kinases/chemistry , Protein-Tyrosine Kinases/blood , Protein-Tyrosine Kinases/chemistry , Proto-Oncogene Proteins c-raf/blood , Respiratory Burst , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/physiology , p38 Mitogen-Activated Protein Kinases
19.
Am J Physiol ; 275(2): L389-97, 1998 08.
Article in English | MEDLINE | ID: mdl-9700101

ABSTRACT

Alveolar macrophages play an important role in host defense and in other types of inflammatory processes in the lung. These cells exhibit many alterations in function compared with their precursor cells, blood monocytes. To evaluate a potential mechanism for these differences in function, we evaluated expression of protein kinase C (PKC) isoforms. We found an increase in Ca2+-dependent PKC isoforms in monocytes compared with alveolar macrophages. We also found differential expression of the Ca2+-independent isoforms in alveolar macrophages compared with monocytes. One consequence of the activation of PKC can be increased expression of mitogen-activated protein (MAP) kinase pathways. Therefore, we also evaluated activation of the MAP kinase extracellular signal-regulated kinase (ERK) 2 by the phorbol ester phorbol 12-myristate 13-acetate (PMA). PMA activated ERK2 kinase in both alveolar macrophages and monocytes; however, monocytes consistently showed a significantly greater activation of ERK2 kinase by PMA compared with alveolar macrophages. Another known consequence of the activation of PKC and subsequent activation of ERK kinase is activation of the transcription factor activator protein-1 (AP-1). We evaluated the activation of AP-1 by PMA in both monocytes and macrophages. We found very little detectable activation of AP-1, as assessed in a gel shift assay, in alveolar macrophages, whereas monocytes showed a substantial activation of AP-1 by PMA. These studies show that the differential expression of PKC isoforms in alveolar macrophages and blood monocytes is associated with important functional alterations in the cells.


Subject(s)
Isoenzymes/biosynthesis , Macrophages, Alveolar/enzymology , Monocytes/enzymology , Protein Kinase C/biosynthesis , Bronchoalveolar Lavage Fluid/cytology , Calcium-Calmodulin-Dependent Protein Kinases/biosynthesis , Calcium-Calmodulin-Dependent Protein Kinases/blood , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Differentiation , Cells, Cultured , Humans , In Vitro Techniques , Isoenzymes/blood , Isoenzymes/isolation & purification , Macrophages, Alveolar/drug effects , Mitogen-Activated Protein Kinase 1 , Monocytes/drug effects , Protein Kinase C/blood , Protein Kinase C/isolation & purification , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factor AP-1/metabolism
20.
Immunology ; 90(4): 502-10, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9176102

ABSTRACT

The activation of MAP kinase in human neutrophils stimulated by both uncoated and plasma-opsonized crystals of triclinic calcium pyrophosphate dihydrate (CPPD) was investigated. The effect of taxol on MAP kinase activation and on the responses of neutrophils stimulated by plasma-opsonized crystals was determined. MAP kinase activation was identified and quantified in Mono Q chromatography separated fractions of neutrophils that had been incubated with CPPD crystals by measuring [gamma-32P]adenosine triphosphate (ATP) phosphorylation of myelin basic protein and using immunoblotting techniques. Human neutrophils were incubated with taxol (0-50 microM), added to plasma-opsonized CPPD (50 mg/ml) and MAP kinase activation, chemiluminescence, superoxide anion generation, lysozyme and myeloperoxidase release were monitored. Both uncoated and plasma coated CPPD crystals induced a large increase in MAP kinase activity in neutrophils over control levels within 1 min of incubation. Pretreatment of neutrophils with taxol was able to suppress this activation of MAP kinase. Taxol produced a concentration-dependent inhibition of opsonized CPPD-induced neutrophil chemiluminescence, superoxide anion production and myeloperoxide release. Taxol at 28 microM also significantly inhibited chemiluminescence, superoxide anion production and myeloperoxidase release from neutrophils stimulated by opsonized zymosan. This is the first report of crystal-induced activation of MAP kinase in neutrophils. Microtubule-associated processes, such as signal transduction, secretion and phagocytosis are involved in particulate-induced neutrophil responses. We have suggested that the inhibitory effect of taxol observed in this work is due to its stabilizing effect on microtubules and disruption of MAP kinase activation associated with microtubules.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Calcium Pyrophosphate/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Neutrophil Activation/drug effects , Neutrophils/drug effects , Paclitaxel/pharmacology , Superoxides/blood , Blotting, Western , Calcium Pyrophosphate/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/blood , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Culture Techniques , Cell Degranulation/drug effects , Crystallization , Humans , Luminescent Measurements , Neutrophil Activation/physiology , Neutrophils/enzymology
SELECTION OF CITATIONS
SEARCH DETAIL
...