1. PATHOGEN È SE DES L É SIONS H É PATIQUES DU VHC : MOD É LISATION ANIMALE Hervé LERAT INSERM U955 Département de Virologie Moléculaire et Immunologie Physiopathologie et Thérapeutique des Hépatites Virales Chroniques (Pr. Pawlotsky) Hôpital Henri Mondor, Créteil, France.
7. The full-length HCV ORF transgenic mouse model FL-N/35 Lerat et al., Gastroenterology 2002 RNA HCV + X C57BL6J Liver micro-injections C57Bl6J x C3H/HeJ X C57BL6J DNA HCV + FL-N E1 E2 core NS4a NS3 NS4b NS5a NS5b NS2 p7 Albumin promoter SV40 PolyA Genotype 1b FL-N/35
8. The full-length HCV ORF transgenic mouse model FL-N/35 Lerat et al., Gastroenterology 2002 RNA HCV + X C57BL6J Liver micro-injections C57Bl6J x C3H/HeJ X C57BL6J DNA HCV + FL-N E1 E2 core NS4a NS3 NS4b NS5a NS5b NS2 p7 Albumin promoter SV40 PolyA Genotype 1b Steatosis Carcinoma Co-fibrogenic +CCl4
9. Hepatocellular Carcinomas A Lerat et al., Gastroenterology 2002. 1 cm 1 cm FL-N/35-170 UNC FL-N/35-171 UNC 1 cm FL-N/35-102 CERFE
10. Visualisation en IRM des CHC chez la souris VHC (H. Lerat, A. Luciani et T. Décaens, données personnelles.) Tumeur
11. Hépatocarcinomes viro-induits : rôle direct ou indirect des protéines virales? Infection VHC Réponse immunitaire de l’hôte Protéines virales Lésions hépatiques Cirrhose CANCER ?
12. Etapes essentielles de la carcinogenèse Negrini et al ., 2010. Nature Mol Cell Biol Rev: 11; 220-28
13. Etapes essentielles de la carcinogenèse Negrini et al ., 2010. Nature Mol Cell Biol Rev: 11; 220-28
14. Inhibition de la voie apoptotique FAS dans le foie des souris VHC APOPTOSE caspase 3 caspase 9 cytochrome C caspase 8 Fas FasL Bid Membrane cellulaire mitochondrie VHC (NS2) CIDE-B fragmentation ADN VHC (NS5A) calpaïne Etape vers le cancer
15. (M. Higgs, H. Lerat, données personnelles) Surcharge en FER CHC ROS Production de ROS dans les souris VHC, implication sur la carcinogenèse hépatique. DNA damage inflammation fibrosis NS3-NS5A Nox4 Electron transport Complex (1) CORE Glutathion
16. GADD45 GADD45 GADD45 GADD153 GADD34 Senseurs de stress Arrêt du cycle cellulaire (G2/M) Lésions de l’ADN – La famille GADD Les protéines du VHC induisent la méthylation du promoteur de GADD45 β: inhibition de sa transcription : évasion du point de contrôle. Q M G 1 G 2 S G 0 CyclinB1 Cdc2 GADD45 β Lésions de l’ADN P P P Histone H3 Histone H1 Lamine VHC
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18. Vers un mécanisme de la carcinogenèse hépatique viro-induite? Chronicité infection CANCER du FOIE Expression VHC Inhibition réponse immune Multiplication cellulaire Surexpression des cyclines et oncogènes Inhibition apoptose Diminution de protéines pro apoptotiques Lésions ADN production ROS Réparation ADN
19. Stéatose induite par le VHC: Mécanismes Moléculaires? Rôle direct des protéines virales?
24. Fibrose induite par le VHC? Mécanismes Moléculaires? Rôle direct des protéines virales?
25. in silico Analysis Non-Tg + CCl4 Tg + CCl4 Picrosirius Red Staining Quantification of fibre deposition 0,0 0,5 1,0 1,5 2,0 2,5 3,0 Tg Non-Tg + CCl4 - CCl4 Tg Non-Tg P=0.0036* Area of fibers (%) n 5 5 13 17 * = Mann Whitney test
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Editor's Notes
The work I will present today has been focussed on one particular protein, Gadd45b, one of a number of related proteins involved in the response to cellular stress. Upon DNA damage, and various other types of cellular stress, expression of these proteins is augmented. CLICK In the case of the Gadd 45 proteins, these can subsequently induce cell cycle arrest CLICK And these proteins are also invovled directly in DNA repair CLICK and have also been implicated in tumour suppression and epigenetic control of gene expression
In order to confirm this observation , the amount of fibers was objectively quantified in each animal from this study. Each lobe of each mouse was analyzed and approximately 800 images were generated from 40 animals included in 3 independent experiments This slide shows two examples of liver sections from a nontransgenic and a transgenic animal, both treated with CCl4, with, below, the image generated in silico for quantification of fiber deposition. The histogram on the right summarizes the results of this analysis. As you can see, there was no difference between non-transgenic and transgenic animals in the absence of CCl4 treatment. In contrast, in CCL4-treated animals, fibrosis was significantly more abundant in the liver of transgenic than non-transgenic animals, objectively confirming the histological observation.