Glycogen synthase kinase-3β negatively regulates group IIA phospholipase A2 expression in human aortic smooth muscle and HepG2 hepatoma cells

AbstractThe present study shows that the IFN-γ-mediated upregulation of secretory phospholipase A2 of group IIA (sPLA2-IIA) in HASMC and HepG2 cells is synergistically increased after simultaneous inhibition of glycogen synthase kinase-3β (GSK-3β) by indirubin-3′-monoxime, 5-iodo or AR-A014418. The effect of GSK-3β inhibition was dose- and time-dependent and can be further augmented by its concomitant incubation with Clostridium difficile toxin B, an inhibitor of small Rho proteins, or H-1152, an inhibitor of Rho-associated kinase. Using AG-490 and caffeic acid phenethyl ester (CAPE), it is further demonstrated that the effect of GSK-3β inhibition on sPLA2-IIA expression depends on Janus kinase-2 and NF-κB-signaling

In situ reverse transcriptase-nested polymerase chain reaction to identify intracellular nucleic acids without the necessity of DNAse pretreatment and hybridisation

In the present study a protocol of in situ reverse transcriptasenested polymerase chain reaction (in situ RT-nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by "DNA repair mechanisms" and "endogenous priming", a two-step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin-labeled nucleotides and intrinsic primers. ing PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT-nested PCR, which in comparison to the method of in situ RT-PCRin situ-hybridisation is simpler and less time-consuming, can be used as an alternative approach to identify intracellular nucleic acids

Expression and shedding of endothelial protein C receptor in prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>Increasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines.</p> <p>Results</p> <p>EPCR expression is up-regulated both at the mRNA and protein levels in invasive prostate DU-145 and PC-3 cells in comparison to normal prostate epithelial cells (PrEC) and less-invasive LNCaP cells. Release of soluble EPCR (sEPCR) is induced by 12-myristate 13-acetate, ionomycin, H₂O₂, and disruptor of lipid rafts in PrEC, DU-145, and PC-3 cells. Furthermore, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interleukin-6 or interferon-γ increase sEPCR release. In LNCaP cells, neither pharmacological agents nor IL-1β or TNF-α result in a significant increase of sEPCR release. The effects of IL-1β and TNF-α on EPCR shedding in DU-145 cells are mediated by MEK/ERK 1/2, JNK, and p38 MAPK signalling cascades. In PC-3 cells, however, the MEK/ERK 1/2 pathway is down-regulated and incubation with cytokines did not elevate the phosphorylated ERK-1/2 fraction as in the case of DU-145 cells. Treatment with 4-aminophenylmercuric acetate (APMA), an activator of metalloproteases, causes a disproportionately large increase of sEPCR release in DU-145 and PC-3 cells, compared to PrEC and LNCaP cells. Finally, an increased release of sEPCR mediated by APMA treatment is shown to be connected with reduced generation of activated protein C indicating the functionality of EPCR in these cells.</p> <p>Conclusions</p> <p>The study demonstrates a number of substantial differences in expression and shedding of EPCR in prostate cancer cell lines in comparison with normal cells that may be relevant for understanding the role of this receptor in carcinogenesis.</p

Involvement of Epigenetic Mechanisms in the Regulation of Secreted Phospholipase A2 Expressions in Jurkat Leukemia Cells

Epigenetic changes provide a frequent mechanism for transcriptional silencing of genes in cancer cells. We previously established that epigenetic mechanisms are important for control of group IIA phospholipase A2 (PLA2G2A) gene transcription in human DU-145 prostate cells. In this study, we analyzed the involvement of such mechanisms in the regulation of five sPLA2 isozymes and the M-type receptor of sPLA2 (sPLA2-R) in human leukemic Jurkat cells. These cells constitutively expressed sPLA2-IB, sPLA2-III, sPLA2-X, and sPLA2-R but not sPLA2-IIA and sPLA2-V. Transcription of sPLA2-IIA and sPLA2-V was, however, detected after exposure of cells to the DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC). Expression of sPLA2-IIA was further enhanced by additional exposure to interferon-γ and blocked by inhibitors of specificity protein 1, nuclear factor κB, and Janus kinase/signal transducer and activator of transcription-dependent pathways. Sequence analysis and methylation-specific polymerase chain reaction of bisulfite-modified genomic DNA revealed two 5′-CpG sites (-111 and -82) in the sPLA2-IIA proximal promoter that were demethylated after 5-aza-dC treatment. These sites may be involved in the DNA binding of specificity protein 1 and other transcription factors. Similar findings after treatment of human U937 leukemia cells with 5-aza-dC indicate that this mechanism of PLA2G2A gene silencing is not restricted to Jurkat and DU-145 cells. These data establish that regulation of sPLA2-IIA and sPLA2-V in Jurkat and other cells involves epigenetic silencing by DNA hypermethylation

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells

10.4252/wjsc.v10.i9.119WORLD JOURNAL OF STEM CELLS109119-13

Methods for comparing multiple digital PCR experiments

The estimated mean copy per partition (λ) is the essential information from a digital PCR (dPCR) experiment because λ can be used to calculate the target concentration in a sample. However, little information is available how to statistically compare dPCR runs of multiple runs or reduplicates. The comparison of λ values from several runs is a multiple comparison problem, which can be solved using the binary structure of dPCR data. We propose and evaluate two novel methods based on Generalized Linear Models (GLM) and Multiple Ratio Tests (MRT) for comparison of digital PCR experiments. We enriched our MRT framework with computation of simultaneous confidence intervals suitable for comparing multiple dPCR runs. The evaluation of both statistical methods support that MRT is faster and more robust for dPCR experiments performed in large scale. Our theoretical results were confirmed by the analysis of dPCR measurements of dilution series. Both methods were implemented in the dpcR package (v. 0.2) for the open source R statistical computing environment

Diagnostic Significance of Prostate-Specific Antigen Velocity at Intermediate PSA Serum Levels in Relation to the Standard Deviation of Different Test Systems

Serial prostate-specific antigen (PSA) measurements (PSA velocity) as an additional instrument to detect prostatic cancer was introduced in 1992. It has previously been reported that PSA increase per year differed in the last 5 years prior to diagnosis in patients with benign prostatic hyperplasia (0.18 ng/ml/year), locally confined (0.75 ng/ml/year) and metastasized (4.4 ng/ml/year) cancer of the prostate (CaP) in contrast to healthy men (0.04 ng/ml/year). The ability of PSA velocity to detect organ-confined CaP in patients with intermediate PSA serum values depends therefore on a reliable and reproducible PSA result. The present study comprised 85 men with PSA values between 3 and 8 ng/ml (Abbott IMx). PSA measurements were repeated with Abbott IMx (n = 85 patients) and Hybritech Tandem-E (n = 59 patients) assays. The PSA serum values differed from one examination to the other from 0.02 to 2.74 ng/ml with the Abbott IMx. Standard deviation amounted to 0.35 ng/ml with the Abbott IMx PSA assay. Using the Hybritech Tandem-E assay, mean standard deviation was 1.15 ng/ml and therefore higher than with the Abbott IMx assay. The difference from one test to the other ranged from 0.05 to 4.05 ng/ml with the Hybritech Tandem-E. Using the Abbott IMx assay, 10.6% of all repeat measurements exceeded 1 ng/ml whereas in the Hybritech Tandem-E assay 62.7% of the second measurements differed >1 ng/ml from the first PSA result. An increase in PSA serum values may therefore be due to intratest variation, physiological day-to-day variation as well as prostatic disease. It is important to notice that the intra-assay variation may be greater than the PSA increase per year in a patient with CaP. Therefore, PSA velocity seems to be of limited value.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Differential Expression of Secretory Phospholipases A2 in Normal and Malignant Prostate Cell Lines: Regulation by Cytokines, Cell Signaling Pathways, and Epigenetic Mechanisms

Upregulation of group IIA phospholipase A2 (sPLA2-IIA) correlates with prostate tumor progression suggesting prooncogenic properties of this protein. Here, we report data on expression of three different sPLA2 isozymes (groups IIA, V, and X) in normal (PrEC) and malignant (DU-145, PC-3, and LNCaP) human prostate cell lines. All studied cell lines constitutively expressed sPLA2-X, whereas sPLA2-V transcripts were identified only in malignant cells. In contrast, no expression of sPLA2-IIA was found in PrEC and DU-145 cells, but it was constitutively expressed in LNCaP and PC-3 cells. Expression of sPLA2-IIA is upregulated in PC-3 and in PrEC cells by IFN-γ in a signal transducer and activator of transcription-1-dependent manner, but not in LNCaP cells. Additional signaling pathways regulating sPLA2-IIA expression include cAMP/protein kinase A, p38 mitogen-activated protein kinase, protein kinase C, Rho-kinase, and mitogen-activated/extracellular response protein kinase / extracellular signal-regulated kinase. No deletions were revealed in the sPLA2-IIA gene from DU-145 cells lacking the expression of sPLA2-IIA. Reexpression of sPLA2-IIA was induced by 5-aza-2′-deoxycytidine demonstrating that DNA methylation is implicated in the regulation of sPLA2-II. Together, these data suggest that sPLA2-IIA and sPLA2-V, but not sPLA2-X, are differentially expressed in normal and malignant prostate cells under the control of proinflammatory cytokines; epigenetic mechanisms appear involved in the regulation of sPLA2-IIA expression, at least in DU-145 cells