- Pancreatic diseases
- Gastrointestinal Cancers
Co-Course Director - Human Health & Disease, Stanford University (2004 - Present)
Member, Stanford School of Medicine Awards Committee, Stanford University (2001 - Present)
Associate Editor, The American Journal of Medicine (2000 - 2004)
Special Sections Editor, Gastroenterology (journal) (2011 - 2016)
Co-Director, Stanford Gastroenterology Training Program, Stanford University (2012 - Present)
Associate Director, Stanford Digestive Disease Center, Stanford University (2001 - 2012)
Honors & Awards
Division Teaching Award, Stanford University (2001, 2004, 2007, 2008, 2012)
AOA inductee, Alpha Omega Alpha Honor Medical Society (1979)
Lester R. Tuchman Award for clinical excellence (graduating class award), Mount Sinai Hospital (1980)
American Gastroenterology Association Fellow, American Gastroenterology Association (2009)
Medical Education:Mt Sinai School of Medicine (1980) NY
Residency:Presbyterian Hospital (1983) NY
Board Certification: Gastroenterology, American Board of Internal Medicine (1987)
Fellowship:UCSF Medical Center (1989) CA
Fellowship:Columbia University School of Public Health (1984) NY
Board Certification: Internal Medicine, American Board of Internal Medicine (1983)
Internship:Presbyterian Hospital (1981) NY
MD, Mt. Sinai Sch. of Med., Medicine (1980)
BA, UC Berkeley, Physiology (1976)
Current Research and Scholarly Interests
The laboratory is focused on human cancers that are dependent on EGFR cell signaling. In particular, we recently established that the AGR2 protein serves an essential role in EGFR presentation to the cell surface, and represents a novel mechanism of regulating cell signaling. Recent work focused on the role of EGFR cell signaling during tissue regeneration in response to injury. Our overall hypothesis is that EGFR signaling serves a vital role in tissue regeneration, and that chronic injury and persistent wound healing lead to the development of preneoplastic lesions and eventually cancer. We hypothesize that this pathway is active in a large number of human cancers. If true, new opportunities for the treatment of preneoplastic and neoplastic diseases are provided, which represents a major focus of the laboratory. Active projects are focused on cancer pathogenesis, tissue regeneration, development of diagnostic assays, and drug development.
Independent Studies (8)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Medicine
MED 299 (Aut, Win, Spr, Sum)
- Early Clinical Experience in Medicine
MED 280 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
MED 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MED 370 (Aut, Win, Spr, Sum)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
MED 199 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
Graduate and Fellowship Programs
Anterior Gradient 2 (AGR2) Induced Epidermal Growth Factor Receptor (EGFR) Signaling Is Essential for Murine Pancreatitis-Associated Tissue Regeneration.
2016; 11 (10)
A recently published study identified Anterior Gradient 2 (AGR2) as a regulator of EGFR signaling by promoting receptor presentation from the endoplasmic reticulum to the cell surface. AGR2 also promotes tissue regeneration in amphibians and fish. Whether AGR2-induced EGFR signaling is essential for tissue regeneration in higher vertebrates was evaluated using a well-characterized murine model for pancreatitis. The impact of AGR2 expression and EGFR signaling on tissue regeneration was evaluated using the caerulein-induced pancreatitis mouse model. EGFR signaling and cell proliferation were examined in the context of the AGR2-/- null mouse or with the EGFR-specific tyrosine kinase inhibitor, AG1478. In addition, the Hippo signaling coactivator YAP1 was evaluated in the context of AGR2 expression during pancreatitis. Pancreatitis-induced AGR2 expression enabled EGFR translocation to the plasma membrane, the initiation of cell signaling, and cell proliferation. EGFR signaling and tissue regeneration were partially inhibited by the tyrosine kinase inhibitor AG1478, but absent in the AGR2-/- null mouse. AG1478-treated and AGR2-/- null mice with pancreatitis died whereas all wild-type controls recovered. YAP1 activation was also dependent on pancreatitis-induced AGR2 expression. AGR2-induced EGFR signaling was essential for tissue regeneration and recovery from pancreatitis. The results establish tissue regeneration as a major function of AGR2-induced EGFR signaling in adult higher vertebrates. Enhanced AGR2 expression and EGFR signaling are also universally present in human pancreatic cancer, which support a linkage between tissue injury, regeneration, and cancer pathogenesis.
View details for DOI 10.1371/journal.pone.0164968
View details for PubMedID 27764193
View details for PubMedCentralID PMC5072742
Epidermal Growth Factor Receptor (EGFR) Signaling Requires a Specific Endoplasmic Reticulum Thioredoxin for the Post-translational Control of Receptor Presentation to the Cell Surface.
journal of biological chemistry
2015; 290 (13): 8016-8027
The epidermal growth factor receptor (EGFR) is a well characterized receptor-tyrosine kinase that functions in development and serves a vital role in many human cancers. Understanding EGFR regulatory mechanisms, and hence approaches for clinical intervention, has focused on ligand-receptor interactions and tyrosine kinase activity. Here, we show using the NCI-H460 lung and A431 epidermoid human cancer cell lines that EGFR binding to anterior gradient homolog 2 (AGR2) in the endoplasmic reticulum is required for receptor delivery to the plasma membrane and thus EGFR signaling. Reduced AGR2 protein levels or mutation of an essential cysteine in the active site result in decreased cell surface EGFR and a concomitant decrease in signaling as reflected by AREG, EGR1, and FOS expression. Similar to previously described EGFR nulls, an AGR2 null also resulted in embryonic lethality. Consistent with its role in regulating EGFR-mediated signaling, AGR2 expression is also enhanced in many human cancers and promotes the transformed phenotype. Furthermore, EGFR-mediated signaling in NCI-H460 cells, which are resistant to the tyrosine kinase inhibitor AG1478, is also disrupted with reduced AGR2 expression. The results provide insights into why cancer prognosis or response to therapy often does not correlate with EGFR protein or RNA levels because they do not reflect delivery to the cell surface where signaling is initiated. AGR2, therefore, represents a novel post-translational regulator of EGFR-mediated signaling and a promising target for treating human cancers.
View details for DOI 10.1074/jbc.M114.623207
View details for PubMedID 25666625
View details for PubMedCentralID PMC4375459
Loss of Anterior Gradient 2 (Agr2) Expression Results in Hyperplasia and Defective Lineage Maturation in the Murine Stomach
JOURNAL OF BIOLOGICAL CHEMISTRY
2013; 288 (6): 4321-4333
Recent studies of epithelial tissues have revealed the presence of tissue-specific stem cells that are able to establish multiple cell lineages within an organ. The stem cells give rise to progenitors that replicate before differentiating into specific cell lineages. The mechanism by which homeostasis is established between proliferating stem or progenitor cells and terminally differentiated cells is unclear. This study demonstrates that Agr2 expression by mucous neck cells in the stomach promotes the differentiation of multiple cell lineages while also inhibiting the proliferation of stem or progenitor cells. When Agr2 expression is absent, gastric mucous neck cells increased in number as does the number of proliferating cells. Agr2 expression loss also resulted in the decline of terminally differentiated cells, which was supplanted by cells that exhibited nuclear SOX9 labeling. Sox9 expression has been associated with progenitor and stem cells. Similar effects of the Agr2 null on cell proliferation in the intestine were also observed. Agr2 consequently serves to maintain the balance between proliferating and differentiated epithelial cells.
View details for DOI 10.1074/jbc.M112.433086
View details for Web of Science ID 000314845000060
View details for PubMedID 23209296
View details for PubMedCentralID PMC3567683
AGR2 Gene Function Requires a Unique Endoplasmic Reticulum Localization Motif
JOURNAL OF BIOLOGICAL CHEMISTRY
2012; 287 (7): 4773-4782
Soluble proteins are enriched in the endoplasmic reticulum (ER) by retrograde transport from the Golgi that is mediated by the KDEL receptors. In addition to the classic carboxyl-terminal KDEL motif, a variety of sequence variants are also capable of receptor binding that result in ER localization. Although different ER localization signals that exhibit varying affinities for the KDEL receptors exist, whether there are functional implications was unknown. The present study determines whether AGR2 requires a specific ER localization signal to be functionally active. AGR2 is expressed in most human adenocarcinomas and serves a role in promoting growth and the transformed phenotype. Using two different cell lines in which AGR2 induces expression of either the EGFR ligand amphiregulin or the transcription factor CDX2, only the highly conserved wild-type carboxyl-terminal KTEL motif results in the appropriate outcome. Deletion of the KTEL motif results in AGR2 secretion and loss of AGR2 function. AGR2 function is also lost when ER residence is achieved with a carboxyl-terminal KDEL or KSEL instead of a KTEL motif. Thus variations in ER localization sequences may serve a specific functional role, and in the case of AGR2, this role is served specifically by KTEL.
View details for DOI 10.1074/jbc.M111.301531
View details for Web of Science ID 000300608500038
View details for PubMedID 22184114
View details for PubMedCentralID PMC3281655
The Human Adenocarcinoma-associated Gene, AGR2, Induces Expression of Amphiregulin through Hippo Pathway Co-activator YAP1 Activation
JOURNAL OF BIOLOGICAL CHEMISTRY
2011; 286 (20): 18301-18310
Anterior Gradient Homolog 2 (AGR2) is expressed by the normal intestine and by most human adenocarcinomas, including those derived from the esophagus, pancreas, lung, breast, ovary, and prostate. Xenografts of human adenocarcinoma cell lines in nude mice previously demonstrated that AGR2 supports tumor growth. In addition, AGR2 is able to induce in vitro a transformed phenotype in fibroblast and epithelial cell lines. The mechanism underlying the growth promoting effects of AGR2 is unknown. The present study shows that AGR2 induces expression of amphiregulin (AREG), a growth promoting EGFR ligand. Induced AREG expression in adenocarcinoma cells is able to rescue the transformed phenotype that is lost when AGR2 expression is reduced. Additional experiments demonstrate that AGR2 induction of AREG is mediated by activation of the Hippo signaling pathway co-activator, YAP1. Thus AGR2 promotes growth by regulating the Hippo and EGF receptor signaling pathways.
View details for DOI 10.1074/jbc.M110.215707
View details for Web of Science ID 000290585200090
View details for PubMedID 21454516
View details for PubMedCentralID PMC3093902
Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response
2009; 462 (7270): 226-U101
The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.
View details for DOI 10.1038/nature08529
View details for Web of Science ID 000271655100044
View details for PubMedID 19907495
The adenocarcinoma-associated antigen, AGR2, promotes tumor growth, cell migration, and cellular transformation
2008; 68 (2): 492-497
The AGR2 gene encodes a secretory protein that is highly expressed in adenocarcinomas of the esophagus, pancreas, breast, and prostate. This study explores the effect of AGR2 expression with well-established in vitro and in vivo assays that screen for cellular transformation and tumor growth. AGR2 expression in SEG-1 esophageal adenocarcinoma cells was reduced with RNA interference. Cellular transformation was examined using NIH3T3 cells that express AGR2 after stable transfection. The cell lines were studied in vitro with assays for density-dependent and anchorage-independent growth, and in vivo as tumor xenografts in nude mice. SEG-1 cells with reduced AGR2 expression showed an 82% decrease in anchorage-independent colony growth and a 60% reduction in tumor xenograft size. In vitro assays of AGR2-expressing NIH3T3 cells displayed enhanced foci formation and anchorage-independent growth. In vivo, AGR2-expressing NIH3T3 cells established tumors in nude mice. Thus, AGR2 expression promotes tumor growth in esophageal adenocarcinoma cells and is able to transform NIH3T3 cells. Immunohistochemistry of the normal mouse intestine detected AGR2 expression in proliferating and differentiated intestinal cells of secretory lineage. AGR2 may be important for the growth and development of the intestine as well as esophageal adenocarcinomas.
View details for DOI 10.1158/0008-5472.CAN-07-2930
View details for Web of Science ID 000252503800021
View details for PubMedID 18199544
Gene Expression Patterns in Pancreatic Tumors, Cells and Tissues
2007; 2 (3)
Cancers of the pancreas originate from both the endocrine and exocrine elements of the organ, and represent a major cause of cancer-related death. This study provides a comprehensive assessment of gene expression for pancreatic tumors, the normal pancreas, and nonneoplastic pancreatic disease.DNA microarrays were used to assess the gene expression for surgically derived pancreatic adenocarcinomas, islet cell tumors, and mesenchymal tumors. The addition of normal pancreata, isolated islets, isolated pancreatic ducts, and pancreatic adenocarcinoma cell lines enhanced subsequent analysis by increasing the diversity in gene expression profiles obtained. Exocrine, endocrine, and mesenchymal tumors displayed unique gene expression profiles. Similarities in gene expression support the pancreatic duct as the origin of adenocarcinomas. In addition, genes highly expressed in other cancers and associated with specific signal transduction pathways were also found in pancreatic tumors.The scope of the present work was enhanced by the inclusion of publicly available datasets that encompass a wide spectrum of human tissues and enabled the identification of candidate genes that may serve diagnostic and therapeutic goals.
View details for DOI 10.1371/journal.pone.0000323
View details for Web of Science ID 000207445200006
View details for PubMedID 17389914
View details for PubMedCentralID PMC1824711
- Detection of pancreatic ductal adenocarcinoma in mice by ultrasound imaging of thymocyte differentiation antigen 1. Gastroenterology 2013; 145 (4): 885-894 e3
- Metabolomic-derived novel cyst fluid biomarkers for pancreatic cysts: glucose and kynurenine GASTROINTESTINAL ENDOSCOPY 2013; 78 (2): 295-?
CITED2 is a novel direct effector of peroxisome proliferator-activated receptor gamma in suppressing hepatocellular carcinoma cell growth
2013; 119 (6): 1217-1226
Previous reports from these authors found that activation of peroxisome proliferator-activated receptor gamma (PPARγ) suppressed hepatocellular carcinoma (HCC). This study sought to identify the molecular target of PPARγ and characterize its antitumor effect in HCC.Optimal PPARγ binding activity was obtained using the PPARγ agonist rosiglitazone (100 μM) as determined by enzyme-linked immunosorbent assay. Under PPARγ activation, 114 PPARγ downstream targets associated with cancer development were identified by oligonucleotide microarray and Gene Ontology analysis. Among them, Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) was the most prominent PPARγ-bound target, as determined by chromatin immunoprecipitation-polymerase chain reaction.CITED2 messenger RNA and protein was significantly down-regulated in primary HCCs compared with their adjacent nontumor tissues. PPARγ induced expression of CITED2 in HCC cell lines after adenovirus-PPARγ transduction. The biological function of CITED2 was evaluated by loss- and gain-of-function assays. CITED2 knockdown in the hepatocyte cell line LO2 and HCC cell line Hep3B significantly increased cell viability and clonogenicity, and promoted G1 -S phase transition in both cell lines. In contrast, ectopic expression of CITED2 in HepG2 and BEL7404 HCC cell lines significantly suppressed cell growth. The tumor suppressive effect of CITED2 was associated with up-regulation of cyclin-dependent kinase inhibitors p15(INK4B) , p21(Wat1/Cip1) , p27(Kip1) , antiproliferative regulator interferon alpha 1, proapoptotic mediators including tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), TNFRSF25, caspase-8, granzyme A, and the tumor suppressor gene maspin. CITED2 was also associated with the down-regulation of cell cycle regulator cyclin D1, oncogene telomerase reverse transcriptase, and proinvasion/metastasis gene matrix metallopeptidase 2.CITED2 is a direct effector of PPARγ for tumor suppression. Cancer 2013. © 2012 American Cancer Society.
View details for DOI 10.1002/cncr.27865
View details for Web of Science ID 000315696600016
View details for PubMedID 23212831
Immunohistochemical panel for distinguishing esophageal adenocarcinoma from squamous cell carcinoma: a combination of p63, cytokeratin 5/6, MUC5AC, and anterior gradient homolog 2 allows optimal subtyping
2012; 43 (11): 1799-1807
Distinguishing adenocarcinoma and squamous cell carcinoma of the esophagus is often based on morphological criteria and can be difficult in small biopsies. We analyzed commonly used immunohistochemical markers (p63, cytokeratin 5/6, cytokeratin 7, CDX2, MUC2, and MUC5AC) and 2 new markers, anterior gradient homolog 2 and SOX2, in esophageal carcinomas to establish the best panel to distinguish these tumors. Tissue microarrays with 69 esophageal adenocarcinomas and 41 whole sections of esophageal squamous cell carcinomas were stained for these markers and semiquantitatively scored. Sensitivities and specificities were calculated for individual markers and select combinations using the morphological diagnosis as a gold standard. All squamous cell carcinomas expressed p63 with 38 of 41 demonstrating reactivity in more than 75% of tumor cells. Cytokeratin 5/6 expression was seen in 40 of 41 squamous cell carcinomas with 39 of 41 demonstrating reactivity in more than 75% of tumor cells. SOX2 expression was present in 35 of 41 of squamous cell carcinomas but also in 24 of 69 of adenocarcinomas, frequently demonstrating extensive reactivity in adenocarcinomas. Anterior gradient homolog 2 was highly sensitive for adenocarcinoma and present in 68 of 69 of cases, but anterior gradient homolog 2 reactivity was also identified in 15 of 41 of squamous cell carcinomas, typically demonstrating focal reactivity in squamous cell carcinoma. MUC5AC expression was seen almost exclusively in adenocarcinomas with only a single squamous cell carcinoma demonstrating focal MUC5AC staining. Overall, the dual expression of both p63 and cytokeratin 5/6 was 99% specific and 98% sensitive for squamous cell carcinoma. In addition, anterior gradient homolog 2 and MUC5AC are useful positive markers of adenocarcinoma in the setting of absent or diminished p63 and cytokeratin 5/6 staining.
View details for DOI 10.1016/j.humpath.2012.03.019
View details for Web of Science ID 000310654500002
View details for PubMedID 22748473
PDX1 regulation of FABP1 and novel target genes in human intestinal epithelial Caco-2 cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2012; 423 (1): 183-187
The transcription factor pancreatic and duodenal homeobox 1 (PDX1) plays an essential role in pancreatic development and in maintaining proper islet function via target gene regulation. Few intestinal PDX1 targets, however, have been described. We sought to define novel PDX1-regulated intestinal genes. Caco-2 human intestinal epithelial cells were engineered to overexpress PDX1 and gene expression profiles relative to control cells were assessed. Expression of 80 genes significantly increased while that of 49 genes significantly decreased more than 4-fold following PDX1 overexpression in differentiated Caco-2 cells. Analysis of the differentially regulated genes with known functional annotations revealed genes encoding transcription factors, growth factors, kinases, digestive glycosidases, nutrient transporters, nutrient binding proteins, and structural components. The gene for fatty acid binding protein 1, liver, FABP1, is repressed by PDX1 in Caco-2 cells. PDX1 overexpression in Caco-2 cells also results in repression of promoter activity driven by the 0.6kb FABP1 promoter. PDX1 regulation of promoter activity is consistent with the decrease in FABP1 RNA abundance resulting from PDX1 overexpression and identifies FABP1 as a candidate PDX1 target. PDX1 repression of FABP1, LCT, and SI suggests a role for PDX1 in patterning anterior intestinal development.
View details for DOI 10.1016/j.bbrc.2012.05.113
View details for Web of Science ID 000307087700031
View details for PubMedID 22640736
Diagnostic accuracy of cyst fluid amphiregulin in pancreatic cysts
Accurate tests to diagnose adenocarcinoma and high-grade dysplasia among mucinous pancreatic cysts are clinically needed. This study evaluated the diagnostic utility of amphiregulin (AREG) as a pancreatic cyst fluid biomarker to differentiate non-mucinous, benign mucinous, and malignant mucinous cysts.A single-center retrospective study to evaluate AREG levels in pancreatic cyst fluid by ELISA from 33 patients with a histological gold standard was performed.Among the cyst fluid samples, the median (IQR) AREG levels for non-mucinous (n = 6), benign mucinous (n = 15), and cancerous cysts (n = 15) were 85 pg/ml (47-168), 63 pg/ml (30-847), and 986 pg/ml (417-3160), respectively. A significant difference between benign mucinous and malignant mucinous cysts was observed (p = 0.025). AREG levels greater than 300 pg/ml possessed a diagnostic accuracy for cancer or high-grade dysplasia of 78% (sensitivity 83%, specificity 73%).Cyst fluid AREG levels are significantly higher in cancerous and high-grade dysplastic cysts compared to benign mucinous cysts. Thus AREG exhibits potential clinical utility in the evaluation of pancreatic cysts.
View details for DOI 10.1186/1471-230X-12-15
View details for Web of Science ID 000301923400002
View details for PubMedID 22333441
View details for PubMedCentralID PMC3305641
The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli type 1 Fimbriae
GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria.An in vitro binding assay was used to assay the binding of recombinant GP2 to defined strains of E. coli that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding.GP2 binds E. coli that express Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues.GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the Enterobacteriacae family.
View details for DOI 10.1186/1471-230X-9-58
View details for Web of Science ID 000269501900001
View details for PubMedID 19627615
View details for PubMedCentralID PMC2726147
- Painless Jaundice and Bilaterally Enlarged Sub-mandibular Glands in an Elderly Man DIGESTIVE DISEASES AND SCIENCES 2009; 54 (3): 488-490
Endoscopic evaluation of esophago-gastro-jejunostomy in rat model of Barrett's esophagus
DISEASES OF THE ESOPHAGUS
2009; 22 (4): 323-330
Endoscopy can be used to monitor the onset of metaplastic transformation and to observe the progression of neoplasia in small animal models of Barrett's esophagus. By avoiding animal sacrifice, the natural history of this disease can be studied in a longitudinal fashion. We aim to characterize the endoscopic features of esophageal mucosa at various stages of the metaplasia-dysplasia-carcinoma sequence in a rat reflux model of Barrett's for comparison with histology. Acid and bile reflux was produced by introducing a side-to-side esophago-gastro-jejunostomy in Sprague-Dawley rats. Endoscopic examination of the distal esophagus was performed in 24 surgically altered and 4 control rats, between weeks 24 and 36 after the operation in 4-week intervals, and all rats were biopsied and sacrificed at 36 weeks. Endoscopic images were classified based on the surface mucosal patterns of the distal esophagus and then compared with histology. The endoscopic appearance was classified as: (i) normal, characterized by a smooth surface; (ii) intestinal metaplasia, defined as elevated plaques/ridges, deep grooves, and thin linear folds; (iii) dysplasia, indicated by coarse folds/grooves, meshlike villi, and foveolar appearance; and (iv) carcinoma, suggested by irregular-shaped mass lesions with ulcerations. The endoscopic criteria for intestinal metaplasia yielded a sensitivity of 100% in comparison with histology. Intestinal metaplasia with high-grade dysplasia was found in two rats and with low-grade dysplasia in three rats. Both focally invasive squamous cell carcinoma and invasive adenocarcinoma were found in one rat. Small animal endoscopy in a rat model of Barrett's esophagus can be used to perform surveillance, classify mucosal patterns, observe the onset of intestinal metaplasia, and monitor the progression of neoplastic transformation, representing a useful tool for studying the natural history of this disease.
View details for DOI 10.1111/j.1442-2050.2008.00909.x
View details for Web of Science ID 000266110800006
View details for PubMedID 19473210
Comprehensive gene expression profiling of Peyer's patch M cells, villous M-like cells, and intestinal epithelial cells
JOURNAL OF IMMUNOLOGY
2008; 180 (12): 7840-7846
Separate populations of M cells have been detected in the follicle-associated epithelium of Peyer's patches (PPs) and the villous epithelium of the small intestine, but the traits shared by or distinguishing the two populations have not been characterized. Our separate study has demonstrated that a potent mucosal modulator cholera toxin (CT) can induce lectin Ulex europaeus agglutinin-1 and our newly developed M cell-specific mAb NKM 16-2-4-positive M-like cells in the duodenal villous epithelium. In this study, we determined the gene expression of PP M cells, CT-induced villous M-like cells, and intestinal epithelial cells isolated by a novel approach using FACS. Additional mRNA and protein analyses confirmed the specific expression of glycoprotein 2 and myristoylated alanine-rich C kinase substrate (MARCKS)-like protein by PP M cells but not CT-induced villous M-like cells. Comprehensive gene profiling also suggested that CT-induced villous M-like cells share traits of both PP M cells and intestinal epithelial cells, a finding that is supported by their unique expression of specific chemokines. The genome-wide assessment of gene expression facilitates discovery of M cell-specific molecules and enhances the molecular understanding of M cell immunobiology.
View details for Web of Science ID 000257404600008
View details for PubMedID 18523247
Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy
2008; 14 (4): 454-458
A combination of targeted probes and new imaging technologies provides a powerful set of tools with the potential to improve the early detection of cancer. To develop a probe for detecting colon cancer, we screened phage display peptide libraries against fresh human colonic adenomas for high-affinity ligands with preferential binding to premalignant tissue. We identified a specific heptapeptide sequence, VRPMPLQ, which we synthesized, conjugated with fluorescein and tested in patients undergoing colonoscopy. We imaged topically administered peptide using a fluorescence confocal microendoscope delivered through the instrument channel of a standard colonoscope. In vivo images were acquired at 12 frames per second with 50-microm working distance and 2.5-microm (transverse) and 20-microm (axial) resolution. The fluorescein-conjugated peptide bound more strongly to dysplastic colonocytes than to adjacent normal cells with 81% sensitivity and 82% specificity. This methodology represents a promising diagnostic imaging approach for the early detection of colorectal cancer and potentially of other epithelial malignancies.
View details for DOI 10.1038/nm1692
View details for Web of Science ID 000254674100034
View details for PubMedID 18345013
The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without ha-ras co-transfection
2008; 47 (3): 827-835
Hepatitis C virus (HCV) is an important cause of chronic liver disease and is complicated by hepatocellular carcinoma (HCC). Mechanisms whereby the virus promotes cellular transformation are poorly understood. We hypothesized that the guanosine triphosphatase activity encoded in the HCV NS4B protein's nucleotide binding motif (NBM) might play a role in the transformation process. Here we report that NS4B can transform NIH-3T3 cells, leading to tumor formation in vivo. This transformation was independent of co-transfection with activated Ha-ras. Detailed analyses of NS4B mutants revealed that this transforming activity could be progressively inhibited and completely abrogated by increasing genetic impairment of the NS4B nucleotide binding motif.NS4B has in vitro and in vivo tumorigenic potential, and the NS4B transforming activity is indeed mediated by its NBM. Moreover, our results suggest that pharmacological inhibition of the latter might inhibit not only HCV replication but also the associated HCC.
View details for DOI 10.1002/hep.22108
View details for Web of Science ID 000253698900009
View details for PubMedID 18081150
Physiological and molecular analysis of acid loading mechanisms in squamous and columnar-lined esophagus
DISEASES OF THE ESOPHAGUS
2008; 21 (6): 529-538
Barrett's esophagus (BE) may be an adaptive cellular response to repeated acid exposure. The aims of this study were to compare intracellular acid loading in BE cells with normal squamous esophageal cells. Primary squamous and BE cells were obtained endoscopically and cultured for up to 24 h. Barrett's adenocarcinoma cell lines TE7 and OE-33 were compared with a normal esophageal (NE) cell line OE-21. Extracellular pH was lowered to 6.0 using HCl; specific ion exchangers were blocked pharmacologically and pH microfluorimetry was performed using 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The effect of prolonged acid preincubations and repeated acid exposure on acid loading and recovery were examined. Acid loading was greater in primary BE than NE cells (DeltapHi -0.22 +/- 0.08 vs.-0.13 +/- 0.01) and maximal in the BE carcinoma cell line TE7 (DeltapHi -0.30 +/- 0.01). Whereas TE7 cells were able to recover fully from repeated acid exposure, OE-21 cells remained profoundly acidic. BE primary and transformed cells utilize DIDS inhibitable sodium-independent chloride/bicarbonate exchange as well as sodium/hydrogen ion exchange for acid loading. In contrast, SE only requires sodium-independent chloride/bicarbonate exchange for acidification. The degree of acid loading is greater in BE than NE cells and it occurs via dual ion exchangers similar to gastric mucosa. Only Barrett's epithelial cells can maintain a physiological pHi following prolonged and repeated reflux exposure, which may confer a teleological advantage.
View details for DOI 10.1111/j.1442-2050.2007.00807.x
View details for Web of Science ID 000258781600009
View details for PubMedID 18840137
Gene expression profiles in gallbladder cancer: The close genetic similarity seen for early and advanced gallbladder cancers may explain the poor prognosis
2008; 29 (1): 41-49
There is currently no data available about the gene expression profiles of gallbladder cancer. The purpose of this study was to identify potential markers for gallbladder cancer and to examine the genetic differences between early and advanced gallbladder cancers. Total RNA was extracted from 17 gallbladder tissue specimens, including 6 advanced gallbladder cancers, 6 early gallbladder cancers and 5 normal control samples. The genes were localized with DNA microarrays and their presence was verified by performing real-time PCR. When the gallbladder cancer isolates were compared to the normal control samples, we identified 4,682 genes, including 2,270 that were overexpressed genes and 2,412 that were underexpressed genes in the gallbladder cancer. We selected 9 overexpressed genes (SERPINB5, BCL10, CD44, ARHGEF11, SERPINB2, RELA, PAK4, PPARD and BUB1B) and 1 underexpressed gene (CAV2) for real-time PCR analysis. When the advanced gallbladder cancer isolates were compared with the early gallbladder cancer isolates, we identified only 12 genes with greater than a 1.7-fold change in gene expression. We have identified several genes that may be tumor markers for gallbladder cancer. The close genetic similarity found between the early and advanced gallbladder cancers may help explain the poor prognosis of this disease.
View details for DOI 10.1159/000132570
View details for Web of Science ID 000256571400006
View details for PubMedID 18497548
Mistaken identity of widely used esophageal adenocarcinoma cell line TE-7
2007; 67 (17): 7996-8001
Cancer of the esophagus is the seventh leading cause of cancer death worldwide. Esophageal carcinoma cell lines are useful models to study the biological and genetic alterations in these tumors. An important prerequisite of cell line research is the authenticity of the used cell lines because the mistaken identity of a cell line may lead to invalid conclusions. Estimates indicate that up to 36% of the cell lines are of a different origin or species than supposed. The TE series, established in late 1970s and early 1980s by Nishihira et al. in Japan, is one of the first esophageal cancer cell line series that was used throughout the world. Fourteen TE cell lines were derived from human esophageal squamous cell carcinomas and one, TE-7, was derived from a primary esophageal adenocarcinoma. In numerous studies, this TE-7 cell line was used as a model for esophageal adenocarcinoma because it is one of the few esophageal adenocarcinoma cell lines existing. We investigated the authenticity of the esophageal adenocarcinoma cell line TE-7 by xenografting, short tandem repeat profiling, mutation analyses, and array-comparative genomic hybridization and showed that cell line TE-7 shared the same genotype as the esophageal squamous cell carcinoma cell lines TE-2, TE-3, TE-12, and TE-13. In addition, for more than a decade, independent TE-7 cultures from Japan, United States, United Kingdom, France, and the Netherlands had the same genotype. Examination of the TE-7 cell line xenograft revealed the histology of a squamous cell carcinoma. We conclude that the TE-7 cell line, used in several laboratories throughout the world, is not an adenocarcinoma, but a squamous cell carcinoma cell line. Furthermore, the cell lines TE-2, TE-3, TE-7, TE-12, and TE-13 should be regarded as one single squamous cell carcinoma cell line.
View details for DOI 10.1158/0008-5472.CAN-07-2064
View details for Web of Science ID 000249406700013
View details for PubMedID 17804709
Gene expression changes associated with Barrett's esophagus and Barrett's-associated adenocarcinoma cell lines after acid or bile salt exposure
Esophageal reflux and Barrett's esophagus represent two major risk factors for the development of esophageal adenocarcinoma. Previous studies have shown that brief exposure of the Barrett's-associated adenocarcinoma cell line, SEG-1, or primary cultures of Barrett's esophageal tissues to acid or bile results in changes consistent with cell proliferation. In this study, we determined whether similar exposure to acid or bile salts results in gene expression changes that provide insights into malignant transformation.Using previously published methods, Barrett's-associated esophageal adenocarcinoma cell lines and primary cultures of Barrett's esophageal tissue were exposed to short pulses of acid or bile salts followed by incubation in culture media at pH 7.4. A genome-wide assessment of gene expression was then determined for the samples using cDNA microarrays. Subsequent analysis evaluated for statistical differences in gene expression with and without treatment.The SEG-1 cell line showed changes in gene expression that was dependent on the length of exposure to pH 3.5. Further analysis using the Gene Ontology, however, showed that representation by genes associated with cell proliferation is not enhanced by acid exposure. The changes in gene expression also did not involve genes known to be differentially expressed in esophageal adenocarcinoma. Similar experiments using short-term primary cultures of Barrett's esophagus also did not result in detectable changes in gene expression with either acid or bile salt exposure.Short-term exposure of esophageal adenocarcinoma SEG-1 cells or primary cultures of Barrett's esophagus does not result in gene expression changes that are consistent with enhanced cell proliferation. Thus other model systems are needed that may reflect the impact of acid and bile salt exposure on the esophagus in vivo.
View details for DOI 10.1186/1471-230X-7-24
View details for Web of Science ID 000248124700001
View details for PubMedID 17597535
View details for PubMedCentralID PMC1925102
Gene expression profiling in lymph node-positive and lymph node-negative pancreatic cancer
2007; 34 (3): 325-334
The purpose of this study was to screen for genes related to lymph node metastasis by comparing the differences in the expression profile between pancreatic cancer with lymph node metastasis and one without, and to predict the invasiveness and the progression of pancreatic cancer on the basis of these findings.The total RNA of the tissues was extracted from 10 pancreatic cancer specimens, including those with lymph node metastasis and those with no metastasis. It was studied by means of a DNA microarray (oligo chip) consisting of 37,842 genes. We screened out 1.5-fold or more differential gene expressions in at least 5 pairs of samples. We classified both samples and genes using a 2-way clustering analysis. The screened-out genes were identified using real-time polymerase chain reaction.We identified 194 genes, including 66 overexpressed and 128 underexpressed genes, in pancreatic cancer with lymph node metastasis. Among them, we identified some genes related to lymph node metastasis in patients with pancreatic cancer: oncogenes, tumor suppressor genes, apoptosis and antiapoptosis genes, tumor angiogenesis factors, and cell cycle regulators. Genes promoting the growth of tumor cells were highly expressed in lymph node-positive pancreatic cancer, whereas genes inducing apoptosis were underexpressed.We have identified genes that may play an important role in metastasis and survival in patients with pancreatic cancer. These results provide new insight into the study of human pancreatic cancer metastasis, including lymph node metastasis, and ultimately may lead to improving early diagnosis and discovering innovative therapeutic approaches for cancer.
View details for Web of Science ID 000246500000007
View details for PubMedID 17414055
The pancreatic stellate cell: a star on the rise in pancreatic diseases
JOURNAL OF CLINICAL INVESTIGATION
2007; 117 (1): 50-59
Pancreatic stellate cells (PaSCs) are myofibroblast-like cells found in the areas of the pancreas that have exocrine function. PaSCs are regulated by autocrine and paracrine stimuli and share many features with their hepatic counterparts, studies of which have helped further our understanding of PaSC biology. Activation of PaSCs induces them to proliferate, to migrate to sites of tissue damage, to contract and possibly phagocytose, and to synthesize ECM components to promote tissue repair. Sustained activation of PaSCs has an increasingly appreciated role in the fibrosis that is associated with chronic pancreatitis and with pancreatic cancer. Therefore, understanding the biology of PaSCs offers potential therapeutic targets for the treatment and prevention of these diseases.
View details for DOI 10.1172/JCI30082
View details for Web of Science ID 000243538400007
View details for PubMedID 17200706
View details for PubMedCentralID PMC1716214
Gene expression profiling reveals stromal genes expressed in common between Barrett's esophagus and adenocarcinoma
2006; 131 (3): 925-933
Barrett's esophagus is a precursor of esophageal adenocarcinoma. DNA microarrays that enable a genome-wide assessment of gene expression enhance the identification of specific genes as well as gene expression patterns that are expressed by Barrett's esophagus and adenocarcinoma compared with normal tissues. Barrett's esophagus length has also been identified as a risk factor for progression to adenocarcinoma, but whether there are intrinsic biological differences between short-segment and long-segment Barrett's esophagus can be explored with microarrays.Gene expression profiles for endoscopically obtained biopsy specimens of Barrett's esophagus or esophageal adenocarcinoma and associated normal esophagus and duodenum were identified for 17 patients using DNA microarrays. Unsupervised and supervised approaches for data analysis defined similarities and differences between the tissues as well as correlations with clinical phenotypes.Each tissue displays a unique expression profile that distinguishes it from others. Barrett's esophagus and esophageal adenocarcinoma express a unique set of stromal genes that is distinct from normal tissues but similar to other cancers. Adenocarcinoma also showed lower and higher expression for many genes compared with Barrett's esophagus. No difference in gene expression was found between short-segment and long-segment Barrett's esophagus.The genome-wide assessment provided by current DNA microarrays reveals many candidate genes and patterns not previously identified. Stromal gene expression in Barrett's esophagus and adenocarcinoma is similar, indicating that these changes precede malignant transformation.
View details for DOI 10.1053/j.gastro.2006.04.026
View details for Web of Science ID 000240561800030
View details for PubMedID 16952561
View details for PubMedCentralID PMC2575112
- The genome is now accessible to the endoscopist GASTROINTESTINAL ENDOSCOPY 2006; 64 (1): 27-28
Absence of the major zymogen granule membrane protein, GP2, does not affect pancreatic morphology or secretion
JOURNAL OF BIOLOGICAL CHEMISTRY
2004; 279 (48): 50274-50279
The majority of digestive enzymes in humans are produced in the pancreas where they are stored in zymogen granules before secretion into the intestine. GP2 is the major membrane protein present in zymogen granules of the exocrine pancreas. Numerous studies have shown that GP2 binds digestive enzymes such as amylase, thereby supporting a role in protein sorting to the zymogen granule. Other studies have suggested that GP2 is important in the formation of zymogen granules. A knock-out mouse was generated for GP2 to study the impact of the protein on pancreatic function. GP2-deficient mice displayed no gross signs of nutrient malab-sorption such as weight loss, growth retardation, or diarrhea. Zymogen granules in the GP2 knock-out mice appeared normal on electron microscopy and contained the normal complement of proteins excluding GP2. Primary cultures of pancreatic acini appropriately responded to secretagogue stimulation with the secretion of digestive enzymes. The course of experimentally induced pancreatitis was also examined in the knock-out mice because proteins known to associate with GP2 have been found to possess a protective role. When GP2 knock-out mice were subjected to two different models of pancreatitis, no major differences were detected. In conclusion, GP2 is not essential for pancreatic exocrine secretion or zymogen granule formation. It is unlikely that GP2 serves a major intracellular role within the pancreatic acinar cell and may be functionally active after it is secreted from the pancreas.
View details for DOI 10.1074/jbc.M410599200
View details for Web of Science ID 000225229500089
View details for PubMedID 15385539
Effects of GP2 expression on secretion and endocytosis in pancreatic AR4-2J cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2004; 322 (1): 320-325
GP2 is the major membrane protein present in secretory granules of the exocrine pancreas. GP2's function is unknown, but a role in digestive enzyme packaging or secretion from secretory granules has been proposed. In addition, GP2 has been proposed to influence endocytosis and membrane recycling following stimulated secretion. Adenovirus-mediated GP2 overexpression in the rat pancreatic cell line AR4-2J was used to study its impact on digestive enzyme secretion and membrane recycling. Immunoelectron microscopy showed that GP2 and amylase co-localized in secretory granules in infected AR4-2J cells. CCK-8 stimulation resulted in a fourfold increase in amylase secretion with or without GP2 expression. GP2 expression also did not influence endocytosis following CCK-8 stimulation. Thus, GP2 expression in AR4-2J cells does not affect amylase packaging in secretory granules or stimulated secretion. GP2 expression also does not influence membrane recycling in response to stimulated stimulation in AR4-2J cells.
View details for DOI 10.1016/j.bbrc.2004.07.120
View details for Web of Science ID 000223581000045
View details for PubMedID 15313209
Determination of plasma glycoprotein 2 levels in patients with pancreatic disease
ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE
2004; 128 (6): 668-674
Blood tests possessing higher diagnostic accuracy are needed for all the major pancreatic diseases. Glycoprotein 2 (GP2) is a protein that is specifically expressed by the pancreatic acinar cell and that has previously shown promise as a diagnostic marker in animal models of acute pancreatitis.This study describes the development of an assay for GP2, followed by the determination of plasma GP2 levels in patients with acute pancreatitis, chronic pancreatitis, and pancreatic cancer.Rabbit polyclonal antisera and mouse monoclonal antibodies were generated against human GP2 and used to develop an enzyme-linked immunosorbent assay. The assay was tested in patients with an admitting diagnosis of pancreatic disease at 2 tertiary care facilities. The diagnosis of acute or chronic pancreatitis and pancreatic cancer was determined using previously established criteria that incorporated symptoms, radiology, pathology, and serology. Plasma GP2 levels were determined in 31 patients with acute pancreatitis, 16 patients with chronic pancreatitis, 36 patients with pancreatic cancer, and 143 control subjects without pancreatic disease. Amylase and lipase levels were also determined in patients with acute pancreatitis.The GP2 assay's sensitivity values were 0.94 for acute pancreatitis, 0.81 for chronic pancreatitis, and 0.58 for pancreatic cancer, which were greater than the 0.71 for acute pancreatitis and 0.43 for chronic pancreatitis (P =.02) observed for amylase. The lipase assay sensitivity for acute pancreatitis was 0.66. The accuracy of the GP2 assay was greater than that of the amylase or lipase assays for acute pancreatitis (GP2 vs lipase, P =.004; GP2 vs amylase, P =.003) when analyzed using receiver operator characteristic curves. When daily serial blood samples were obtained for 13 patients with acute pancreatitis, GP2 levels remained abnormally elevated for at least 1 day longer than the amylase or lipase levels.The GP2 assay is a useful new marker for acute and chronic pancreatitis.
View details for Web of Science ID 000221753000010
View details for PubMedID 15163232
Exploration of global gene expression patterns in pancreatic adenocarcinoma using cDNA microarrays
AMERICAN JOURNAL OF PATHOLOGY
2003; 162 (4): 1151-1162
Pancreatic cancer is the fifth leading cause of cancer death in the United States. We used cDNA microarrays to analyze global gene expression patterns in 14 pancreatic cancer cell lines, 17 resected infiltrating pancreatic cancer tissues, and 5 samples of normal pancreas to identify genes that are differentially expressed in pancreatic cancer. We found more than 400 cDNAs corresponding to genes that were differentially expressed in the pancreatic cancer tissues and cell lines as compared to normal pancreas. These genes that tended to be expressed at higher levels in pancreatic cancers were associated with a variety of processes, including cell-cell and cell-matrix interactions, cytoskeletal remodeling, proteolytic activity, and Ca(++) homeostasis. Two prominent clusters of genes were related to the high rates of cellular proliferation in pancreatic cancer cell lines and the host desmoplastic response in the resected pancreatic cancer tissues. Of 149 genes identified as more highly expressed in the pancreatic cancers compared with normal pancreas, 103 genes have not been previously reported in association with pancreatic cancer. The expression patterns of 14 of these highly expressed genes were validated by either immunohistochemistry or reverse transcriptase-polymerase chain reaction as being expressed in pancreatic cancer. The overexpression of one gene in particular, 14-3-3 sigma, was found to be associated with aberrant hypomethylation in the majority of pancreatic cancers analyzed. The genes and expressed sequence tags presented in this study provide clues to the pathobiology of pancreatic cancer and implicate a large number of potentially new molecular markers for the detection and treatment of pancreatic cancer.
View details for Web of Science ID 000181748300013
View details for PubMedID 12651607
View details for PubMedCentralID PMC1851213
Hyposmotic stress induces cell growth arrest via proteasome activation and cyclin/cyclin-dependent kinase degradation
JOURNAL OF BIOLOGICAL CHEMISTRY
2002; 277 (22): 19295-19303
Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.
View details for DOI 10.1074/jbc.M109654200
View details for Web of Science ID 000175894800009
View details for PubMedID 11897780
Processing of the major pancreatic zymogen granule membrane protein, GP2
2002; 24 (4): 336-343
The pancreatic exocrine secretory granule, the zymogen granule, releases digestive enzymes into the intestine. GP2 is the most abundant zymogen granule membrane protein. Coincident with exocrine secretion, GP2 is released from the membrane and secreted into the pancreatic duct.To characterize changes in the structure of GP2 as it progresses through the secretory pathway.Polarized MDCK cells that express the rat GP2 gene were used to examine the sequential processing of the polypeptide backbone.Within the cell, GP2 is initially proteolytically processed from a 55- to a 53-kd form at or before the trans-Golgi network. The protein is then processed to a 51-kd form, which is found on the apical plasma membrane and in secretions. Similar processing was also observed in primary rat pancreatic cultures and in MDCK cells that express human GP2. The amino-terminal sequence of human GP2 derived from pancreatic secretions was determined for two human patients and began at Gly39, revealing a potential processing site.In contrast to other digestive enzymes secreted by the pancreas that are activated by proteolysis in the intestine, GP2 undergoes sequential intracellular cleavage. Alterations in GP2 structure by proteolysis may regulate GP2 function at specific sites within the pancreatic cell.
View details for Web of Science ID 000175266300003
View details for PubMedID 11961485
Effects of keratin filament disruption on exocrine pancreas-stimulated secretion and susceptibility to injury
EXPERIMENTAL CELL RESEARCH
2000; 255 (2): 156-170
Disruption or absence of hepatocyte keratins 8 and 18 is associated with chronic hepatitis, marked hepatocyte fragility, and a significant predisposition to stress-induced liver injury. In contrast, pancreatic keratin disruption in transgenic mice that express keratin 18 Arg89 --> Cys (K18C) is not associated with an obvious pancreatic pathology. We compared the effects of keratin filament disruption on pancreatic acini or acinar cell viability, and on cholecystokinin (CCK)-stimulated secretion, in transgenic mice that overexpress wild-type keratin 18 and harbor normal extended keratin filaments (TG2) and K18C mice. We also compared the response of these mice to pancreatitis induced by a choline-deficient ethionine-supplemented diet or by caerulein. Despite extensive cytoplasmic keratin filament disruption, the apicolateral keratin filament bundles appear intact in the acinar pancreas of K18C mice, as determined ultrastructurally and by light microscopy. No significant pancreatitis-associated histologic, serologic, or F-actin/keratin apicolateral redistribution differences were noted between TG2 and K18C mice. Acinar cell viability and yield after collagenase digestion were lower in K18C than in TG2 mice, but the yields of intact acini and their (125)I-CCK uptake and responses to CCK-stimulated secretion were similar. Our results indicate that keratin filament reorganization is a normal physiologic response to pancreatic cell injury, but an intact keratin cytoplasmic filament network is not as essential in protection from cell injury as in the liver. These findings raise the possibility that the abundant apicolateral acinar keratin filaments, which are not as evident in hepatocytes, may play the cytoprotective role that is seen in liver and other tissues. Alternatively, identical keratins may function differently in different tissues.
View details for Web of Science ID 000085987200003
View details for PubMedID 10694432
Characterization of an alternatively spliced isoform of rat vesicle associated membrane protein-2 (VAMP-2)
1999; 451 (2): 209-213
VAMPs are vesicle associated membrane proteins that are essential for secretion. A spliced isoform of rat VAMP-2, called VAMP-2B, is characterized in this study. The VAMP-2B transcript is the result of alternative RNA splicing in which an intron is retained. The predicted amino acid sequence of VAMP-2B differs from VAMP-2 at its carboxy-terminal end. Because recent studies have shown that VAMP's carboxy-terminal end influences the protein's sorting, the location of myc-epitope tagged VAMP-2B in PC12 cells was determined. Subcellular fractionation showed colocalization of myc-VAMP-2B and endogenous VAMP-2. Thus alternative RNA splicing does not affect VAMP-2 sorting in PC12 cells.
View details for Web of Science ID 000080596800023
View details for PubMedID 10371166
Tissue-specific alternative RNA splicing of rat vesicle-associated membrane protein-1 (VAMP-1)
1997; 199 (1-2): 173-179
The vesicle-associated membrane protein (VAMP) family is essential to vesicle-mediated protein transport. Three mammalian isoforms, VAMP-1, VAMP-2, and cellubrevin, play a role in protein transport to the plasma membrane. In this study, we describe a new rat VAMP-1 isoform produced by alternative pre-mRNA splicing. Only one VAMP-1 isoform dominates in each tissue. Analysis of the nucleotide sequence for the newly discovered isoform, VAMP-1b, reveals that its expression is determined by whether an intron is retained or removed. The predicted amino acid sequences for the VAMP-1 isoforms differ at the carboxy-terminal end of the protein. A similar process has been described for VAMPs in Drosophila melanogaster and suggests a conserved function for the carboxy-terminal domain that can be modulated.
View details for Web of Science ID A1997YB73200022
View details for PubMedID 9358054
Sequence of the cDNA encoding human GP-2, the major membrane protein in the secretory granule of the exocrine pancreas
1996; 171 (2): 311-312
GP-2 is the major membrane protein in the pancreatic secretory granule of the exocrine pancreas. The nucleotide sequence of the human GP-2 gene was determined from cDNA clones isolated from a lambda gt11 pancreatic library.
View details for Web of Science ID A1996UR51800034
View details for PubMedID 8666297
Polarized GP2 secretion in MDCK cells via GPI targeting and apical membrane-restricted proteolysis
AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY
1996; 270 (1): G176-G183
The major zymogen granule membrane protein in the exocrine pancreas is glycoprotein 2 (GP2), a glycosyl phosphatidylinositol (GPI)-linked membrane protein. Despite its GPI anchor, GP2 is secreted into the pancreatic duct. We examined the mechanism underlying the secretion of GP2 in isolated pancreatic acini and transfected Madin-Darby canine kidney (MDCK) cells (MDCK-GP2). MDCK-GP2 cells release GP2 almost exclusively (> 95%) from the apical membrane. Using GP2 as a model, we defined a novel mechanism of polarized protein secretion in which a secretory protein is targeted via a GPI anchor to the apical plasma membrane, whereupon the mature form is released by proteolysis. Furthermore, we described two features of MDCK cells that enhance the polarized release of GP2: an apical plasma membrane-restricted distribution of the protease responsible for GP2 membrane cleavage, and a transcytotic pathway to reroute basolateral plasma membrane GP2 to the apical cell surface.
View details for Web of Science ID A1996TT39400023
View details for PubMedID 8772516
HIERARCHY OF MECHANISMS INVOLVED IN GENERATING NA/K-ATPASE POLARITY IN MDCK EPITHELIAL-CELLS
JOURNAL OF CELL BIOLOGY
1995; 130 (5): 1105-1115
We have studied mechanisms involved in generating a polarized distribution of Na/K-ATPase in the basal-lateral membrane of two clones of MDCK II cells. Both clones exhibit polarized distributions of marker proteins of the apical and basal-lateral membranes, including Na/K-ATPase, at steady state. Newly synthesized Na/K-ATPase, however, is delivered from the Golgi complex to both apical and basal-lateral membranes of one clone (II/J), and to the basal-lateral membrane of the other clone (II/G); Na/K-ATPase is selectively retained in the basal-lateral membrane resulting in the generation of complete cell surface polarity in both clones. Another basal-lateral membrane protein, E-cadherin, is sorted to the basal-lateral membrane in both MDCK clones, demonstrating that there is not a general sorting defect for basal-lateral membrane proteins in clone II/J cells. A glycosyl-phosphatidylinositol (GPI)-anchored protein (GP-2) and a glycosphingolipid (glucosylceramide, GlcCer) are preferentially transported to the apical membrane in clone II/G cells, but, in clone II/J cells, GP-2 and GlcCer are delivered equally to both apical and basal-lateral membranes, similar to Na/K-ATPase. To examine this apparent inter-relationship between sorting of GlcCer, GP-2 and Na/K-ATPase, sphingolipid synthesis was inhibited in clone II/G cells with the fungal metabolite, Fumonisin B1 (FB1). In the presence of FB1, GP-2 and Na/K-ATPase are delivered to both apical and basal-lateral membranes, similar to clone II/J cells; FB1 had no effect on sorting of E-cadherin to the basal-lateral membrane of II/G cells. Addition of exogenous ceramide, to circumvent the FB1 block, restored GP-2 and Na/K-ATPase sorting to the apical and basal-lateral membranes, respectively. These results show that the generation of complete cell surface polarity of Na/K-ATPase involves a hierarchy of sorting mechanisms in the Golgi complex and plasma membrane, and that Na/K-ATPase sorting in the Golgi complex of MDCK cells may be regulated by exclusion from an apical pathway(s). These results also provide new insights into sorting pathways for other apical and basal-lateral membrane proteins.
View details for Web of Science ID A1995RR84100009
View details for PubMedID 7657695
THE LEVEL OF THE ZYMOGEN GRANULE PROTEIN GP2 IS ELEVATED IN A RAT MODEL FOR ACUTE-PANCREATITIS
1994; 107 (6): 1819-1827
GP2 is the major membrane protein in pancreatic zymogen granules. It is linked to the membrane via a glycosyl-phosphatidylinositol linkage. After cleavage, a significant fraction of GP2 becomes soluble. The present study assessed whether GP2 is a useful serum marker for acute pancreatitis.Using an anti-GP2 monoclonal antibody, an enzyme-linked immunosorbent assay was developed to measure the serum levels of GP2 in rats with cerulein-induced acute pancreatitis.The anti-GP2 antibody was specific because it did not cross-react with uromodulin, a structurally similar protein to GP2, or to protein extracts from nonpancreatic tissues. Eight hours after the induction of pancreatitis, the serum levels of amylase, lipase, and GP2 peaked. Peak GP2 levels were 4.2 times higher than those of controls. At 24 hours, GP2 was still 70% of the peak level, whereas amylase and lipase were 5.5% and 0.5%, respectively, of their peak levels.GP2 may serve as a potentially valuable marker for clinical acute pancreatitis.
View details for Web of Science ID A1994PU54300032
View details for PubMedID 7525398
APICAL PLASMA-MEMBRANE PROTEINS ARE NOT OBLIGATORILY STORED IN SECRETORY GRANULES IN EXOCRINE CELLS
JOURNAL OF CELL SCIENCE
1994; 107: 2271-2277
Exocrine cells are epithelial cells in which secretory granules undergo fusion with the apical plasma membrane upon secretagogue stimulation. Several apical plasma membrane proteins have been found in secretory granules in cells from pancreas and salivary glands raising the possibility that incorporation into secretory granules followed by exocytosis of the granules accounts for their insertion into the apical plasma membrane. To test this hypothesis, we have expressed the influenza hemagglutinin (HA) in pancreatic AR42J cells, which make zymogen-like granules upon incubation with dexamethasone. The influenza virus HA is known to be specifically targeted to the apical plasma membrane of epithelial cells that lack a regulated pathway and is also known to be excluded from secretory granules in virally-infected pituitary AtT20 cells. Localization of the protein by immunofluorescence microscopy revealed that it accumulated at the plasma membrane of the transfected AR42J cells. HA was not observed in the amylase-rich secretory granules. By immunolabeling of ultrathin cryosections of the transfected cells, HA was also found exclusively on the cell surface, with label over secretory granules not exceeding that seen in control, untransfected cells. In addition, in cell fractionation experiments performed on radiolabeled AR42J cell transformants, HA was not detectable in the secretory granule fractions. These results indicate that HA is not efficiently stored in mature secretory granules and is likely to reach the cell surface via constitutive transport pathways.
View details for Web of Science ID A1994PD47500022
View details for PubMedID 7983185
IDENTIFICATION OF A VESICLE-ASSOCIATED MEMBRANE-PROTEIN (VAMP)-LIKE MEMBRANE-PROTEIN IN ZYMOGEN GRANULES OF THE RAT EXOCRINE PANCREAS
JOURNAL OF BIOLOGICAL CHEMISTRY
1994; 269 (7): 5328-5335
Zymogen granules of the exocrine pancreas are the secretory organelles responsible for the regulated secretion of digestive enzymes. Several proteins are associated with or are integral components of the lipid bilayer that forms the zymogen granule membrane. These proteins likely represent important components in the regulated secretion of digestive enzymes. VAMPs (vesicle-associated membrane proteins)/synaptobrevins are a family of 18-kDa integral membrane proteins originally characterized in synaptic vesicles. Polyclonal antisera raised against either a VAMP/glutathione S-transferase (GST) fusion protein or rat brain synaptic vesicles, detected an 18-kDa immunoreactive protein in zymogen granule membranes that co-migrates electrophorectically with rat brain synaptic vesicle VAMP. Rat brain synaptic vesicle VAMP was detected by both antisera. Botulinum-B toxin treatment of zymogen granule membranes did not result in cleavage of zymogen granule membrane VAMP, indicating that exocrine pancreatic VAMP is either VAMP1 or a novel VAMP-isoform. Immunofluorescent studies demonstrated that exocrine pancreatic VAMP localized with GP2, a zymogen granule membrane protein, to the apical region of pancreatic acinar cells. No significant labeling was observed in basolateral regions of pancreatic acinar cells. These results establish the presence of a VAMP protein in the zymogen granule of the rat pancreas and suggest that VAMPs have a role in exocrine secretion.
View details for Web of Science ID A1994MX57100095
View details for PubMedID 8106518
ANTISENSE OLIGODEOXYNUCLEOTIDES TO THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR INHIBIT CAMP-ACTIVATED BUT NOT CALCIUM-ACTIVATED CHLORIDE CURRENTS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1992; 89 (15): 6785-6789
Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway.
View details for Web of Science ID A1992JF85600026
View details for PubMedID 1379720