Accumulation of neoplastic traits prior to spontaneous in vitro transformation of rat cholangiocytes determines susceptibility to activated ErbB-2/Neu.

Cholangiocarcinoma, a severe form of biliary cancer, has a high mortality rate resulting partially from the advanced stage of disease at earliest diagnosis. A better understanding of the progressive molecular and cellular changes occurring during spontaneous cholangiocarcinogenesis is needed to identify potential biomarkers for diagnosis/prognosis or targets for novel therapeutics. Here, we show that with continued passage (p) in vitro, rat bile duct epithelial cells (BDEC) accumulated neoplastic characteristics that by mid-passage (p31-85) included alterations in morphology, increased growth rate, growth factor independence, decreased cell adhesion, loss of cholangiocyte markers expressed at low passage (p<30), and onset of aneuploidy. At high passage (p>85), BDEC cultures showed increasing numbers of cells expressing activated, tyrosine phosphorylated ErbB-2/Neu, a receptor tyrosine kinase previously reported to be at elevated levels in cholangiocarcinomas. Enrichment for high passage ErbB-2/Neu-positive cells yielded several anchorage-independent sub-lines with elevated levels of activated ErbB-2/Neu and increased expression of cyclooxygenase-2 (COX-2). When injected into immunodeficient beige/nude/xid mice, these sub-lines formed poorly differentiated cystic tumors strongly positive for rat cholangiocyte markers, a finding consistent with a previous report showing the susceptibility of high passage, non-tumorigenic BDEC to transformation by activated ErbB-2/Neu. Mid passage BDEC, in contrast, were resistant to the transforming activity of activated ErbB-2/Neu and remained anchorage dependent in vitro and non-tumorigenic in vivo following stable transfection. Based on these findings, we concluded that during progression to high passage, cultured BDEC undergo preneoplastic changes that enhance their susceptibility to transformation by ErbB-2/Neu. The ability to generate cells at different points in the process of spontaneous neoplastic transformation offers a valuable model system for identifying molecular features that determine whether over-expression of activated ErbB-2/Neu is necessary and sufficient to induce neoplastic conversion.

Monoclonal antibody to novel cell surface epitope on Hsc70 promotes morphogenesis of bile ducts in newborn rat liver.

We previously described a cell surface reactive monoclonal antibody, MAb OC.10, which recognizes an epitope shared by rat fetal liver ductal cells, hepatic progenitor cells, mature cholangiocytes, and hepatocellular carcinomas (HCC). Here, intrasplenic injection of MAb OC.10 into newborn rats was shown by immunofluorescence microscopy to strongly label intrahepatic bile ducts. Furthermore, the in situ labeling of intrahepatic cholangiocytes by injecting MAb OC.10 increased the number of intraportal and intralobular bile ducts with well-defined lumens when compared to IgM-injected control animals. The antigen for MAb OC.10 was identified by mass spectrometry as Hsc70, a constitutively expressed heat shock protein belonging to the HSP70 family. Immunoblot analysis demonstrated that MAb OC.10 reacted with recombinant bovine Hsc70 protein, with protein immunoprecipitated from rat bile duct epithelial (BDE) cell lysates with monoclonal anti-Hsc70 antibody, and with Hsc70-FLAG protein over-expressed in human 293T cells. In addition, Hsc70-specific small interfering RNA reduced the amount of OC.10 antigen expressed in nucleofected BDE cells. Consistent with the specificity of MAb OC.10 for Hsc70, heat shock did not induce OC.10 expression in BDE cells, a characteristic of Hsp70. Immunofluorescence with BDE cells further suggested that MAb OC.10 binds a novel cell surface epitope of Hsc70. This was in contrast to a commercially available monoclonal anti-Hsc70 antibody that showed strong cytosolic reactivity. These findings demonstrate that presentation of the OC.10 epitope differs between cytosolic and surface forms of Hsc70 and may suggest distinct differences in protein conformation or epitope availability determined in part by protein-protein or protein-lipid interactions. Phage display and pepscan analysis mapped the epitope for MAb OC.10 to the N-terminal 340-384 amino acids of the ATPase domain of rat Hsc70. These findings suggest that MAb OC.10 recognizes an epitope on rat Hsc70 when presented on the cell surface that promotes morphogenic maturation of bile ducts in newborn rat liver. Furthermore, since we have shown previously that the OC.10 antigen is expressed on HCC subpopulations with oval cell characteristics, our current results indicate that Hsc70 has the potential to be expressed on the surface of certain tumor cells.

Engraftment of syngeneic and allogeneic endothelial cells, hepatocytes and cholangiocytes into partially hepatectomized rats previously treated with mitomycin C.

BACKGROUND: Pretreatment with retrorsine crosslinks host hepatocyte DNA and prevents proliferation after partial hepatectomy (PH), allowing selective expansion of transplanted progenitors. Shortcomings are length of protocol and carcinogenicity of retrorsine. METHODS: This report describes a rapid liver repopulation protocol using mitomycin C (MMC) to block proliferation of rat hepatocytes in response to PH. One week post-MMC treatment, dipeptidyl peptidase IV negative host rats were given a PH followed by injection of late gestation, newborn, or adult total liver isolates from dipeptidyl peptidase IV positive rats. For allogeneic transplantation, host rats received injections of anti-CD3 antibody before and after PH. RESULTS: Host liver staining 2 to 9 weeks posttransplantation revealed well-defined donor hepatocyte colonies with strong canalicular dipeptidyl peptidase IV activity. At the same cell dose, fetal and newborn isolates produced more colonies than adult liver isolates. Hepatocyte colonies also coexpressed marker proteins characteristic of adult hepatocytes and showed polarized localization of plasma membrane proteins. Host livers contained large clusters of sinusoids lined by dipeptidyl peptidase IV positive endothelial cells coexpressing the endothelial cell marker, RECA-1, but lacked the canalicular marker leucine aminopeptidase. Colonies containing donor hepatocytes, endothelial cells, and bile ducts were also observed. Similar levels of engraftment and expansion were achieved with allogeneic liver cell isolates by using anti-CD3 antibody treatment. CONCLUSIONS: The MMC transplantation model provides a rapid method for engraftment and expansion of hepatocytes, endothelial cells, and cholangiocytes and should be applicable to investigations centering on the role of endothelial cells in liver regeneration and the identification and characterization of putative endothelial, hepatocyte, and cholangiocyte progenitors.