Multiple Adaptive Mechanisms to Chronic Liver Disease Revealed at Early Stages of Liver Carcinogenesis in the Mdr2-Knockout Mice Mark Katzenellenbogen, 1 Orit Pappo, 2 Hila Barash, 1 Naama Klopstock, 1 Lina Mizrahi, 1 Devorah Olam, 1 Jasmine Jacob-Hirsch, 4 Ninette Amariglio, 4 Gidi Rechavi, 4 Leslie Ann Mitchell, 1 Ron Kohen, 3 Eytan Domany, 5 Eithan Galun, 1 and Daniel Goldenberg 1 1 Goldyne Savad Institute of Gene Therapy and 2 Department of Pathology, Hadassah University Hospital; 3 Department of Pharmacology, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel; 4 Pediatric Hemato-Oncology Department and Institute of Hematology, Chaim Sheba Medical Center, Tel-Hashomer, Tel-Aviv University, Tel-Aviv, Israel; and 5 Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, Israel Abstract Molecular events preceding the development of hepatocellular carcinoma were studied in the Mdr2-knockout (Mdr2-KO) mice. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane. Portal inflammation ensues at an early age followed by hepatocellular carcinoma development after the age of 1 year. Liver tissue samples of Mdr2-KO mice in the early and late precancerous stages of liver disease were subjected to histologic, biochemical, and gene expression profiling analysis. In an early stage, multiple protective mechanisms were found, including induction of many anti- inflammatory and antioxidant genes and increase of total antioxidant capacity of liver tissue. Despite stimulation of hepatocyte DNA replication, their mitotic activity was blocked at this stage. In the late stage of the disease, although the total antioxidant capacity of liver tissue of Mdr2-KO mice was normal, and inflammation was less prominent, many protec- tive genes remained overexpressed. Increased mitotic activity of hepatocytes resulted in multiple dysplastic nodules, some of them being steatotic. Expression of many genes regulating lipid and phospholipid metabolism was distorted, including up-regulation of choline kinase A, a known oncogene. Many other oncogenes, including cyclin D1, Jun, and some Ras homologues, were up-regulated in Mdr2-KO mice at both stages of liver disease. However, we found no increase of Ras activation. Our data suggest that some of the adaptive mechanisms induced in the early stages of hepatic disease, which protect the liver from injury, could have an effect in hepatocarcinogenesis at later stages of the disease in this hepatocellularcarcinomamodel. (Cancer Res 2006; 66(8): 4001-10) Introduction Hepatocarcinogenesis has many features that are common in mice and humans. Various mouse models of hepatocellular carcinoma, including knockout and transgenic animals, have been used to study the molecular mechanisms of liver cancer. Compar- ative analysis of global gene expression patterns of different mouse hepatocellular carcinoma models and human hepatocellular carcinoma samples showed that most mouse hepatocellular carcinomas are similar to specific subgroups of human hepatocel- lular carcinomas and thus may be useful in modeling studies (1). To study the molecular mechanisms of hepatocellular carcinoma development and to find candidate genes for gene therapy intervention in the early stages of the disease, we did intensive analysis of liver tissues of the Mdr2-knockout (Mdr2-KO) mice in the precancerous stages of liver disease. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane (2). The absence of phospholipids from bile leads to bile regurgitation into the portal tracts (3), causing portal inflammation that ensues at an early age followed by slowly developing hepatocellular carcinoma, which, in this regard, mimics the human disease (4). In the present study, we investigated different signaling pathways associated with precancerous stages of liver disease in the Mdr2- KO model. Liver tissue samples of Mdr2-KO and control Mdr2- heterozygous mice at ages 3 and 12 months were subjected to histologic, biochemical, and gene expression profiling analysis. Results revealed complex interactions between signaling pathways that control inflammatory reactions, response to oxidative stress, lipid metabolism, and cell proliferation. These findings suggest that induction of multiple adaptive mechanisms protecting liver tissue in the early stages of the disease as well as progressively disturbed lipid and phospholipid metabolism may have an effect in hepatocellular carcinoma development in the later stages of liver disease in Mdr2-KO mice. Materials and Methods Mice. Founders of the FVB.129P2-Abcb4 tm1Bor (Mdr2-KO; old name FVB.129P2-Pgy2 tm1Bor ) and the wild-type FVB/NJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Colonies of both strains were maintained under specific pathogen-free conditions in the Animal Facility of The Hebrew University Medical School. The F 1 hybrids produced by breeding of a FVB.129P2-Abcb4 tm1Bor male and a FVB/NJ female were used as age-matched controls. Harvesting of mouse liver tissue. Nonfasting male Mdr2-KO or control heterozygous mice were anesthetized with isoflurane and sacrificed by cervical dislocation. Livers were rapidly excised and weighed; part of the left lobe was fixed in 4% buffered formaldehyde for histologic analysis, and the remaining liver tissue was quickly frozen in liquid nitrogen and stored at Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). E. Galun is the Sam and Ellie Fishman Chair in Gene Therapy. E. Domany is the incumbent of the Henry J. Leir Professorial Chair. Requests for reprints: Daniel Goldenberg, Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, P.O. Box 12000, Jerusalem 91120, Israel. Phone: 972-2-677-8108; Fax: 972-2-643-0982; E-mail: goldenberg@hadassah.org.il. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-2937 www.aacrjournals.org 4001 Cancer Res 2006; 66: (8). April 15, 2006 Research Article Research. on January 25, 2016. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from