SV40 transgenic mice developing hepatocellular carcinoma

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126

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INCREASED LUNG METASTASIS INCIDENCE IN DOUBLE nm23-NULL/SV40 TRANSGENIC MICE DEVELOPING HEPATOCELLULAR C A R C I N O M A

M. Boissan I , D. Wendurn2, S. Anmud-Dabernat3, A. Munier 1, I. Lascu 4, J.Y. Daniel a, M.L. Lacombe 1. 2 Unitg INSERM 402, Facultg de

M£decine Saint-Antoine, Universitd Pierre et Marie Curie, Paris, France," 2Laboratoire d'Anatomie Pathologique, H@ital Saint-Antoine, AP-HP, Paris, France, 3Laboratoire de Biologic de la Diff~renciation et du D£veloypement, EA DRED 3674, Universiti de Bordeaux 2, Bordeaux, France, 4IBGC-CNRS, Universit~ de Bordeaux 2, Bordeaux, France Background and Ahns: nm23-H1 was proposed as a metastasis suppressor gene based on 1) an inverse correlation between its expression and the metastatic potential of several types of human tumors including hepatocellular carcinoma (HCC) and 2) a decrease in the metastatic potential of highly aggressive cell lines after its enforced expression. With tile aim to further analyze tile role ofnm23 in primary tumor development and metastatic dissemination, we studied hepatic tumoral progression using Wansgenic mice models invalidated for nm23-M 1 (nm23-M 1-/-) resuming all steps of tumor progression. Methods: Two mechanistically distinct models of hepatocarcinogenesis were used in nm23-Ml+/+ and nrn23-M1-/- mice: one was chemically induced by injection of diethylnitrosamine and file other was obtaining by breeding with ASV mice, which express tile SV40 T antigen in tile liver and spontaneously develop hepatocellular carcinoma. Tumor progression and expression of nm23 isoforms (M1 and M2) and of several markers (ERK1/2, c-myc, cyclins, Rho A, Rho C) were analyzed in primary tumor and metastases by microscopic examination, immunohistochemistty, Western blotting and RT-PCR. Finally, we investigated whether the nm23-M1 disruption iitfluenced tile formation of metastases at a distant organ using an experimental metastasis assay. Results: In the two models, the lack of nm23-M 1 had no effect on primary tumor formation. Only tile ASV model exhibited pulmonary metastases positive for tile Hep Par-1 hepatocyte marker. The nm23-M1 ~ mice presented a highly significant increase in metastatic incidence as compared to nm23-Ml+/+ mice (69% vs 37%; p < 0.01). In nm23-Ml+/+ mice, the Nm23-M 1 labeling was highly heterogeneous in primary tumors and was low or negative in hing metastases suggesting that the loss of nm23-M1 favors metastatic spread. Interestingly, HCC o f ASV/nrn23-M1-/- mice presented a lower cyclin A level than tumors o fASV/nm23-M 1+/+. Finally, a similar number of pulmonary nodules were observed in nrn23-M 1+/+ and n m 2 3 - M 1 - / - mice after intravenous injection of the metastatic B16F10 cell line showing that the lack of nm23 does not influence homing and growth of metastases in tile target orgarl. Conclusions. This work is tile first demonstration of tile metastatic suppressor role of nm23 in an in vivo model of carcinogenesis.

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C H E M O P R E V E N T I O N OF HEPATOCELLULAR CARCINOMA. A S S E S S M E N T OF THE EFFICACY OF PIOGLITAZONE AND LANREOTIDE IN A CARCINOGENIC RAT MODEL I. Borbath I , M Lebrun 1, J. Abarca I , P. Moulin 2, C. Sempoux 2, I. Leclercq t , 5~ Horsmans I . ~Laboratoire de Gastro-Entdrologie, Service

de Gastro-Entd~vlogie, Cliniques St-Lue, Bruxelles, Belgium," 2Service d'Anatomie Pathologique, Cliniques St-Luc, Bruxelles, Belgium Pioglitazone (PGZ) and lanreotide (LAN) have been shown to reduce proliferation of HCC cell lines. Their use as chemoprophylactic agents deserves consideration. Object|yes: to analyse, in a carcinogenic rat model, tile effect, of PGZ and LAN on the development of preneoplastic foci. Methods: Preneoplasia was induced in 30 Wistar rats by 2 IP injections of diethylnitrosamine, 2 weeks apart, followed by garage with 2-acetylaminofluorene, for either 3 (group A) or 6 weeks (group B). Rats from groups

A and B were immediately subdivided into 3 groups receiving either PGZ 0.01% mixed with food, LAN 3 mg/kg IM/2 weeks or nothing (CTL). Immunohistochemislry was performed on paraffin-embedded sections using rat glutathione S-transferase (GSTp), a marker of preneoplastic foci, Ki67 for proliferation assessernent and anti-cleaved caspase 3 (Casp3) for apoptosis assessment. GSTp(+) area was measured in all sections, expressed as the ratio of labelled area/total area of the section. GSTp was quantified by western blotting (WB) on liver homogenates. Proliferation and apoptosis were assessed by counting Ki67(+) and Casp3(+) nuclei in 1000 GSTp(+) and (-) cells. Results were expressed as % of CTL for WB and as % of labelled cells for immunohistochemistry. Results: In group A, 41.64-33.6% of liver section was GSTp(+) in CTL. PGZ significantly decreased the size of GSTp(+) area to 6.954-4.3%, but not LAN (19.7:t=9.7%, NS). These data were confirmed by WB. Ki67(+) cells were significantly fewer in GSTp(+) foci in PGZ compared to CTL (11.8±3.5% vs 25.5±1.8%, p=0.027), but not in LAN. Casp3(+) cells in GSTp(+) area was similar in all groups [0.53% (PGZ), 0.33% (LAN) and 0.53% (CTL), NS]. In group B, 65.24-13.5% of the section was GSTp(+) in CTL. PGZ and LAN significantly reduced GSTp(+) area to 40.6±12.7% and 43.5±16.4% respectively. This was confmned by WB only for PGZ (43.6% of CTL). The nmnber of Ki67(+) or Casp3(+) cells were not significantly different in PGZ, LAN and CTL. Conclusion: PGZ only, reduces tile size of preneoplastic foci in this carcinogenic model. The mechanism involves inhibition of proliferation rather than induction of apoptosis. Further studies are performed to assess this effect on tile development of HCC.

FACTOR-DEPENDENT HEPATOGENIC [•0] GROWTH DIFFERENTIATION OF HUMAN BONE MARROW DERIVED M E S E N C H Y M A L STEM CELLS I. Aurich I , L. Mitller2, K. Tisljar I , H. Aurich I , W.E. Fleig I , B. ChristI .

;First Department of Internal Medicine, Martin Luther University of Halle V~ttenberg, V~ttenberg, Germany," 2Fourth Department of Internal Medicine, Martin Luther University of Halle Wittenberg, Wittenberg, Germany The use of human hepatocytes for the cell therapy of liver diseases is hampered by the limited availabilifiy of donor organs for tile isolation of tile cells. The multipotent differentiation and proliferation capacity of mesenchymal stem cells might offer the perspective to generate functional hepatocytes from these cells. Therefore, it was the aim of the study to establish hepatocytes from human bone marrow-derived mesenchymal stem cells. Mononuclear cells from human adult bone marrow were purified by density gradient centrifugation and mesenchymal stem cells selected by plastic adherence. Cells (lid not express CD45, CD34 and CD 14 and displayed multipotent mesenchymal differentiation capacity thus representing an enriched stem cell fraction largely void of hematopoietic cells. Hepatogenic differentiation was achieved by appropriate culture conditions and cells subsequently characterised by immunohistochernistry, Western blot and PCR analyses. In the presence of growth factors HGF and EGF or FGF-4, respectively, cultured cells lost their mesenchymal character and gained epithelial features instead, as shown by changes in cell morphology (decrease in cytoplasm/nucleus ratio, polygonal shape) and the expression of the epithelial marker CK18. While hepatocyte-specific properties such as phosphoenolpyruvate carboxykinase, carbamylphosphate synthetase, transferrin, Hep Par 1 and cytochrome P450 type 1A1 were expressed both in the presence of EGF and FGF-4, hepatocyte progenitor cell markers cytokecatin 7, cytokecatin 19 and 0~-fetoprotoin were detectable only in cells treated with FGF-4. The differentiated hepatocyte marker cennexin 32 was expressed only in cells treated with EGE In these cells the expression of a red fluorescent transgene was driven by the hepatocyte-specific promoter of the phosphoenolpyruvate carboxyldnase gene indicating in part proper hepatocyte function. Hence, mesenchymal stern cells from human bone marrow may gain epithelial attributes. FGF-4