Makoto Satake

Hepatic Oval Cells Have the Side Population Phenotype Defined by Expression of ATP-Binding Cassette Transporter ABCG2/BCRP1

Organ-specific stem cells can be identified by the side population (SP) phenotype, which is defined by the property to effectively exclude the Hoechst 33342 dye. The ATP-binding cassette transporter ABCG2/BCRP1 mediates the SP phenotype. Because hepatic oval cells possess several characteristics of stem cells, we examined whether they have the SP phenotype using the 2-acetylaminofluorene/partial hepatectomy (PH) model. Fluorescence-activated cell sorting analysis showed that a population of non-parenchymal cells containing oval cells, prepared on day 7 after PH, carried a significant number of SP cells, whereas that of non-parenchymal cells without oval cells, prepared on day 0 after PH, did not. Northern blot analysis using total liver RNA obtained on various days after PH showed that the expression of ABCG2/BCRP1 mRNA increased after PH, reaching the highest level on day 7, and then gradually decreased. This pattern of changes in the ABCG2/BCRP1 mRNA level was well correlated to that in the number of oval cells. Furthermore, in situ hybridization revealed that oval cells were the sites of expression of ABCG2/BCRP1 mRNA. These results indicate that oval cells have the SP phenotype defined by expression of ABCG2/BCRP1, suggesting that oval cells may represent stem cells in the liver.

Oncostatin M Inhibits Proliferation of Rat Oval Cells, OC15-5, Inducing Differentiation into Hepatocytes

Oval cells of the liver participate in liver regeneration when hepatocytes are prevented from proliferating in response to liver damage. To clarify the role of oncostatin M (OSM) in the liver regeneration involving oval cells, we examined the expression of OSM and OSM-specific receptor (OSM-R) in the liver undergoing regeneration in the 2-acetylaminofluorene/partial hepatectomy model. Expression levels of OSM-R changed in correlation to the number of oval cells, and its expression was exclusively observed in oval cells. On the other hand, OSM was expressed in both oval cells and Kupffer cells. To examine the effect of OSM on the growth and differentiation of oval cells, rat oval cells (OC15-5) were incubated in conditioned medium of 293T cells expressing rat OSM cDNA. This resulted in suppression of growth, changes in morphology (microvilli and large cytoplasm with developed organelles), and expression of hepatocyte markers (albumin, tyrosine amino transferase, and tryptophan oxygenase). The effects of the conditioned medium with rat OSM were abrogated by introducing a small interfering RNA specifically targeting rat OSM-R into OC15-5 cells. These results indicate that OSM is a key mediator for inducing differentiation of OC15-5 cells into hepatocytes and suggest that the OSM/OSM-R system is pivotal in the differentiation of oval cells into hepatocytes, thereby promoting liver regeneration.

Comparison between mucinous cystic neoplasm and intraductal papillary mucinous neoplasm of the branch duct type of the pancreas with respect to expression of CD10 and cytokeratin 20.

Objective: Mucinous cystic neoplasm (MCN) and intraductal papillary mucinous neoplasm of the branch duct type (IPMN-BD) differ in biological and clinical behaviors, but MCN is often misdiagnosed as IPMN-BD. The purpose of this study was to find useful markers for the differential diagnosis of MCN and IPMN-BD. Methods: Immunohistochemically, the expression of the 2 types of mucin (MUC) 1 (MUC1/DF3 and MUC1/CORE), MUC2, MUC5AC, MUC6, human gastric mucin (HGM), caudal-related homeobox transcription factor 2 (CDX2), CD10, cytokeratin (CK) 7, and CK20 was examined in 7 cases of MCN and 16 cases of IPMN-BD. Results: Expression frequencies in MCN and IPMN-BD were 100% versus 44% for MUC1/DF3, 86% versus 31% for MUC1/CORE, 57% versus 19% for MUC2, 86% versus 100% for MUC5AC, 57% versus 88% for MUC6, 86% versus 100% for HGM, 57% versus 0% for CDX2, 71% versus 0% for CD10, 100% versus 69% for CK7, and 86% versus 6% for CK20. Conclusions: Mucin 1/DF3, MUC1/CORE, CDX2, CD10, and CK20 were expressed significantly more frequently in MCN than in IPMN-BD. In particular, CD10 and CK20 showed marked differences in immunohistochemical sensitivity and specificity between MCN and IPMN-BD. It is therefore proposed that CD10 and CK20 may be used for the differential diagnosis of MCN and IPMN-BD.

Role of c-kit receptor tyrosine kinase-mediated signal transduction in proliferation of bile epithelial cells in young rats after ligation of bile duct: a study using Ws/Ws c-kit mutant rats

BACKGROUND/AIMS Recently, it has been shown that the c-kit receptor tyrosine kinase (KIT) is expressed in the liver of young rats, and its expression is up-regulated in bile epithelial cells (BEC) after ligation of the common bile duct (BDL). To clarify a role of KIT in BEC, we examined whether BEC of Ws/Ws rats, whose KIT kinase activity was severely impaired, could proliferate in response to bile stasis after BDL. METHODS/RESULTS When 2-week-old control normal (+/+) and Ws/Ws mutant rats underwent BDL, only a few BEC were found in the portal field of livers of Ws/Ws rats, whereas many BEC were found in that of +/+ rats. Furthermore, Ki-67 immunostaining showed that the proliferative activity of BEC in 2-week-old Ws/Ws rats was much lower than that in +/+ rats of the same age. In contrast, when 6-week-old +/+ and Ws/Ws rats underwent BDL, BEC similarly proliferated in the livers of +/+ and Ws/Ws rats, and the proliferative activity of BEC was comparable. CONCLUSIONS The mechanism whereby BEC proliferate in response to bile stasis after BDL may be different between 2- and 6-week-old rats, and KIT mediated-signal transduction plays a crucial role in the proliferation of immature BEC in young rats.

A Case of Biliary Cystadenocarcinoma with Ovarian Like Stroma

症例は63歳の女性で，約8か月前より肝嚢胞を指摘され経過観察されていた．2009年6月，CA19-9が上昇したため精査目的にて入院となった．腹部超音波,CT，MRIにて肝左葉に嚢胞性病変を認め，嚢胞壁の一部に壁肥厚を認めた．FDG-PETを施行したところ嚢胞内部に異常集積像を認めた．腫瘍マーカーの上昇およびFDG-PETの異常集積像より胆管嚢胞腺癌と診断し，肝左葉切除術を施行した．切除標本では嚢胞の一部に壁肥厚を認め，嚢胞内部に血腫および粘液を認めた．病理組織学的検査では胆管嚢胞腺癌であり，卵巣様間質を伴っていた．術後合併症は認めず，術後12か月の現在，再発なく健在である．卵巣様間質を伴う胆管嚢胞腺癌は極めてまれな疾患であり，若干の文献的考察を加え報告する．

Fascin, an Actin Bundlling Protein, Can Be a Novel Marker of Human Hepatic Stellate Cells and May Regulate Functions of Hscs Through FAK-PI3K-AKT Pathway

Objective: Fascin is a component of actin bundles and may regulate various cellular events. Expression and function of fascin in human hepatic stellate cells (HSCs) has remained largely unknown. Methods: Expression of fascin in human liver tissues was studied by immunohistochemistry. To identify cells expressing fascin, double immunofluorostaining with vimentin, heat shock protein 47 (HSP47), neural cell adhesion molecule (NCAM), or fibulin-2 was performed under confocal microscopy. In culture experiments, expression of fascin and phosphorylation of focal adhesion kinase (FAK) and Akt in LX-2 cells, a cell line of human HSCs, was investigated by western blot. Transfection of specific siRNAs was used to reduce the expression of fascin in LX-2 cells. Proliferation and migration ability of LX-2 cells were assayed by CyQuant assay kit and Matrigel-coated culture insert system, respectively. Expression of mRNAs of MMP-2 and collagens was examined by quantitative RT-PCR. Results: Immunohistochemistry indicated that fascin was found along the sinusoids and overlapped with vimentin, HSP47, or NCAM, while it was absent in periportal myofibroblastic cells and did not co-localize with fibulin-2, a marker of portal myofibroblasts, in both non-fibrotic and fibrotic livers. In addition, fascin immunoreactivity was undetectable in septa of fibrotic human livers. Expression of fascin in LX-2 cells was confirmed by westrn blot. Two different specific siRNAs against fascin significantly reduced the number of viable LX-2 cells to 65% of control and downregulated the expression of mRNAs of types I and III collagens and MMP-2 to 62, 65, and 70% of control, respectively. This condition also lowered their migration activity to 46% of control and the level of phosphorylation of FAK and Akt. Conclusion: Fascin may be an excellent novel marker of human HSCs that distinguishes HSCs from periportal myofibroblasts. Fascin may regulate functions of human HSCs through FAK-phosphoinositide 3-kinase-Akt pathway.

S2026 Heat Shock Protein 47 Regulates Expression of Collagen V and TIMP2 in Pancreatic Cancer Cells

(Introduction) Heat Shock Protein 47 (HSP 47) is a collagen-specific (collagen type I-V) chaperone molecular residing in the endoplasmic reticulum (ER) and it has been thought to be a marker of fibroblasts. It was reported that HSP 47 was expressed in cancer cells as well as fibroblasts of pancreatic cancer tissues. However, its function in cancer cells has not been investigated. (Materials and Methods) In order to investigate the presence of HSP 47 and collagen type I-V in human pancreatic cancer tissues, immunohistochemistry was performed. Expression of HSP 47 in pancreatic cancer cell lines, KMP-4,-5 and -6cells, was examined by western blot. Colocalization of HSP 47 and collagens ( type I-V ) were investigated by double immunofluorostaining. To deplete the expression of HSP 47 in cells, two kinds of siRNAs were transfected. At 120 h after transfection, attached cell number were assayed by CyQuant assay kit. Cell invasion ability was assay with Matrigel coated culture insert system. Expression of MMP -2, -9, TIMP-1, and -2 and collagens were examined by QRT-PCR. (Results) In 3 of 4 pancreatic cancer tissues, cancer cells were HSP47 positive. In one of 4 tissues, HSP47 was negative. In one metastatic lymph node, cancer cells was HSP 47 negative. Among collagens, immunoreactivity for collagen V was found in pancreatic cancer cells. Immunofluorostaining in cells showed that HSP 47 colocalize with collagen type V. At 120 h after transfection of siRNA into KMP-6 cells, attached cell number was not affected by depletion of HSP47. Cell invasion assay showed that depletion of HSP47 reduce migration cells. Expression of MMP -2, -9 and TIMP-1 at the RNA levels were not affected by depletion of HSP 47, but that of collagen type V and TIMP-2 was reduced by 70% and 40% of control, respectively. (Conclusion) HSP47 colocalize with collagen type V and regulates gene expression of collagen V in pancreatic cancer cells. HSP47 depletion downregulated expression of TIMP2 of cancer cells, but not affect cell survaival. These data indicates that HSP47 may be a chaperone molecular for collagen type V and inhibit cell invasion by regulating expression of TIMP2.