Eric R. Lemmer

Dual functions of E2F-1 in a transgenic mouse model of liver carcinogenesis

Deregulation of E2F transcriptional control has been implicated in oncogenic transformation. Consistent with this idea, we recently demonstrated that during hepatocarcinogenesis in c-myc/TGFα double transgenic mice, there is increased expression of E2F-1 and E2F-2, as well as induction of putative E2F target genes. Therefore, we generated transgenic mice expressing E2F-1 under the control of the albumin enhancer/promoter to test the hypothesis that E2F family members may contribute to liver tumor development. Overexpression of E2F-1 resulted in mild but persistent increases in cell proliferation and death during postnatal liver growth, and no increases in hepatic regenerative growth in response to partial hepatectomy. Nevertheless, from 2 months postnatally E2F-1 transgenic mice exhibited prominent hepatic histological abnormalities including preneoplastic foci adjacent to portal tracts and pericentral large cell dysplasia. From 6 to 8 months onward, there was an abrupt increase in the number of neoplastic nodules (‘adenomas’) with 100% incidence by 10 months. Some adenomas showed evidence of malignant transformation, and two of six mice killed at 12 months showed trabecular hepatocellular carcinoma. Endogenous c-myc was up-regulated in the early stages of E2F-1 hepatocarcinogenesis, whereas p53 was overexpressed in the tumors, suggesting that both E2F-1-mediated proliferation and apoptosis are operative but at different stages of hepatocarcinogenesis. In conclusion, E2F-1 overexpression in the liver causes dysplasia and tumors and suggests a cooperation between E2F-1 and c-myc oncogenes during liver oncogenesis.

Intact signaling by transforming growth factor β is not required for termination of liver regeneration in mice

Transforming growth factor β (TGF‐β) is a potent inhibitor of hepatocyte proliferation in vitro and is suggested to be a key negative regulator of liver growth. To directly address the role of TGF‐β signaling in liver regeneration in vivo, the TGF‐β type II receptor gene (Tgfbr2) was selectively deleted in hepatocytes by crossing “floxed” Tgfbr2 conditional knockout mice with transgenic mice expressing Cre under control of the albumin promoter. Hepatocytes isolated from liver‐specific Tgfbr2 knockout (R2LivKO) mice were refractory to the growth inhibitory effects of TGF‐β1. The peak of DNA synthesis after 70% partial hepatectomy occurred earlier (36 vs. 48 hours) and was 1.7‐fold higher in R2LivKO mice compared with controls. Accelerated S‐phase entry by proliferating R2LivKO hepatocytes coincided with the hyperphosphorylation of Rb protein and the early upregulation of cyclin D1 and cyclin E. However, by 120 hours after partial hepatectomy, hepatocyte proliferation was back to baseline in both control and R2LivKO liver. Regenerating R2LivKO liver showed evidence of increased signaling by activin A and persistent activity of the Smad pathway. Blockage of activin A signaling by the specific inhibitor follistatin resulted in increased hepatocyte proliferation at 120 hours, particularly in R2LivKO livers. In conclusion, TGF‐β regulates G1 to S phase transition of hepatocytes, but intact signaling by TGF‐β is not required for termination of liver regeneration. Increased signaling by activin A may compensate to regulate liver regeneration when signaling through the TGF‐β pathway is abolished, and may be a principal factor in the termination of liver regeneration. (HEPATOLOGY 2004.)

Isolation from human fetal liver of cells co-expressing CD34 haematopoietic stem cell and CAM 5.2 pancytokeratin markers

BACKGROUND/AIMS Ductal plate and bile duct cells in developing human liver express haematopoietic stem cell markers, such as c-kit and CD34, in association with cytokeratin markers CAM 5.2 and CK 18. The identification of such ductal plate cells as likely progenitors for both bile duct epithelial cells and hepatocytes and their possible reappearance as oval cells in the regenerating liver have generated much interest in their pluripotential capacities. This study aimed to isolate cells from human fetal liver that co-express haematopoietic stem cell and epithelial cell markers. METHODS Human fetal liver was harvested following legal termination of pregnancy at week 14-22. CD34+ mononuclear cells were isolated from liver cell suspensions with immunomagnetic beads. Immunofluorescent staining, using anticytokeratin CAM 5.2 against CK 8 and 18, was performed on permeabilised CD34+ cells for flow cytometry and fluorescent microscopy. CD34+ cells were also stained for other stem cell markers (HLA-DR, c-kit) and committed haematopoietic cell markers (CD33, CD38). RESULTS Approximately 0.9% (range 0.07-4.0%) of the mononuclear cells isolated were CD34+ cells. The number of mononuclear cells isolated correlated with fetal liver weight (r=0.508). About 3-8% of these CD34+ cells stained positively for CAM 5.2. In addition, CD34+ cells were positive for HLA-DR, but only a small percentage was positive for c-kit. Staining for the committed haematological markers, CD33 and CD38, was consistently negative. CONCLUSIONS This study describes an immunoaffinity method for the enrichment from human fetal liver of cells that co-express haematopoietic stem cell and epithelial cell markers. Such cellular subsets may correspond to pluripotential ductal plate and bile duct cells.

Hydatid Cyst in the Head of the Pancreas with Obstructive Jaundice

We report a rare case of obstructive jaundice caused by an intrapancreatic hydatid cyst in a 17-year-old black girl. Ultrasonography and computed tomography demonstrated the obstructing cyst in the head of the pancreas. Cyst aspiration produced clear fluid with a low amylase content and no hydatid hooklets or protoscolices. Pancreaticoduodenectomy was performed for a presumed cystic neoplasm of the pancreas, but histology showed the true diagnosis. Pancreatic hydatidosis should be considered in the differential diagnosis of obstructing pancreatic cysts in the appropriate epidemiological setting.

Prolonged Survival following Portoenterostomy for Biliary Atresia

We report a case of prolonged survival following hepatic portoenterostomy for biliary atresia, which was performed 3 years before Kasai described the operation. To our knowledge he represents the olde