We investigated the mechanism of phenotypic plasticity of hepatocytes in a three-dimensional organoid culture system, in which hepatocytic spheroids were embedded within a collagen gel matrix. Hepatocytes expressed several bile duct markers including cytokeratin (CK) 19 soon after culture and underwent branching morphogenesis within the matrix in the presence of insulin and epidermal growth factor. Cultured hepatocytes did not express Delta-like, a specific marker for oval cells and hepatoblasts. Furthermore, hepatocytes isolated from c-kit mutant rats (Ws/Ws), which are defective in proliferation of oval cells, showed essentially the same phenotypic changes as those isolated from control rats. The bile duct-like differentiation of hepatocytes was associated with increased expression of Jagged1, Jagged2, Notch1, and several Notch target genes. CK19 expression and branching morphogenesis were inhibited by dexamethasone, a mitogen-activated protein kinase kinase 1 (MEK1) inhibitor (PD98059), and a phosphatidyl inositol 3-kinase inhibitor (LY294002). After being cultured for more than 3 weeks within the gels, hepatocytes transformed into ductular structures surrounded by basement membranes. Our results suggest that hepatocytes might have the potential to transdifferentiate into bile duct-like cells without acquiring a stem-like phenotype and that this is mediated through specific protein tyrosine phosphorylation pathways.

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A growing body of evidence indicates that the gap junction (GJ) plays a pivotal role in tumor suppression by exerting cell-cell communication. It has, however, been reported that expression of connexin26 (Cx26) protein is induced in human ductal carcinomas of the breast and that its amount increases in proportion to the grade of malignancy. We thus examined the effects of overexpressed Cx26 on growth characteristics in GJ-deficient human MCF-7 breast cancer cells that maintain the phenotype of earlystage cancers. MCF-7 cells were transfected with Cx26 cDNA, and several clones of stable transformants exhibiting a high level of cell-cell communication were established. When they were examined in terms of various growth characteristics in vitro, the proliferation rate and the saturation density were drastically reduced in Cx26-transfected clones compared with the mock-transfectant. The anchorage-independent growth capacity was also decreased by 50-75% after transfection of Cx26. Furthermore, the cell migration toward growth factors and cell invasion into Matrigel in a Boyden chamber were suppressed to 5-10% and 20-60%, respectively, of the control in Cx26-transfected clones. When implanted into the mammary fat pads of nude mice in the presence of an excess of 17β β β β-estradiol, Cx26-transfected clones tended to show slower tumor growth than the mock-transfectant, although the difference was not statistically significant. Our results strongly suggest that the induction of Cx26 protein observed in human breast cancers, reported previously, may not be very relevant to the development of breast cancers, and that Cx26 can function as a tumor suppressor in breast cancer cells. (Cancer Sci 2003; 94: 501-507) omeostasis in cellular society is an important factor for maintenance of tissue function, and its disorder often results in dysfunction of organs and development of diseases including cancers. Among cellular apparatuses that contribute to tissue homeostasis, the gap junction (GJ) mediates gap junctional intercellular communication (GJIC) and is unique in that tiny water-soluble molecules (M r <1000), such as inorganic ions, small metabolites and some second messengers, can travel directly between two adjacent cytoplasms through the junction.1) A gap junctional channel consists of two membrane-integrated hemichannels provided by each of two adjacent cells, and each hemichannel comprises a hexameric complex of connexin protein. The connexin multigene family is composed of at least 20 members in mammals. 2)Many studies have so far proved that down-regulation of the GJ is involved in carcinogenic pathways and that connexin proteins can function as tumor-suppressors. 3,4) In almost all tumors, the function of the GJ is down-regulated through one or more of a variety of mechanisms, including no or reduced expression, aberrant localization, and aberrant phosphorylation or dephosphorylation of connexin protein. Moreover, enforced expression of connexin protein very often abolishes the growth capacity of transformed cells su...

Growing evidence revealed that liver sinusoidal endothelial cells (SEC) play several important roles in physiology and pathology of the liver. It has been well understood that their structural characteristics, such as the membrane sieve and lack of basement membrane, facilitate direct contact of soluble and insoluble serum substances with hepatic parenchymal cells, resulting in enhancement of hepatic metabolic activity. In addition, SEC is now regarded as a member of the scavenger endothelial cells, which have potential to eliminate a variety of macromolecules from the blood circulation by receptor-mediated endocytosis. It is reported that molecules preferentially eliminated by SEC are denatured or modified proteins such as advanced glycation end products, extracellular matrix components including hyaluronic acid, and some lipoproteins. The nature of the scavenger receptors corresponding to these molecules remains to be clarified. Recently, it was noted that SEC has an antigen-presenting function similar to dendritic cells. Taken together, it is suggested that SEC, cooperating with Kupffer cells and hepatic dendritic cells, may partake of immunoregulatory functions in the liver. SEC also plays a pivotal role in the pathological process of ischemia-reperfusion injury following liver surgery and liver transplantation. Thus, it is of importance to elucidate the mechanisms of apoptosis and proliferation of SEC. Recent results on the regulation of growth and apoptotic signaling of SEC are discussed.

To elucidate the mechanism of apoptosis of liver sinusoidal endothelial cells (SECs), we examined the phosphorylation status of Bad and its upstream signaling molecules during apoptosis in culture and after ischemia-reperfusion injury. Rat SECs were isolated by the immunomagnetic method, and 2 days after culture, most SECs underwent apoptosis, which was associated with decreased tyrosine phosphorylation of cellular proteins. Addition of orthovanadate (OV), a protein tyrosine phosphatase inhibitor, sustained cellular protein phosphorylation and strongly inhibited apoptosis. Bad was dephosphorylated at Ser-112 and Ser-136 during apoptosis, but the phosphorylation status of Bad was maintained in the presence of OV. OV activated the Akt, extracellular signalregulated protein kinase, and p38 mitogen-activated protein kinase pathways, which are involved in Bad phosphorylation. In the absence of OV, depletion of Bad by RNA interference conferred resistance to apoptosis. Hepatic injury after ischemia-reperfusion was alleviated by OV treatment, with significant inhibition of SEC apoptosis. SEC apoptosis in vivo was associated with dephosphorylation of Bad, Akt, and extracellular signal-regulated protein kinase, which was blocked by OV treatment. Our data suggest that maintenance of Bad phosphorylation is important in the prevention of SEC apoptosis and that the anti-apoptotic property of OV might have therapeutic utility.

Metastasis from renal cell carcinoma (RCC) to the testis is rare. This case report presented an extremely rare case of simultaneous bilateral testicular metastases from RCC in a 65-year-old man who had experienced indolent scrotal enlargement over a period of several months. Scrotal ultrasonography showed 4.0- and 2.0-cm-sized masses in the left and right testes, respectively. Contrast-enhanced computed tomography identified multiple tumors in the kidneys, the pancreas and the left adrenal gland. Left orchiectomy and pathological examination were performed and indicated testicular metastasis from clear cell RCC. The patient underwent complete surgical resection of all residual lesions. Postoperative follow-up examination without adjuvant therapy identified no recurrence over 11 months. This study also reviewed existing literature and determined that retrograde venous spread from the primary kidney tumor to the testis may be an important pathway for testicular metastasis from RCC. In conclusion, RCC can result in testicular metastases not only unilaterally, but also bilaterally, as was observed in the present case.

Embryonic development of the liver is closely associated with vascular organization. However, little is known about the mechanisms of vascular differentiation during liver development. Our previous study showed that the maturation of sinusoidal endothelial cells (SECs) occurred during embryonic day 13.0 (E13.0) to E15.0. To improve our understanding of SEC differentiation, we examined here the expression of maturation markers, SE-1 and stabilin-2, in fetal livers and also attempted to establish an in vitro SEC differentiation system by culturing E13.5 fetal liver cells. Immunohistochemical examination of SE-1 and stabilin-2 expression during fetal rat liver development revealed that these differentiation markers were co-expressed in SECs in the late stage of liver development, although stabilin-2 was expressed in almost all vascular endothelial cells in the early stage. Liver cells from the E13.5 rat fetus were cultured in EBM-2 medium containing vascular endothelial growth factor (VEGF), transforming growth factor beta1 (TGF-beta1) and VEGF plus SB-431542 (an inhibitor of the TGF-beta1 receptor, activin receptor-like kinase 5 [ALK-5]). In vitro SEC differentiation, as indicated by the appearance of cells co-expressing SE-1 and stabilin-2 and of cells with cytoplasmic fenestrae in endothelial sheets, was induced by the addition of both VEGF and SB-431542, an inhibitor of the phosphorylation of Smad2/3 but not that of Smad1/5/8 in the cultured cells. These results indicate for the first time that both VEGF signaling and the blocking of the ALK-5-Smad2/3 signal pathway are important for the fetal differentiation of SECs.

There have only been a few reports on lansoprazoleassociated collagenous colitis. Colonic mucosa of collagenous colitis is known to be endoscopically normal. We present a case of collagenous colitis where the mucosa showed diffuse cloudiness mimicking ulcerative colitis. A 70-year-old woman developed watery diarrhea four to nine times a day. She had interstitial pneumonia at 67 and reflux esophagitis at 70 years. Lansoprazole 30 mg/d had been prescribed for reflux esophagitis for nearly 6 mo. Lansoprazole was withdrawn due to its possible side effect of diarrhea. Colonoscopy disclosed diffuse cloudiness of the mucosa which suggested ulcerative colitis. Consequently sulfasalazine 2 g/d was started. The patient's diarrhea dramatically disappeared on the following day. However, biopsy specimens showed subepithelial collagenous thickening and infiltration of inflammatory cells in the lamina propria, confirming the diagnosis of collagenous colitis. One month after sulfasalazine therapy was initiated, colonoscopic and histological abnormalities resolved completely. Five months later the diarrhea recurred. The findings on colonoscopy and histology were the same as before, confirming a diagnosis of collagenous colitis relapse. We found that the patient had begun to take lansoprazole again 3 mo ahead of the recent diarrhea. Withdrawal of lansoprazole promptly resolved the diarrhea. Endoscopic and histological abnormalities were also completely resolved, similar to the first episode. Retrospectively, the date of commencement of sulfasalazine and discontinuation of lansoprazole in the first episode was found to be the same. We conclude that this patient had lansoprazole-associated collagenous colitis.