Expression patterns of epiplakin1 in pancreas, pancreatic cancer and regenerating pancreas

Epiplakin1 (Eppk1) is a plakin family gene with its function remains largely unknown, although the plakin genes are known to function in interconnecting cytoskeletal filaments and anchoring them at plasma membrane-associated adhesive junction. Here we analyzed the expression patterns of Eppk1 in the developing and adult pancreas in the mice. In the embryonic pancreas, Eppk1+/Pdx1+ and Eppk1+/Sox9+ pancreatic progenitor cells were observed in early pancreatic epithelium. Since Pdx1 expression overlapped with that of Sox9 at this stage, these multipotent progenitor cells are Eppk1+/Pdx1+/Sox9+ cells. Then Eppk1 expression becomes confined to Ngn3+ or Sox9+ endocrine progenitor cells, and p48+ exocrine progenitor cells, and then restricted to the duct cells and a cells at birth. In the adult pancreas, Eppk1 is expressed in centroacinar cells (CACs) and in duct cells. Eppk1 is observed in pancreatic intraepithelial neoplasia (PanIN), previously identified as pancreatic ductal adenocarcinoma (PDAC) precursor lesions. In addition, the expansion of Eppk1-positive cells occurs in a caerulein-induced acute pancreatitis, an acinar cell regeneration model. Furthermore, in the partial pancreatectomy (Px) regeneration model using mice, Eppk1 is expressed in "ducts in foci", a tubular structure transiently induced. These results suggest that Eppk1 serves as a useful marker for detecting pancreatic progenitor cells in developing and regenerating pancreas.     

Nestin、CK19及insulin等在胚胎胰腺发育中的表达

目的:研究Nestin、CK19、胰岛素、胰高血糖素及生长抑素在胚胎胰腺发育中的表达,探讨胰岛细胞分化发育的机制。方法:采用免疫组化SABC法及免疫组化双染法(LAB SA),对30例6～14wk人胚胎胰腺中,Nestin、CK19、胰岛素,胰高血糖素及生长抑素阳性的细胞进行定位。结果:(1)胚胎胰腺发育中Nestin阳性细胞存在于胰腺的间质,数量极少;CK19在胰腺导管上皮分化中持续表达,呈强阳性;(2)7wk胰腺导管上皮开始分化出胰岛细胞,胰岛素、胰高血糖素及生长抑素表达阳性,三者的表达并无时段差异性;随着胎龄的增加,阳性细胞数增加,14wk时胰岛逐渐形成,表达达到高峰。结论:胚胎胰腺的发育中,胰腺间质存在胰腺干细胞;胚胎早期胰腺导管上皮细胞开始分化并分泌胰岛素,其自分泌的激素可能参与调节胰岛细胞的迁移和胰岛的形成。     

Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by the E6E7 gene of human papilloma virus 16.

Pancreatic cancer is one of the most lethal cancers in humans. The majority of these cancers arise from the pancreatic duct epithelium. Research into the pathogenesis of pancreatic carcinoma has largely relied on animal models. In vitro models of pancreatic carcinogenesis using propagable cultured epithelial cells derived from the pancreatic ducts of rats and hamsters have been described. A human model, however, has been nonexistent due to the unavailability of propagable cultured duct epithelial cells derived from normal human pancreas. We report here a reproducible method for the long-term culture of pancreatic duct epithelial cells derived from normal and benign adult human pancreata by infection with a retrovirus containing the E6 and E7 genes of the human papilloma virus 16. One of these cell lines has become immortal and has propagated continuously for more than 20 passages. They remain anchorage dependent in their growth and nontumorigenic in nude mice. These cell lines and the methodology described here to establish them may provide new avenues for in vitro studies of the roles played by duct epithelium in human pancreatic diseases and cancers.     

Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells

The emergence of cells with hepatocellular properties in the adult pancreas has been described in several experimental models. To determine whether adult pancreas contains cells that can give rise to therapeutically useful and biochemically normal hepatocytes, we transplanted suspensions of wild-type mouse pancreatic cells into syngeneic recipients deficient in fumarylacetoacetate hydrolase and manifesting tyrosinemia. Four of 34 (12%) mutant mice analyzed were fully rescued by donor-derived cells and had normal liver function. Ten additional mice (29%) showed histological evidence of donor-derived hepatocytes in the liver. Previous work has suggested that pancreatic liver precursors reside within or close to pancreatic ducts. We therefore performed additional transplantations using either primary cell suspensions enriched for ducts or cultured ducts. Forty-four mutant mice were transplanted with cells enriched for pancreatic duct cells, but only three of the 34 (9%) recipients analyzed displayed donor-derived hepatocytes. In addition, 28 of the fumarylacetoacetate hydrolase-deficient mice were transplanted with cultured pancreatic duct cells, but no donor-derived hepatocytes were observed. Our results demonstrate for the first time that adult mouse pancreas contains hepatocyte progenitor cells capable of significant therapeutic liver reconstitution. However, contrary to previous reports, we were unable to detect these cells within the duct compartment.     

Intraperitoneal transplantation of pancreatic anlagen

To determine whether embryonic pancreatic anlagen transplanted to an intraperitoneal site in adult hosts grow, differentiate, and function, we implanted pancreas from embryonic day (E) 12.5 Lewis rat embryos into the omentum of adult Lewis rats or C57Bl/6J mice. E12.5 pancreatic anlagen were relatively undifferentiated except for the presence of condensing tubuloacinar cords. By 2 weeks after implantation, pancreatic anlagen transplanted into rats had enlarged and differentiated such that islets of Langerhans that stained positive for insulin could be delineated. Continued differentiation, as reflected by the presence of "ductal" islets connected to the duct epithelium, was observed at 6 weeks after implantation. At 15 weeks after implantation, "mature" islets had separated from the ducts. Electron microscopy showed eccentric dense bodies within clear vacuoles consistent with insulin granules. Little or no acinar tissue was present in developed anlagen. Within 5 weeks of pancreatic anlagen transplantation, levels of glucose in rats rendered diabetic by an injection of streptozotocin were normalized compared with levels in nontransplanted diabetic controls. Rat pancreatic anlagen underwent growth and development in the peritoneum of C57Bl/61 mice that received costimulatory blocking agents but not in the absence of costimulatory blockade. We concluded that whole E12.5 pancreatic anlagen undergo growth, differentiation, and function after intraperitoneal placement. Implantation of the embryonic pancreas, a "cellular" transplant, is followed by selective differentiation of islet compared with acinar components.     

Comparative Phenotypic Studies of Duct Epithelial Cell Lines Derived from Normal Human Pancreas and Pancreatic Carcinoma

We have investigated the mRNA/protein expression of several tyrosine kinase receptors, growth factors, and p16^INK4A cyclin inhibitor in cell lines derived from normal human pancreatic duct epithelium (HPDE) and compared them with those of five pancreatic ductal carcinoma cell lines. Cultured HPDE cells express low levels of epidermal growth factor receptor (EGFR), erbB2, transforming growth factor (TGF)-α, Met/hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor (VEGF), and keratinocyte growth factor (KGF). They also expressed high levels of amphiregulin but did not express EGF and cripto. The expression levels were similar in primary normal HPDE cells and those expressing transfected E6E7 genes of human papilloma virus-16, but their immortalization appeared to enhance the expression of EGFR and Met/HGFR. In comparison, pancreatic carcinoma cell lines commonly demonstrated overexpression of EGFR, erbB2, TGF-α, Met/HGFR, VEGF, and KGF, but they consistently showed marked down-regulation of amphiregulin mRNA expression. In contrast to all carcinoma cell lines that showed deletions of the p16 gene, HPDE cells consistently demonstrated normal p16 genotype and its mRNA expression. This is the first report that compares the phenotypic expression of cultured pancreatic ductal carcinoma cells with epithelial cell lines derived from normal human pancreatic ducts. The findings confirm that malignant transformation of human pancreatic duct cells commonly results in a deregulation of expression of various growth factors and receptors.     

Hex expression suggests a role in the development and function of organs derived from foregut endoderm.

Hex is a divergent homeobox gene expressed as early as E4.5 in the mouse and in a pattern that suggests a role in anterior-posterior patterning. Later in embryogenesis, Hex is expressed in the developing thyroid, lung, and liver. We now show Hex expression during thymus, gallbladder, and pancreas development and in the adult thyroid, lung, and liver. At E10.0, Hex is expressed in the 3rd pharyngeal pouch, from which the thymus originates, the endodermal cells of liver that are invading the septum transversum, the thyroid, the dorsal pancreatic bud, and gallbladder primoridum. At E13.5, expression is maintained at high levels in the thyroid, liver, epithelial cells lining the pancreatic and extrahepatic biliary ducts and is present in both the epithelial and mesenchymal cells of the lung. Expression in the thymus at this age is less than in the other organs. In the E16.5 embryo, expression persists in the thyroid, pancreatic, and bile duct epithelium, lung, and liver, with thymic expression dropping to barely detectable levels. By E18.5, expression in the thyroid and bile ducts remains high, whereas lung expression is markedly decreased. At this age, expression in the pancreas and thymus is no longer present. Finally, we show the cell types in the adult thyroid, lung, and liver that express Hex in the mature animal. Our results provide more detail on the potential role of Hex in the development of several organs derived from foregut endoderm and in the maintenance of function of several of these organs in the mature animal.     

Insulinoma of the pancreas with insular-ductular differentiation in its liver metastasis--indication of a common stem-cell origin of the exocrine and endocrine components

We describe an insulinoma of the pancreas in a 56-year-old patient, which showed insular-ductular differentiation in its liver metastasis. Although the primary tumor was uniformly endocrine in nature with insulin production, the metastasis contained two distinct cell types in organoid arrangement. One cell type was insulin-positive and was arranged in islet-like structures; the other was insulin-negative but distinctly pan-cytokeratin and cytokeratin 7 positive and arranged in ducts. In the primary tumor and the metastasis, the tumor cells were surrounded by a desmoplastic stroma. As to the histogenesis of the tumor and its metastasis, we discuss the following possibilities: (1) the tumor cells might derive from a common stem cell that matures into two phenotypically different cell lines, resembling the situation in embryogenesis and (2) one tumor cell type originates from the other by transdifferentiation (metaplasia). We conclude that the parallel occurrence of endocrine and ductal differentiation supports the concept that, under certain conditions, islet cells and ductular cells may also originate from islets and that mixed endocrine/exocrine pancreatic tumors do not necessarily arise from totipotent duct cells but might also have a primary endocrine cell origin.     

Developmental expression of metalloproteases ADAM 9, 10, and 17 becomes restricted to divergent pancreatic compartments.

The A Disintegrin And Metalloprotease (ADAM) family of metalloproteases affects a variety of proteins with important roles in development and disease, including growth factors and adhesion molecules. We have analyzed the expression patterns of ADAMs 9, 10, and 17 during pancreas ontogeny. All ADAMs investigated were expressed in the pancreatic anlagen but invariably became restricted to divergent pancreatic compartments. ADAM9 and 17 became restricted to the insulin-producing beta-cells and all islet cells, respectively. During embryogenesis, ADAM10 was detected predominantly in acinar cells, but in the adult, it was localized to the cell surface membrane of both endocrine and exocrine cells. In addition to ADAM9, a potential prognostic factor for ductal cancers, we describe the expression of ADAM10 and ADAM17 in the pancreatic ductal epithelium. Altogether, the dynamic expression profile of the ADAM proteases described here may reflect a functional divergence of these as mediators of pancreas biology.     

Acinar cells contribute to the molecular heterogeneity of pancreatic intraepithelial neoplasia

A number of studies have shown that pancreatic ductal adenocarcinoma develops through precursor lesions termed pancreatic intraepithelial neoplasia (PanIN). PanINs are thought to initiate in the small ducts of the pancreas through activating mutations in the KRAS proto-oncogene. What remains unanswered is the identification of the individual cell type(s) that contributes to pancreatic ductal adenocarcinoma formation. To follow the cellular and molecular changes that occur in acinar and duct cell properties on Kras(G12D) expression, we took advantage of LSL-Kras(G12D/+)/p48(Cre/+) mice, which faithfully mimic the human disease. In young animals (4 weeks), the predominant cellular alteration in the exocrine pancreas was acinar metaplasia in which individual acini consisted of acinar cells and duct-like cells. Metaplastic acinar structures were highly proliferative, expressed Notch target genes, and exhibited mosaic expression patterns for epidermal growth factor receptor, ErbB2, and pErk. This expression pattern paralleled the expression pattern detected in mouse PanINs, suggesting that mouse PanINs and acinar-ductal metaplasia follow similar molecular pathways. Indeed, immunofluorescence studies confirmed the presence of acinar cells within mPanIN lesions, raising the possibility that Kras(G12D)-induced mPanINs develop from acinar cells that undergo acinar-ductal metaplasia. Identification of an acinar contribution to PanIN formation offers new directions for successful targeted therapeutic approaches to combat this disease.     

Intermediate cells in the pancreas and cell transformation

The paper reviews the medical literature concerning the occurrence and several possible explanations of the origin nature of pancreatic intermediate cells present. Electron microscopic studies of the human pancreas and results of animal experiments in the last years strongly suggest that cells in the wall of small pancreatic ducts (ductules) may develop into duct cells, exocrine pancreatic cells (acinar cells), endocrine islet cells, and hepatocytes even in the adult organism. This suggests that transitional cells arise from the known types of pancreatic intermediate cells during this process. The development of the different cells may start from a pool of omnipotent cells (entodermal stem cells of the foregut) present in the walls of the ductules, or from undifferentiated cells, reflecting their embryonic potential as a result of proliferation of duct cells (i.e. indirect cellular transformation = metaplasia of duct cells).     

Connexin 30.2 is expressed in exocrine vascular endothelial and ductal epithelial cells throughout pancreatic postnatal development

Previously we have demonstrated that the GJ protein connexin 30.2 (Cx30.2) is expressed in pancreatic beta cells and endothelial cells (ECs) of the islet. In the present study, we address whether Cx30.2 is expressed in the exocrine pancreas, including its vascular system. For this, adult mouse pancreatic sections were double labeled with specific antibodies against Cx30.2 and CD31, an endothelial cell marker, or with anti-α-actin smooth muscle, a smooth muscle cell (SMC) marker or anti-mucin-1, a marker of epithelial ductal cells, using immunofluorescence (IF) studies. Cx30.2-IF hot spots were found at junctional membranes of exocrine ECs and SMCs of blood vessels. Furthermore, Cx30.2 was localized in mucin-1 positive cells or epithelial ductal cells. Using immunohistochemistry (IHC) studies, it was found that in vessels and ducts of different diameters, Cx30.2 was also expressed in these cell types. In addition, it was found that Cx30.2 is already expressed in these cell types in pancreatic sections of 3, 14 and 21 days postpartum. Moreover, this cell specific pattern of expression was also found in the adult rat, hamster and guinea pig pancreas. Expression of Cx30.2 mRNA and protein in the pancreas of all these species was confirmed by RT-PCR and Western blot studies. Overall, our results suggest that intercellular coupling mediated by Cx30.2 intercellular channels may synchronize the functional activity of ECs and SMCs of vascular cells, as well as of epithelial ductal cells after birth.     

Hnf6 and Tcf2 (MODY5) are linked in a gene network operating in a precursor cell domain of the embryonic pancreas

During pancreatic organogenesis endocrine cells arise from non self-renewing progenitors that express Ngn3. The precursors that give rise to Ngn3+ cells are presumably located within duct-like structures. However, the nature of such precursors is poorly understood. We show that, at E13-E18, the embryonic stage during which the major burst of beta-cell neogenesis takes place, pancreatic duct cells express Hnf1beta, the product of the maturity-onset diabetes of the young type 5 (MODY5) gene. Ngn3+ cells at this stage invariably cluster with mitotically competent Hnf1beta+ cells, and are often intercalated with these cells in the epithelium that lines the lumen of primitive ducts. We present several observations that collectively indicate that Hnf1beta+ cells are the immediate precursors of Ngn3+ cells. We furthermore show that Hnf1beta expression is markedly reduced in early pancreatic epithelial cells of Hnf6-deficient mice, in which formation of Ngn3+ cells is defective. These findings define a precursor cellular stage of the embryonic pancreas and place Hnf1beta in a genetic hierarchy that regulates the generation of pancreatic endocrine cells.     

Keratinocyte growth factor induces pancreatic ductal epithelial proliferation.

Keratinocyte growth factor (KGF) causes a proliferation of pancreatic ductal epithelial cells in adult rats after daily systemic administration for 1 to 2 weeks. Even before the proliferation of intralobular ducts is histologically evident, KGF also induces proliferating cell nuclear antigen expression within the ductal epithelium of intercalated, intralobular, and interlobular ducts. KGF also causes incorporation of 5-bromodeoxyuridine in ductal epithelial cells. Epithelial cell proliferation is histologically most prominent at the level of the intralobular ducts adjacent to and within the islets of Langerhans. Pancreatic ductal proliferation is not histologically apparent in rats sacrificed 7 to 10 days after the cessation of KGF administration. The pancreatic hormones insulin, glucagon, somatostatin, and pancreatic polypeptide are normally distributed within islets that demonstrate intrainsular ductal proliferation. The proliferating ductal epithelium does not show endocrine differentiation as evidenced by the lack of immunoreactivity for pancreatic hormones. KGF is a potent in vivo mitogen for pancreatic ductal epithelial cells.     

Intraductal acinar cell carcinoma of the pancreas

We describe a purely intraductal acinar cell carcinoma involving branch ducts of the pancreas in a 74-year-old man, which presented as recurrent episodes of acute pancreatitis. Endoscopic ultrasound examination revealed an intraductal mass bulging into the main pancreatic duct suggesting, pre-operatively, an intraductal mucinous papillary tumour. Gross examination showed several dilated branch ducts that contained haemorrhagic tumour material without any solid or true cystic formation within the pancreatic parenchyma. Using histology, a purely intraductal acinar cell carcinoma was observed, involving branch ducts only, associated with foci of carcinoma in situ in adjacent exocrine parenchyma. The main pancreatic duct was free of disease except for its communication with a cancerous branch duct. A concomitant neuroendocrine microadenoma was incidentally found during slide screening. Immunohistochemistry performed on the intraductal proliferation confirmed zymogen secretion with positive staining for alpha-1 anti-chymotrypsin and anti-trypsin and the persistence of diastase-periodic acid-Schiff positive granules in the apical pole of the tumour cells. Neuroendocrine markers were negative in the acinar cell carcinoma and positive in the neuroendocrine microadenoma. To our knowledge, this is the first report of an intraductal acinar cell carcinoma of the pancreas involving branch ducts and sparing the main pancreatic duct.     

A comparison between hyperplasia of ductal epithelium and mucin producing tumor of the pancreas]

A comparison between papillary proliferation of the ductal epithelium with goblet cell metaplasia (Hyperplasia) and mucin producing tumor (MPT) of the pancreas was studied clinicopathologically. Hyperplasia was found in 35 out of 56 autopsied cases (62.5%) and revealed an age-related frequency. Hyperplasia was situated chiefly in the branches of pancreatic duct, whereas MPT in both the main and branches of pancreatic duct. The ducts with hyperplasia frequently contained secretions in their lumens and were found in the proximity of isolated islets of Langerhans, while MPT consisted of high columnar and mucin producing cells, arranged in cyst-papillary pattern, filled with mucin and accompanied by disappearance of the surrounding parenchyma. Both Hyperplasia and MPT were frequently occurred in an elderly age group. Therefore, papillary hyperplasia of the ductal epithelium with goblet cell metaplasia and mucin producing tumor showed similar appearance, except for cellular atypism.     

The epigenetic regulators Bmi1 and Ring1B are differentially regulated in pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up‐regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K‐Ras^G12V conditional knock‐in and caerulein‐treated K‐Ras^G12V mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells—but not acinar cells—in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar–ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up‐regulated in high‐grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.