Analysis of the potential of cancer cell lines to release tissue factor-containing microvesicles: correlation with tissue factor and PAR2 expression

BackgroundDespite the association of cancer-derived circulating tissue factor (TF)-containing microvesicles and hypercoagulable state, correlations with the incidence of thrombosis remain unclear.MethodsIn this study the upregulation of TF release upon activation of various cancer cell lines, and the correlation with TF and PAR2 expression and/or activity was examined. Microvesicle release was induced by PAR2 activation in seventeen cell lines and released microvesicle density, microvesicle-associated TF activity, and phoshpatidylserine-mediated activity were measured. The time-course for TF release was monitored over 90 min in each cell line. In addition, TF mRNA expression, cellular TF protein and cell-surface TF activities were quantified. Moreover, the relative expression of PAR2 mRNA and cellular protein were analysed. Any correlations between the above parameters were examined by determining the Pearson’s correlation coefficients.ResultsTF release as microvesicles peaked between 30–60 min post-activation in the majority of cell lines tested. The magnitude of the maximal TF release positively correlated with TF mRNA (c = 0.717; p

Calmodulin-dependent nuclear import of HMG-box family nuclear factors: importance of the role of SRY in sex reversal

The HMG (high-mobility group)-box-containing chromatin-remodelling factor SRY (sex-determining region on the Y chromosome) plays a key role in sex determination. Its role in the nucleus is critically dependent on two NLSs (nuclear localization signals) that flank its HMG domain: the C-terminally located ‘β-NLS’ that mediates nuclear transport through Impβ1 (importin β1) and the N-terminally located ‘CaM-NLS’ which is known to recognize the calcium-binding protein CaM (calmodulin). In the present study, we examined a number of missense mutations in the SRY CaM-NLS from human XY sex-reversed females for the first time, showing that they result in significantly reduced nuclear localization of GFP (green fluorescent protein)–SRY fusion proteins in transfected cells compared with wild-type. The CaM antagonist CDZ (calmidazolium chloride) was found to significantly reduce wild-type SRY nuclear accumulation, indicating dependence of SRY nuclear import on CaM. Intriguingly, the CaM-NLS mutants were all resistant to CDZ's effects, implying a loss of interaction with CaM, which was confirmed by direct binding experiments. CaM-binding/resultant nuclear accumulation was the only property of SRY found to be impaired by two of the CaM-NLS mutations, implying that inhibition of CaM-dependent nuclear import is the basis of sex reversal in these cases. Importantly, the CaM-NLS is conserved in other HMG-box-domain-containing proteins such as SOX-2, -9, -10 and HMGN1, all of which were found for the first time to rely on CaM for optimal nuclear localization. CaM-dependent nuclear translocation is thus a common mechanism for this family of important transcription factors

FGF2 Translationally Induced by Hypoxia Is Involved in Negative and Positive Feedback Loops with HIF-1α

BACKGROUND: Fibroblast growth factor 2 (FGF2) is a major angiogenic factor involved in angiogenesis and arteriogenesis, however the regulation of its expression during these processes is poorly documented. FGF2 mRNA contains an internal ribosome entry site (IRES), a translational regulator expected to allow mRNA expression during cellular stress. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we have developed a skin ischemia model in transgenic mice expressing a reporter transgene under the control of the FGF2 IRES. The results reveal that FGF2 is induced at the protein level during ischemia, concomitant with HIF-1alpha induction and a decrease in FGF2 mRNA. In addition, the FGF2 IRES is strongly activated under these ischemic conditions associated with hypoxia, whereas cap-dependent translation is repressed by 4E-BP hypophosphorylation. We also show that up-regulation of FGF2 protein expression in response to hypoxia correlates with the increase of FGF2 IRES activity in vitro, in human retinoblasts 911. The use of siRNAs targeting HIF or FGF2 indicates that FGF2 and HIF-1alpha reciprocally regulate their expression/accumulation, by a negative feedback loop in early hypoxia, followed by a positive feedback loop in late hypoxia. CONCLUSION/SIGNIFICANCE: FGF2 expression is up-regulated in vivo and in vitro in response to hypoxia. Strikingly, this up-regulation is not transcriptional. It seems to occur by an IRES-dependent mechanism, revealing new mechanistic aspects of the hypoxic response. In addition, our data show that FGF2 interacts with HIF-1alpha in a unique crosstalk, with distinct stages in early and late hypoxia. These data reveal the physiological importance of IRES-dependent translation during hypoxic stress and underline the complexity of the cellular response to hypoxia, suggesting a novel role of FGF2 in the regulation of HIF-1alpha during the induction of angiogenesis

Platelet RNA sequencing for cancer screening in patients with unprovoked venous thromboembolism: a prospective cohort study

BackgroundPlatelet RNA sequencing has been shown to accurately detect cancer in previous studies.ObjectivesTo compare the diagnostic accuracy of platelet RNA sequencing with standard-of-care limited cancer screening in patients with unprovoked venous thromboembolism (VTE).MethodsPatients aged ≥40 years with unprovoked VTE were recruited at 13 centers and followed for 12 months for cancer. Participants underwent standard-of-care limited cancer screening, and platelet RNA sequencing analysis was performed centrally at study end for cases and selected controls. Sensitivity and specificity were calculated, using the predefined primary positivity threshold of 0.54 for platelet RNA sequencing aiming at 86% test sensitivity, and an additional predefined threshold of 0.89 aiming at 99% test specificity.ResultsA total of 476 participants were enrolled, of whom 25 (5.3%) were diagnosed with cancer during 12-month follow-up. For each cancer patient, 3 cancer-free patients were randomly selected for the analysis. The sensitivity of limited screening was 72% (95% CI, 52-86) at a specificity of 91% (95% CI, 82-95). The area under the receiver operator characteristic for platelet RNA sequencing was 0.54 (95% CI, 0.41-0.66). At the primary positivity threshold, all patients had a positive test, for a sensitivity estimated at 100% (95% CI, 87-99) and a specificity of 8% (95% CI, 3.7-16.4). At the secondary threshold, sensitivity was 68% (95% CI, 48-83; p value compared with limited screening 0.71) at a specificity of 36% (95% CI, 26-47).ConclusionPlatelet RNA sequencing had poor diagnostic accuracy for detecting occult cancer in patients with unprovoked VTE with the current algorithm.Thrombosis and Hemostasi

La grotte ornée de Villars (Dordogne).: Revision of the decoration and new datas

National audienceThe decorated cave of Villars, a hundred meters long, is a limited sector of a labyrinthine subterranean network of about ten kilometers. The whole system was discovered and mapped by the Speleo-Club of Périgueux from 1953 onwards. Parietal vestiges, discovered in January 1958, were the subject of a first methodical publication in 1974. The study was completed from 2009 to 2011 within the framework of the ANR MADAPCA through the revision of the parietal decoration, prior to a dozen radiometric dates being obtained from charcoal marks on the walls, and from reindeer bones and antler). The figures are small in size, generally placed within the frames provided naturally by concretions. The decoration, drawn in black, except for 3 signs and red dots, is dominated by the classic horse-bison association. Among the identifiable figures, there are 2 horses, 1 bison and 1 bovine in the Salle des Cierges or its surroundings, while in the Salle des Peintures there are 6 horses (including one vertically drawn), 2 bison, 2 or 3 ibex, 1 rhinoceros, 1 man and a few outlines of unidentified species. The anatomical details and the animation of the animal silhouettes, as well as the association, at the back of the sanctuary, of 4 elements (man-bison scene, horse-head and rhinoceros), recall the style and themes of Lascaux.The accompanying signs consist of dots, isolated or in clusters, long lines, and some more differentiated signs: curved (including 2 schematic feminine figures), grid, diagonal cross, barbed sign. About 100 intact or fragmentary objects were recovered during the exploration and development of the cave, either by humans (flints, calcite cups, pigments, reindeer antler and bones) or by bears which preceded them and left many traces of their hibernations (clawmarks, paw-prints and bones).Thirteen 14C dates have been obtained, the results of which broadly confirm the estimates presented by archaeologists on the basis of stylistic analyses, comparing the works of Villars with those of Lascaux.La grotte ornée de Villars, longue d’une centaine de mètres, est un secteur limité d’un réseau souterrain labyrinthique d’une dizaine de kilomètres. L’ensemble a été découvert et topographié par le Spéléo-Club de Périgueux à partir de 1953. Les vestiges pariétaux, découverts en janvier 1958, ont fait l’objet d’une première publication méthodique en 1974. L’étude a été complétée de 2009 à 2011 dans le cadre de l’ANR MADAPCA par la révision de la décoration pariétale, préalable à une douzaine de datations radiométriques (témoins charbonneux pariétaux, bois et os de renne).Les figures sont de petite taille, en général inscrites dans le cadre fourni naturellement par les concrétions. Le décor, dessiné en noir, sauf 3 signes et des points rouges, est dominé par l’association classique cheval-bison. Parmi les figures identifiables, on compte, dans la Salle des Cierges ou ses abords, 2 chevaux, 1 bison et 1 bovin, et, dans la Salle des Peintures, 6 chevaux (dont un verticalement dessiné), 2 ou 3 bisons, 2 ou 3 bouquetins, 1 rhinocéros, 1 homme et quelques silhouettes d’espèce non identifiée. Les détails anatomiques et l’animation des silhouettes animales, de même que l’association, au fond du sanctuaire, de 4 éléments (scène homme-bison, avant-train de cheval et rhinocéros) rappelle le style et les thèmes de Lascaux. L’accompagnement sémiologique est fait de ponctuations, isolées ou en nappe, de longs traits et quelques signes plus différenciés : en accolade (dont 2 figures féminines schématiques), grille, croix diagonale, barbelé, triangle.Une centaine d’objets entiers ou fragmentaires ont été recueillis lors de l’exploration et de l’aménagement de la caverne, provenant soit des hommes (silex, godets de calcite, pigments, bois et os de renne) soit des ours qui les ont précédés et ont laissé de nombreuses traces de leurs séjours hivernaux (griffures, empreintes et os). Treize datations 14C ont été effectuées, dont les résultats confirment, dans les grandes lignes, les estimations données par les archéologues à partir d’analyses stylistiques, rapprochant les œuvres de Villars de celles de Lascaux