Lysophosphatidic acid via LPA-receptor 5/protein kinase D-dependent pathways induces a motile and pro-inflammatory microglial phenotype.

BACKGROUND: Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1-6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed "LPA" throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways. METHODS: Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits. RESULTS: LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors' phosphorylation (NF-κB, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production. CONCLUSIONS: In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of pro-inflammatory mediators.

Overexpression of KCNJ3 gene splice variants affects vital parameters of the malignant breast cancer cell line MCF-7 in an opposing manner.

BACKGROUND: Overexpression the KCNJ3, a gene that encodes subunit 1 of G-protein activated inwardly rectifying K(+) channel (GIRK1) in the primary tumor has been found to be associated with reduced survival times and increased lymph node metastasis in breast cancer patients. METHODS: In order to survey possible tumorigenic properties of GIRK1 overexpression, a range of malignant mammary epithelial cells, based on the MCF-7 cell line that permanently overexpress different splice variants of the KCNJ3 gene (GIRK1a, GIRK1c, GIRK1d and as a control, eYFP) were produced. Subsequently, selected cardinal neoplasia associated cellular parameters were assessed and compared. RESULTS: Adhesion to fibronectin coated surface as well as cell proliferation remained unaffected. Other vital parameters intimately linked to malignancy, i.e. wound healing, chemoinvasion, cellular velocities / motilities and angiogenesis were massively affected by GIRK1 overexpression. Overexpression of different GIRK1 splice variants exerted differential actions. While GIRK1a and GIRK1c overexpression reinforced the affected parameters towards malignancy, overexpression of GIRK1d resulted in the opposite. Single channel recording using the patch clamp technique revealed functional GIRK channels in the plasma membrane of MCF-7 cells albeit at very low frequency. DISCUSSION: We conclude that GIRK1d acts as a dominant negative constituent of functional GIRK complexes present in the plasma membrane of MCF-7 cells, while overexpression of GIRK1a and GIRK1c augmented their activity. The core component responsible for the cancerogenic action of GIRK1 is apparently presented by a segment comprising aminoacids 235-402, that is present exclusively in GIRK1a and GIRK1c, but not GIRK1d (positions according to GIRK1a primary structure). CONCLUSIONS: The current study provides insight into the cellular and molecular consequences of KCNJ3 overexpression in breast cancer cells and the mechanism upon clinical outcome in patients suffering from breast cancer.

Endothelin (ET)-1 and ET-3 promote expression of c-fos and c-jun in human choriocarcinoma via ET(B) receptor-mediated G(i)- and G(q)-pathways and MAP kinase activation.

BACKGROUND AND PURPOSE: Endothelins (ETs) and their G protein-coupled receptors exert key physiological functions during normal and aberrant placental development. Trophoblast cells mediate the contact between the embryo and the mother, by establishing a transient organ, the placenta. Choriocarcinoma cells display many of the biochemical and morphological characteristics of in utero invasive trophoblast cells and may therefore be used as a suitable model to study epithelial tumour progression of foetal-derived cells. EXPERIMENTAL APPROACH: The present study aimed at investigating ET receptor-mediated activation of the mitogen-activated protein kinase (MAPK) pathway in human choriocarcinoma. KEY RESULTS: Both JAR and Jeg-3 choriocarcinoma cell lines expressed ET receptor subtype B (ET(B)) but not ET(A) receptor transcripts. ET(B) receptor engagement by ET-1 and ET-3 resulted in a similar time- and concentration-dependent phosphorylation of p42/44 MAPK, also known as extracellular regulated kinase 1/2. Using specific pharmacological antagonists/inhibitors, we showed that ET-1/-3-mediated signal transduction by the ET(B) receptor is transmitted via G(i)- and G(q)-dependent pathways through activation of the Src (G(i)) and protein kinase C (G(q)) axis that converge at Ras/Raf, leading to downstream activation of p42/44. On a functional level, ET(B) engagement and subsequent phosphorylation of p42/44 resulted in enhanced transcription of the immediate early response genes c-fos and c-jun, a process commonly assumed to be mediated by the ET(A) receptor, and increased cell growth and relative cell area. CONCLUSIONS AND IMPLICATIONS: As human choriocarcinoma cells secrete ETs, pharmacological antagonism of ETs and/or ET(B) receptor-mediated signal transduction could represent a likely target therapy for choriocarcinoma.

Carcinoma of the larynx: predictors of diagnostic delay.

OBJECTIVES: In tumors of the upper aerodigestive tract a dose-response relationship between tumor stage and its prognosis raises the chance of a reduction in the diagnostic delay. The interval between the first symptom and the start of tumor-specific therapy and the influencing factors is not well known. The goals of this study were to investigate the diagnostic delay and the influencing factors and to predict the factors that prolong the diagnostic process. STUDY DESIGN: Prospective, nonrandomized study. METHODS: The intervals between first symptom and first consultation of a physician (patient delay) and between first consultation and first tumor-specific therapy (doctor delay) were investigated. Predictors for increased patient or doctor delay were evaluated, and a multiple logistic regression model for increased doctor delay was developed. RESULTS: The total diagnostic delay (sum of patient and doctor delays) was 3 to 4 months. A multifactorial model for the ratio of odds revealed that for women and glottic cancers (1.73) the chance was twice as high for a doctor delay greater than 30 days, whereas subjects with an increased patient delay (>30 d) had reduced odds of being delayed by the physician. CONCLUSIONS: The higher chance of extended doctor delay for glottic tumors compared with supraglottic tumors should be reflected in differential therapeutic strategies. Even unspecific lesions of the vocal folds should be followed up at the end of the therapy, at least. Therapies of glottic lesions without final control have the chance of resulting in delays in treatment. Women, especially, should be included in a follow-up examination. On the other hand, patients with increased patient delay have lower odds of longer doctor delay, which may be the result of tumor growth and, consequently, easier diagnosis.

Cross-talk reduction in confocal images of dual fluorescence labelled cell spheroids.

Dual fluorescence labelling is an advanced method to separate two individual specimens in a biological system using confocal microscopy. An inherent problem of this method is fluorescence channel cross-talk, which causes problems for the exact spatial determination and separation of the specimens. Using a parallel fluorescence detection and an image processing technique, based on an image subtraction method, we have developed a very straight forward method for correcting the dual channel fluorescence images. We successfully applied this method to a 3-dimensional cancer spheroid invasion assay and controlled the cross-talk compensation efficiency by a quality parameter.

Fractal dimension of K1735 mouse melanoma clones and spheroid invasion in vitro.

An in vitro tumour-host confrontation method to investigate the invasion behaviour of cancer has been applied to K1735 mouse melanomas. Fluorescently labelled spheroids of cancer cells and host cells were confronted and the temporal course of cancer invasion into the host was investigated using confocal laser scanning microscopy. To improve the quantitative data of this method, the boundary images of the fluorescently labelled confrontation pairs were treated as fractals. The physical and mathematical framework for determination of the fractal capacity dimension is widely used in biology and medicine and has proved to be a very useful tool for describing the cancer invasion process. The fractal capacity dimension determination was carried out by dilation of the binary boundaries of the objects, which were treated as an estimate of the Minkowski-Bouligand dimension. The fractal dimension correlated well with the degree of invasion of the K1735-M2 clone. Control experiments, with host-host confrontations and various K1735 clones with reduced invasiveness, support these results.

[Technical errors in the application of Graf's hip sonography method]. / Untersuchungsfehler durch Schallkopfkippung bei der Hüftsonographie nach Graf.

AIM: The aim of the study is to point out possible examination errors because of a titled transducer, and to analyse their sources. METHODS: The results obtained by experienced examiners using the correct examination technique were compared to results obtained under defined deviations from the standard. With the use of a computer model the course taken by the ultrasound waves in the neonatal hip was simulated and the sonographic image reconstructed. RESULTS: Twisted as well as tilted positions of the transducer lead to significant errors in alpha, the acetabular roof angle. This is in accordance with the computer model predictions. Caudo-cranially tilted probes lead to false-positive, therapeutically relevant incorrect diagnoses. CONCLUSION: The computer simulation predicts a high degree of accuracy of hip sonography in Graf's technique under standard conditions. The effectiveness of sonographic infant hip screening largely depends on the ability to adhere to the standardized approach, minimizing possible errors. A mechanical transducer guide prevents tilting errors by reducing the degrees of variability in positioning the transducer.