Publications (60) View all
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Article: Deregulated Expression of Urokinase and its Inhibitor Type 1 in Prostate Cancer Cells: Role of Epigenetic Mechanisms.
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ABSTRACT: Plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) play a crucial role in cancer progression. In the present study we examined the regulation of PAI-1 and uPA expressions in normal prostate epithelial cells (PrEC) and the prostate cancer cell lines LNCaP, DU-145, and PC-3. The antigen and mRNA levels of PAI-1 were down-regulated in cancer cells, especially in LNCaP and DU-145. In the presence of proinflammatory cytokines, an increase of PAI-1 mRNA levels was observed in PrEC, LNCaP and PC-3, but not in DU-145 cells. Treatment with demethylating agent, 5-aza-2´-deoxycytidine increased the level of PAI-1 transcript in DU-145 cells and restored the inducing effect of cytokines on PAI-1 expression. An aberrant methylation of PAI-1 promoter in DU-145 and LNCaP cells was shown by methylation-sensitive high resolution melting (MS-HRM) analysis. PAI-1 methylation was also significantly increased in tumor samples (23.2±1.7%) in comparison to adjacent non-tumor tissue (6.0±0.8%). Furthermore, the expression of uPA was increased in high invasive cell lines DU-145 and PC-3 in comparison to PrEC and low invasive LNCaP cells. MS-HRM analysis revealed aberrant methylation of uPA promoter in LNCaP cells, but not in PrEC, DU-145 and PC-3 cells, as well as in normal and prostate cancer tissue samples. In conclusion, the study shows that PAI-1 and uPA expression was changed in opposite directions in high invasive prostate cancer cell lines resulting in a strong decrease of PAI-1/uPA ratio, which may indicate a shift towards proteolytic activities. Methylation of the PAI-1 gene is suggested as one of the molecular mechanisms involved in the cancer-associated down-regulation of the PAI-1 expression.Experimental and Molecular Pathology 03/2013; · 2.42 Impact Factor -
Article: Analysis of plasma 3-methoxytyramine, normetanephrine and metanephrine by ultraperformance liquid chromatography-tandem mass spectrometry: utility for diagnosis of dopamine-producing metastatic phaeochromocytoma.
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ABSTRACT: Measurements of plasma normetanephrine (NMN) and metanephrine (MN) provide a sensitive test for diagnosis of phaeochromocytomas and paragangliomas (PPGLs), but do not allow detection of dopamine-producing tumours. Here we introduce a novel mass spectrometric based method coupled to ultraperformance liquid chromatography (LC-MS/MS) for measuring NMN, MN and 3-methoxytyramine (MTY), the O-methylated metabolite of dopamine. Specific collision-induced fragment ions assessed by multireaction monitoring transitions were used for identification, with quantification according to signal intensities of analytes relative to stable isotope labelled internal standards. Results for solid-phase extracted samples from 196 subjects analysed by LC-MS/MS were compared with those analysed by liquid chromatography with electrochemical detection (LC-ECD). Concentration ranges in 125 volunteers were compared with those from 63 patients with PPGLs, including 14 with metastatic disease. The LC-MS/MS method showed linearity over four orders of magnitude with analytical sensitivity sufficient to measure to 0.02 nmol/L. Intra- and inter-assay coefficients of variation ranged from 2.8% to 13.5%. NMN and MN were respectively measured 17% and 10% higher and MTY 26% lower by LC-MS/MS than by LC-ECD. Medians and ranges for 3-methoxytramine, NMN and MN were respectively 0.08 (0.03-0.13), 0.35 (0.13-0.95) and 0.15 (0.07-0.33) nmol/L in volunteers. Among patients with PPGLs, plasma methoxytyramine was six-fold higher in patients with than without metastastases (1.09 versus 0.19 nmol/L) and in three patients was the only metabolite increased. The LC-MS/MS method enables accurate, selective and sensitive measurements of catecholamine O-methylated metabolites that should be particularly useful for screening and management of dopamine-producing metastatic PPGLs.Annals of Clinical Biochemistry 03/2013; 50(Pt 2):147-55. · 2.17 Impact Factor -
Article: Simultaneous liquid chromatography tandem mass spectrometric determination of urinary free metanephrines and catecholamines, with comparisons of free and deconjugated metabolites.
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ABSTRACT: OBJECTIVE: We introduce a novel liquid chromatographic tandem-mass spectrometric method for simultaneous measurements of urinary catecholamines and their free O-methylated metabolites, which we compare to the deconjugated metabolites. METHODS: Method performance was validated for recovery, linearity, precision and accuracy, analyte stability, ion suppression and carry over. Results from 53 patients with and 138 volunteers without pheochromocytoma were compared. RESULTS: Analyte recoveries ranged from 60 to 96% and intra- and inter-assay coefficients of variation from 2.7 to 13.2%. The method showed excellent linearity over 3 orders of magnitude with analytical sensitivity sufficient to measure to 1.2 nmol/L. Free O-methylated metabolites were excreted at less than 20% the rates of the deconjugated metabolites, but were easily measureable. Increases in urinary normetanephrine in pheochromocytoma patients relative to volunteers were higher for free than deconjugated metabolites and higher for both than for norepinephrine (10 vs 5.5 vs 3.7 fold increases). In contrast, relative increases in urinary free versus deconjugated metanephrine (2.7 and 3.2) and methoxytyramine (2.1 and 1.9) did not differ, but for methoxytyramine were larger than for dopamine (1.2). CONCLUSION: Measurements of urinary catecholamines and their free O-methylated metabolites by our method provide potential advantages over urinary deconjugated metanephrines for diagnosis of pheochromocytoma.Clinica chimica acta; international journal of clinical chemistry 01/2013; · 2.54 Impact Factor -
Article: Double stable isotope ultra performance liquid chromatographic-tandem mass spectrometric quantification of tissue content and activity of phenylethanolamine N-methyltransferase, the crucial enzyme responsible for synthesis of epinephrine.
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ABSTRACT: Here, we describe a novel method utilizing double stable isotope ultra performance liquid chromatography-tandem mass spectrometry to measure tissue contents and activity of phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for synthesis of the stress hormone, epinephrine. The method is based on measurement of deuterium-labeled epinephrine produced from the reaction of norepinephrine with deuterium-labeled S-adenosyl-L-methionine as the methyl donor. In addition to enzyme activity, the method allows for determination of tissue contents of PNMT using human recombinant enzyme for calibration. The calibration curve for epinephrine was linear over the range of 0.1 to 5,000 pM, with 0.5 pM epinephrine representing the lower limit of quantification. The calibration curve relating PNMT to production of deuterium-labeled epinephrine was also linear from 0.01 to 100 ng PNMT. Intra- and inter-assay coefficients of variation were respectively 12.8 % (n = 10) and 10.9 to 13.6 % (n = 10). We established utility of the method by showing induction of the enzyme by dexamethasone in mouse pheochromocytoma cells and strong relationships to PNMT gene expression and tissue epinephrine levels in human pheochromocytomas. Development of this assay provides new possibilities for investigations focusing on regulation of PNMT, the crucial final enzyme responsible for synthesis of epinephrine, the primary fight-or-flight stress hormone.Analytical and Bioanalytical Chemistry 12/2012; · 3.78 Impact Factor -
Article: Aberrant methylation of the M-type phospholipase A2 receptor gene in leukemic cells.
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ABSTRACT: BACKGROUND: The M-type phospholipase A2 receptor (PLA2R1) plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS) or acute leukemia. METHODS: Sites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM) analysis was then carried out to quantify PLA2R1 methylation at 5`-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA. RESULTS: Expression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2 -deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% +/- 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia. CONCLUSIONS: The study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis.BMC Cancer 12/2012; 12(1):576. · 3.01 Impact Factor
