[Fracture and embolization of implantable subclavian venous catheter fragments due to costoclavicular compression: pinch-off syndrome]. / Sección y embolización de catéter permanente por síndrome de pinzamiento costoclavicular (pinch-off).

The fracture of an implantable subclavian venous access device and the subsequent embolization of a catheter fragment is a known complication that is usually associated with a set of clinical and radiologic signs of costoclavicular compression. This scenario is also known as pinch-off syndrome. We describe 2 cases of venous port fracture which led us to review the efficacy of follow-up procedures used in our hospital. As a result, we added instructions for radiologic and clinical verification of catheter placement, taking into consideration the dynamic nature of compression. We also established protocols for coordinating the involvement of different services.

Sección y embolización de catéter permanente por síndrome de pinzamiento costoclavicular (pinch-off) / Fracture and embolization of implantable subclavian venous catheter fragments due to costoclavicular compression: pinch-off syndrome

Una de las complicaciones de los dispositivos venososimplantables subclavios es la rotura y posterior embolizaciónde un fragmento de catéter, que habitualmente seasocia a signos clínicos y radiológicos de pinzamientocostoclavicular, también llamado síndrome de “pinchoff”.Describimos dos casos de rotura ocurridas en nuestrohospital que nos llevaron a revisar la eficacia delseguimiento aplicado. Se añadieron instrucciones para lacomprobación radiológica y clínica del catéter, teniendoen cuenta el carácter dinámico del pinzamiento y se establecieronprotocolos de coordinación entre los serviciosimplicados)(AU)

The fracture of an implantable subclavian venousaccess device and the subsequent embolization of acatheter fragment is a known complication that is usuallyassociated with a set of clinical and radiologic signs ofcostoclavicular compression. This scenario is also knownas pinch-off syndrome. We describe 2 cases of venousport fracture which led us to review the efficacy offollow-up procedures used in our hospital. As a result, weadded instructions for radiologic and clinical verificationof catheter placement, taking into consideration thedynamic nature of compression. We also establishedprotocols for coordinating the involvement of differentservices(AU)

Different regulation of the Trio Dbl-Homology domains by their associated PH domains.

Guanine nucleotide exchange factors for Rho-GTPases (Rho-GEFs) invariably share a catalytic Dbl-Homology (DH) domain associated with a Pleckstrin Homology (PH) domain, whose function in Rho-GEF activation is not well understood. Trio is the first member of an emerging family of Dbl proteins containing two Rho-GEF domains (GEFD1 and GEFD2). TrioGEFD1 activates the GTPases RhoG and Rac1, while TrioGEFD2 acts on RhoA. In this study, we have investigated the roles of the two PH domains of Trio in Rho-GEF activity. We show that TrioPH1 is required for GEFD1-mediated induction of actin cytoskeleton remodeling and JNK activation. TrioPH1 is involved both in the catalytic activity and in the subcellular localization of its associated DH domain, by acting as a cytoskeletal targeting signal. Moreover, TrioPH1 in association with DH2 activates the JNK pathway, by an unknown mechanism independent of DH2 catalytic activity. TrioPH2 does not behave as a targeting module in intact cells. TrioPH2 inhibits DH2-dependent stress fiber formation, which correlates with the TrioPH2-mediated inhibition of DH2 GEF activity. In addition, expression in the neuron-like PC12 cell line of the intact Trio protein deleted of each PH domain shows that only TrioPH1 is required for Trio-induced neurite outgrowth. Taken together, these data demonstrate that the two PH domains play a different role in the control of Trio Rho-GEF function.

Raf-MEK-Erk cascade in anoikis is controlled by Rac1 and Cdc42 via Akt.

Signals 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.

Extinction of rac1 and Cdc42Hs signalling defines a novel p53-dependent apoptotic pathway.

Apoptosis is a normal physiological process which eliminates cells that do not receive adequate extracellular signals. One of the pathways signalling apoptosis is controlled by the small GTPases of the Rho family, also involved in cell proliferation, differentiation and motility. Another major apoptosis signalling pathway involves the p53 tumour suppressor which is activated by a variety of stress and mediates growth arrest or apoptosis in normal cells. We show here that upon detachment from the extracellular matrix, fibroblasts undergo rapid apoptosis that can be rescued by constitutive activation of Rac1 and Cdc42Hs GTPases. Conversely, inhibition of Rac1 and Cdc42Hs efficiently triggers apoptosis in adherent cells. Interestingly, apoptosis is not observed in p53-/- cells either cultured in suspension or inhibited for Rac1 and Cdc42Hs activity. Moreover, Rac1 and Cdc42Hs extinction in normal cells activates endogenous p53. Using specific inhibitors of MAPK pathways, we demonstrate that, in our experimental system, p38 signals survival, while ERK activity is required for apoptosis. Our data constitute the first demonstration that Rac1 and Cdc42Hs control pathways that require simultaneous signalling through MAPK ERK and p53 to induce apoptosis.

pRB binds to and modulates the transrepressing activity of the E1A-regulated transcription factor p120E4F.

The retinoblastoma protein pRB is involved in the transcriptional control of genes essential for cell cycle progression and differentiation. pRB interacts with different transcription factors and thereby modulates their activity by sequestration, corepression, or activation. We report that pRB, but not p107 and p130, binds to and facilitates repression by p120(E4F), a ubiquitously expressed GLI-Kruppel-related protein identified as a cellular target of E1A. The interaction involves two distinct regions of p120(E4F) and the C-terminal part of pRB. In vivo pRB-p120(E4F) complexes can only be detected in growth-arrested cells, and accordingly contain the hypophosphorylated form of pRB. Repression of an E4F-responsive promoter is strongly increased by combined expression of p120(E4F) and pRB, which correlates with pRB-dependent enhancement of p120(E4F) binding activity. Elevated levels of p120(E4F) have been shown to block growth of mouse fibroblasts in G(1). We find this requires pRB, because RB(-/-) fibroblasts are significantly less sensitive to excess p120(E4F).

Anti-apoptotic activity of p53 maps to the COOH-terminal domain and is retained in a highly oncogenic natural mutant.

The tumour suppressor p53 plays a complex role in the regulation of apoptosis. High levels of wild type p53 potentiate the apoptotic response, while physiological range, low levels of the protein have an anti-apoptotic activity in serum starved immortalized fibroblasts. Here we report that primary fibroblast-like cells that show normal growth control are also efficiently protected from apoptosis by the endogenous p53 activity. The capacity to inhibit apoptosis is not restricted to the wild type protein: the R-->H175 p53 mutant fully retains the anti-apoptotic activity of the wild type p53, providing a possible explanation for its high oncogenicity. Using a series of point and deletion mutants of p53 under the control of tetracycline-regulated promoter we show that certain mutants, like the wild type, protect cells at low levels but lead to apoptosis when overexpressed. This latter effect is lost upon deletion of a proline-rich domain in the NH2 part of the protein. The anti-apoptotic activity can be mapped to the extreme carboxy-terminal part of the protein and is therefore independent of other well characterized p53 activities. Our results add a new level of complexity to the network of interactions mediated by p53 in normal physiology and pathology.

[Role of the multifunctional Trio protein in the control of the Rac1 and RhoA gtpase signaling pathways]. / Rôle de la protéine multifonctionnelle Trio dans le contrôle des voies de signalisation des GTPases Rac1 et RhoA.

The small GTPases Cdc42, Rac and RhoA have important regulatory roles in mediating cytoskeletal rearrangements, MAP kinase cascades and induction of G1 cell cycle progression. The activity of the GTPases is regulated by guanine nucleotide exchange factors (GEFs) which accelerate their GDP/GTP exchange rate, and thereby activate them. All the GEFs for the Rho-GTPases family share two conserved domains: the DH domain (for Dbl-homology domain) responsible for the enzymatic activity, and the PH domain, probably responsible for the proper localization of the molecule. Trio is a multifunctional protein that is comprised of two functional Rho-GEFs domains and a serine/threonine kinase domain. We have shown in vitro and in vivo that the first GEF domain (GEFD1) activates Rac1, while the second GEF domain (GEFD2) acts on RhoA. Moreover, the co-expression of both domains induces simultaneously the activation of both GTPases. To our knowledge, this is the first example of a member of the Rho-GEF family, that contains two functional exchange factor domains, with restricted and different specificity. We are currently investigating how these GEF domains are activated, by addressing the role of the PH domains in GTPases activation by Trio. We have shown that: 1) the PH1 of Trio is necessary for Rac activation by the GEFD1; 2) the PH1 of Trio targets the molecule to the cytoskeleton; 3) the GEFD1 domain of Trio binds, in a two-hybrid screen, the actin binding protein filamin. These data suggest that the PH1 targets Trio to the cytoskeleton close to Rac and its effectors, probably via interaction with the actin-binding protein filamin, consistent with a role of Trio in actin cytoskeleton remodeling.