D'importants progres ont ete faits au cours de ces derniers mois dans la determination de la fonction cellulaire des petites GTPases de la famille Rho. Il vient en effet d'etre montre que deux membres de cette famille (Rac et Rho) sont impliques dans le controle de la reorganisation des reseaux d'actine cytosquelettique provoquee par les facteurs de croissance. En outre, la decouverte de plusieurs facteurs multifonctionnels , possedant un domaine GAP capable de moduler l'activite des proteines Rho , ainsi que des activites extremement diverses telles que serine kinase ou represseur transcription ne}, suggere que les proteines de la famille Rho sont reglees de facon concertee avec les proteines Ras, et interviennent dans la transduction des signaux mitogenes. La traque des facteurs GAP ainsi que la determination de leurs cibles permettront-elles d'obtenir une vision integree des processus de division et de reorganisation de l'architecture cellulaire ?
AbstractSignals from the extracellular matrix are essential for the survival of many cell types. Dominant-negative mutants of two members of Rho family GTPases, Rac1 and Cdc42, mimic the loss of anchorage in primary mouse fibroblasts and are potent inducers of apoptosis. This pathway of cell death requires the activation of both the p53 tumor suppressor and the extracellular signal-regulated mitogen-activated protein kinases (Erks). Here we characterize the proapoptotic Erk signal and show that it differs from the classically observed survival-promoting one by the intensity of the kinase activation. The disappearance of the GTP-bound forms of Rac1 and Cdc42 gives rise to proapoptotic, moderate activation of the Raf-MEK-Erk cascade via a signaling pathway involving the kinases phosphatidlyinositol 3-kinase and Akt. Moreover, concomitant activation of p53 and inhibition of Akt are both necessary and sufficient to signal anoikis in primary fibroblasts. Our data demonstrate that the GTPases of the Rho family control three major components of cellular signal transduction, namely, p53, Akt, and Erks, which collaborate in the induction of apoptosis due to the loss of anchorage. ACKNOWLEDGMENTSWe are grateful to A. Brunet, P. Chavrier, A. Hall, P. Lenormand, C. Marshall, C. Norbury, S. Roche, M. Vandromme, M. White, and E. Yonish-Rouach for various plasmids used in this work and to P. Boquet for the generous gift of C. difficile toxin B. We thank Damien Gregoire for help with the anoikis experiments, Pierre Travo for help with immunofluorescence microscopy, and Bob Hipskind for invaluable comments on the manuscript.This work was supported by INSERM, CNRS, and Association pour la Recherche contre le Cancer (support given to U.H.). T.F.F. is the recipient of Career Development Award DAMD17-00-1-0214 and O.Z. is the recipient of a fellowship from Association pour la Recherche contre le Cancer.
Wolbachia are maternally inherited endosymbiotic bacteria that infect many arthropod species and may induce cytoplasmic incompatibility (CI) resulting in abortive embryonic development. Among all the described host species, mosquitoes of the Culex pipiens complex display the highest variability of CI crossing types. Paradoxically, searches for polymorphism in Wolbachia infecting strains and field populations hitherto failed or produced very few markers. Here, we show that an abundant source of the long-sought polymorphism lies in WO prophage sequences present in multiple copies dispersed in the genome of Wolbachia infecting C. pipiens (wPip). We identified up to 66 different Wolbachia variants in C. pipiens strains and field populations and no occurrence of superinfection was observed. At least 49 different Wolbachia occurred in Southern Europe C. pipiens populations, and up to 10 different Wolbachia were even detected in a single population. This is in sharp contrast with North African and Cretan samples, which exhibited only six variants. The WO polymorphism appeared stable over time, and was exclusively transferred maternally. Interestingly, we found that the CI pattern previously described correlates with the variability of Gp15, a prophage protein similar to a bacterial virulence protein. WO prophage sequences thus represent variable markers that now open routes for approaching the molecular basis of CI, the host effects, the structure and dynamics of Wolbachia populations.
Dihydrofolate reductase isolated from the liver of normal adult mice was focused on a LKB analytic column. Separation was carried out in a sucrose gradient and a pH gradient was formed within limits of pH 7-10 by means of 1% ampholin. 48 hours' separation yielded two enzyme fractions focusing at pH 8.1 and pH 8.7 respectively, 80% of the total activity being found in the latter fraction. If the original enzyme was preincubated with NADPH, a further activity zone, at pH 7.5, was formed after isoelectric focusing and the other two peaks were depressed. The form of the enzyme focusing at pH 8.7 had higher specific activity than the form focusing at pH 8.1. Neither of these forms of the enzyme was as pure as the enzyme prepared by affinity chromatography. This was also confirmed by double radial immunodiffusion. Degree of Methotrexate inhibition and potassium chloride activation of the two fractions isolated by isoelectric focusing was the same.
Quantitative differences in signal transduction are to date an understudied feature of tumour heterogeneity. The MAPK Erk pathway, which is activated in a large proportion of human tumours, is a prototypic example of distinct cell fates being driven by signal intensity. We have used primary hepatocyte precursors transformed with different dosages of an oncogenic form of Ras to model subclonal variations in MAPK signalling. Orthotopic allografts of Ras-transformed cells in immunocompromised mice gave rise to fast-growing aggressive tumours, both at the primary location and in the peritoneal cavity. Fluorescent labelling of cells expressing different oncogene levels, and consequently varying levels of MAPK Erk activation, highlighted the selection processes operating at the two sites of tumour growth. Indeed, significantly higher Ras expression was observed in primary as compared to secondary, metastatic sites, despite the apparent evolutionary trade-off of increased apoptotic death in the liver that correlated with high Ras dosage. Analysis of the immune tumour microenvironment at the two locations suggests that fast peritoneal tumour growth in the immunocompromised setting is abrogated in immunocompetent animals due to efficient antigen presentation by peritoneal dendritic cells. Furthermore, our data indicate that, in contrast to the metastatic-like outgrowth, strong MAPK signalling is required in the primary liver tumours to resist elimination by NK (natural killer) cells. Overall, this study describes a quantitative aspect of tumour heterogeneity and points to a potential vulnerability of a subtype of hepatocellular carcinoma as a function of MAPK Erk signalling intensity.
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