Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

<p>Abstract</p> <p>Background</p> <p>Neurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells.</p> <p>Methods</p> <p>Colon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF). DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting.</p> <p>Results</p> <p>Neurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK) and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC), whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K), TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR) transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells.</p> <p>Conclusions</p> <p>While acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116 cells. In these cells, neurotensin-induced activation of ERK and stimulation of DNA synthesis was PKC-dependent, whereas activation of the PI3K/Akt pathway was mediated by stimulation of metalloproteinases and subsequent transactivation of the EGFR. Thus, the data show that the signalling mechanisms mediating the effects of neurotensin involve multiple pathways and are cell-dependent.</p

Méthode alternative de détermination du tritium organiquement lié (TOL) dans les échantillons de l'environnement

National audienceDepuis plusieurs années la surveillance environnementale du tritium organiquement lié (TOL) auxabords des sites industriels nucléaires connait un regain d’intérêt. Le tritium est l’un desradionucléides les plus rejetés par l’industrie nucléaire, le calcul de dose associé nécessite doncd’être précis. Actuellement la méthode de référence pour la détermination du TOL à bas niveau dansl’environnement est basée sur l’utilisation d’un four tubulaire pour la combustion de l’échantillon.Bien que cette méthode soit très performante en termes de limite de détection, elle est constituéede nombreuses étapes comme la détermination du pourcentage d’hydrogène de l’échantillon ouencore le traitement de l’eau de combustion et nécessite l’achat d’un four spécifique.L’optimisation des coûts et du temps d’analyse fait l’objet d’une attention particulière dans unlaboratoire d’analyse. Dans ce contexte le laboratoire de surveillance de l’environnement du CEAMarcoule a mis au point une méthode alternative de détermination du TOL dans les échantillons del’environnement. Cette méthode basée sur la combustion d’un échantillon dans une chambre sousflux d’oxygène, a été mise au point rapidement au laboratoire et n’a nécessité aucun investissementsupplémentaire

Biokinetic models for rats exposed to repeated inhalation of uranium: implications for the monitoring of nuclear workers

For dose assessment following chronic or accidental inhalation of radioactive aerosols, the dosimetric models of the International Commission on Radiological Protection (ICRP) provide dose coefficients, retention and excretion functions. Unknown date or dates of intake is the major source of uncertainty in dose assessment during routine monitoring of nuclear workers. The two assumptions commonly made in dose assessment from an unknown time pattern of intake have been tested experimentally with a model of repeated inhalation by rats. The hypothetical intake derived from lung measurement was relatively reliable under the two hypotheses. The hypothetical intake derived from excreta measurement depended on the choice of hypothesis and on the real time pattern of intake

The effect of repeated inhalation on the distribution of uranium in rats

For the assessment of doses after inhalation of airborne uranium compounds by workers, the International Commission on Radiological Protection (ICRP) developed compartmental models that are used to calculate reference dose coefficients and retention and excretion functions. It is assumed that each acute intake has no effect on the biokinetics of later intakes. Consequently, retention and excretion after multiple or chronic exposure are predicted using the same models as after acute exposure. This assumption was tested here on rats exposed to repeated inhalation of uranium dioxide (UO 2 ). First, excretion and organ retention were determined after a single inhalation of UO 2 . The follow-up of incorporated activity was used to design a biokinetic model for uranium inhaled by rats. Second, the biokinetics of uranium were monitored in two experiments of repeated inhalations of uranium dioxide under different intake patterns. For these two experiments, the organs' retention and excretion after repeated UO 2 inhalation were predicted using the biokinetic model and compared to the experimental measurement. Under the two sets of experimental conditions considered, the prediction of the biokinetic model based on acute exposure data was consistent with the biokinetics observed after repeated UO 2 inhalations, with the possible exception of retention in the skeleton. Copyright© Taylor & Francis Group, LLC

Interprétation des données physico-chimiques et biocinétiques pour le calcul de dose : exemple d'un composé industriel UO2 appauvri fabriqué pour le combustible MOX

La Commission Internationale de Protection Radiologique (CIPR), dans ses nouvelles recommandations, propose l'utilisation de paramètres déterminés expérimentalement pour effectuer un calcul de dose efficace par unité d'incorporation (DPUI) spécifique pour chaque composé. Le but de cette étude est de définir une méthodologie (techniques d'analyse, test in vitro de dissolution et test in vivo sur rats) permettant d'accéder à ces données et de présenter les méthodes de calcul utilisées, comme le logiciel GIGAFIT pour l'interprétation des données de transfert sanguin, et le logiciel LUDEP pour le calcul de dose. Ce travail a permis de déterminer pour un composé UO2 (uranium appauvri), utilisé dans la fabrication du combustible MOX, les principaux paramètres physico-chimiques et biologiques qui entrent dans le calcul de dose : le Diamètre Aérodynamique Médian en Activité ou DAMA de 6,5 microns; la Surface Spécifique SS= 2,68 m2.g-1; et les fraction et taux de transfert sanguin respectivement fr, entre 2,5% et 44,4%, Sr, entre 0,09 et 1,7 j-1, et Ss, entre 6,4x10-4 et 1,5x10-3 j-1. Ces résultats intégrés dans LUDEP conduisent à une DPUI spécifique par inhalation pour ce composé UO2 égale à 2,84x10-6 Sv.Bq-1

Interprétation des données physico-chimiques et biocinétiques pour le calcul de dose : exemple d'un composé industriel UO

La Commission Internationale de Protection Radiologique (CIPR), dans ses nouvelles recommandations, propose l'utilisation de paramètres déterminés expérimentalement pour effectuer un calcul de dose efficace par unité d'incorporation (DPUI) spécifique pour chaque composé. Le but de cette étude est de définir une méthodologie (techniques d'analyse, test in vitro de dissolution et test in vivo sur rats) permettant d'accéder à ces données et de présenter les méthodes de calcul utilisées, comme le logiciel GIGAFIT pour l'interprétation des données de transfert sanguin, et le logiciel LUDEP pour le calcul de dose. 
Ce travail a permis de déterminer pour un composé UO2 (uranium appauvri), utilisé dans la fabrication du combustible MOX, les principaux paramètres physico-chimiques et biologiques qui entrent dans le calcul de dose : le Diamètre Aérodynamique Médian en Activité ou DAMA de 6,5 μm; la Surface Spécifique SS= 2,68 m2.g-1; et les fraction et taux de transfert sanguin respectivement fr, entre 2,5% et 44,4%, Sr, entre 0,09 et 1,7 j-1 , et Ss, entre 6,4 10-4 et 1,5 10-3 j-1. Ces résultats intégrés dans LUDEP conduisent à une DPUI spécifique par inhalation pour ce composé UO2 égale à 2,84 10-6 Sv.Bq-1

L'ICP-MS: Un outil pour le suivi des contaminations internes à l'uranium

Inductively coupled plasma mass spectrometry (ICP-MS) is considered as a very convenient technique for radionuclide measurement in the nuclear field owing to its high sensitivity and accuracy and to the possibility measuring a large range of masses. To study the different biokinetic behaviours of two different uranium oxides, insoluble UO2 and soluble UO4' in rats after successive inhalations, an analytical procedure to measure the quantity of uranium coming from each oxide was developed. The number of biological matrices studied was limited to three: two ways of the airborne uranium entry, i.e. the set lung/trachea and the gastrointestinal tract, and one way of excretion with urines. After repeated inhalations of UO2 followed by one acute inhalation of UO4' total uranium measurement in the different matrices was performed either with a kinetic phosphorescence analyzer (KPA), ICP-MS or with α- spectrometry. In addition, the amount of uranium coming from UO4' UO2 or natural uranium, respectively, was calculated by ICP-MS and α-spectrometry. All the results were compared and found to be similar. As a conclusion, the analytical procedure developed with ICP-MS was validated and ICP-MS was confirmed to be an efficient tool for the study of successive internal contaminations with uranium

L'ICP-MS: Un outil pour le suivi des contaminations internes à l'uranium

Inductively coupled plasma mass spectrometry (ICP-MS) is considered as a very convenient technique for radionuclide measurement in the nuclear field owing to its high sensitivity and accuracy and to the possibility measuring a large range of masses. To study the different biokinetic behaviours of two different uranium oxides, insoluble UO2 and soluble UO4' in rats after successive inhalations, an analytical procedure to measure the quantity of uranium coming from each oxide was developed. The number of biological matrices studied was limited to three: two ways of the airborne uranium entry, i.e. the set lung/trachea and the gastrointestinal tract, and one way of excretion with urines. After repeated inhalations of UO2 followed by one acute inhalation of UO4' total uranium measurement in the different matrices was performed either with a kinetic phosphorescence analyzer (KPA), ICP-MS or with α- spectrometry. In addition, the amount of uranium coming from UO4' UO2 or natural uranium, respectively, was calculated by ICP-MS and α-spectrometry. All the results were compared and found to be similar. As a conclusion, the analytical procedure developed with ICP-MS was validated and ICP-MS was confirmed to be an efficient tool for the study of successive internal contaminations with uranium

Genotoxic and inflammatory effects of depleted uranium particles inhaled by rats

Depleted uranium (DU) is a radioactive heavy metal coming from the nuclear industry and used in numerous military applications. Uranium inhalation can lead to the development of fibrosis and neoplasia in the lungs. As little is known concerning the molecular processes leading to these pathological effects, some of the events in terms of genotoxicity and inflammation were investigated in rats exposed to DU by inhalation. Our results show that exposure to DU by inhalation resulted in DNA strand breaks in broncho-alveolar lavage (BAL) cells and in increase of inflammatory cytokine expression and production of hydroperoxides in lung tissue suggesting that the DNA damage was in part a consequence of the inflammatory processes and oxidative stress. The effects seemed to be linked to the doses, were independent of the solubility of uranium compounds and correlating with the type of inhalation. Repeated inhalations seemed to induce an effect of potentiation in BAL cells and also in kidney cells. Comet assay in neutral conditions revealed that DNA damage in BAL cells was composed partly by double strands breaks suggesting that radiation could contribute to DU genotoxic effects in vivo. All these in vivo results contribute to a better understanding of the pathological effect of DU inhalation. © The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved