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[Cancer Research 63, 7032-7037, November 1, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

Increased Expression and Processing of the Alzheimer Amyloid Precursor Protein in Pancreatic Cancer May Influence Cellular Proliferation

Donna E. Hansel1, Ayman Rahman, Sven Wehner, Volker Herzog, Charles J. Yeo and Anirban Maitra

Departments of Pathology [D. E. H., A. R., A. M.], Surgery [C. J. Y.], and Oncology [C. J. Y.], The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the Institute of Cell Biology and Bonner Forum Biomedizin, University of Bonn, D-53115 Bonn, Germany [S. W., V. H.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Abnormal cleavage of amyloid precursor protein (APP) in the central nervous system has been linked to the development of Alzheimer’s disease. Recent work has identified additional roles for APP in peripheral tissue, such as cellular proliferation and motility. APP undergoes proteolytic processing to release a soluble NH2-terminal ectodomain fragment (sAPP), an Aß or p3 peptide, and cytosolic COOH-terminal fragments. We have identified the up-regulation of APP expression in pancreatic cancer cells both in vitro and in vivo. APP undergoes high levels of proteolytic processing in pancreatic cancer cells, and sAPP can be detected in collected medium in vitro. Inhibition of sAPP signaling reduces pancreatic cancer cell number via a reduction in cellular proliferation. We propose that APP may function to promote growth in pancreatic cancer cells via signaling through sAPP and may therefore represent a novel therapeutic target in pancreatic cancer.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The Alzheimer APP2 /protease nexin II, located on chromosome locus 21q21.3, was originally identified as a key component in plaque formation in the brains of patients with Alzheimer’s disease and Down’s syndrome (1) . APP contains a single transmembrane domain and undergoes two-step, {gamma}-secretase-dependent proteolytic processing on the cell surface to release sAPP, Aß or p3 peptide, and CTFs (Fig. 1ACitation ; Refs. 2 and 3 ). Although the abnormal cleavage of APP at altered sites leads to amyloid deposition in the nervous system, the physiological function of APP has not been well defined. APP is expressed at high levels in the central nervous system and peripheral tissues, including epidermis, thyroid, and pancreas (4, 5, 6) . Both the NH2- and COOH-terminal domains of APP have been proposed to mediate various functions, including cell growth and process extension (7) . Because these processes are critical in cancer growth, we examined APP expression and processing in pancreatic cancer, which has been proposed to arise from alterations in the epithelium of small intralobular ducts and represents a rapidly growing form of epithelium-derived cancer. We have identified the novel expression and high-level processing of APP in pancreatic cancer cells and a putative role for the sAPP fragment in pancreatic cancer cell proliferation.



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  		Fig. 1. Expression of APP, processing enzymes components, and APP interactors in pancreatic cancer cells. A, diagramatic representation of the full-length, membrane-spanning APP protein, proteolytic products, and downstream signaling cascades. B, RT-PCR for APP and interactors in HPDE cells and APP in PancI cells. C, RT-PCR on PancI and HS766T cells for APP and interactors. In B and C, GAPDH was performed as a standardization, and water was used as a negative control.

 

   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
RT-PCR.
RNA was prepared using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). cDNA was prepared per the manufacturer’s instructions (Invitrogen, Inc., Carlsbad, CA). PCR was performed in a final mixture of 1x Platinum PCR Supermix (Invitrogen, Inc.), 400 nM forward and reverse primer, and 2 µl of cDNA. PCR reactions were performed in a Thermo Hybaid MBS 0.2S cycler as follows: 96°C for 5 min, 35 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 3 min, incubation at 72°C for 5 min, and a final hold temperature of 4°C. Samples were run on a 1% agarose gel in Tris-buffered saline. Primers were as follows: APP foward primer, 5'-TCCATTCATCATGGTGTGGT-3'; APP reverse primer, 5'-ACACCGATGGGTAGTGAAGC-3'; PS-1 forward primer, 5'-AGATGCCTCCTCTGTCCTCA-3'; PS-1 reverse primer, 5'-CCGTCTTTGGGCATACATCT-3'; JIP-1 forward primer, 5'-TGGAGGTGAAGTCCCTGTTC-3'; JIP-1 reverse primer, 5'-AGGGTGACAGGACCAAGATG-3'; Fe65 forward primer, 5'-TCCCCAGAGGACACAGATTC-3'; Fe65 reverse primer, 5'-GTGAGCTGGGACTCCTCTTG-3'; SHCA forward primer, 5'-GCATGGACTCTGGGTTTCAT-3'; SHCA reverse primer, 5'-TCACCACAGGACATTTTCCA-3'; APPBP1 forward primer, 5'-TACCATTGCTGCATTCTTGG-3'; and APPBP1 reverse primer, 5'-ACAACCCGAAGGCAATTACA-3'. GAPDH primers were used for standardization in PCR reactions.

Cell Culture, Antibody Incubation, and BrdUrd Labeling.
Pancreatic cancer cell lines PancI, HS766T, and BxPC3 were purchased from the American Type Culture Collection cell line bank (Manassas, VA). HPDE cells represent an immortal cell line from normal pancreatic duct epithelium (HPDE) transfected by the E6 and E7 gene of human papillomavirus 16 (8) . Cells were grown on 10-cm Falcon dishes (Becton Dickinson Labware, Franklin Lakes, NJ) in DMEM (GIBCO, Invitrogen Corp., Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO). Twenty-four h before antibody or sAPP addition, the cell culture medium was replaced with DMEM/0.5% fetal bovine serum. Antibody incubation was performed with the appropriate dilution of mouse control IgG1 (Cymbus Technology, Chandlers Ford, United Kingdom) or monoclonal APP A4/22C11 (NH2-terminal antibody; Chemicon International, Temecula, CA) for 24 h. Cells were subsequently labeled for 6 h using the BrdUrd Labeling Kit I (Roche Diagnostics, Indianapolis, IN) and stained per protocol. sAPP was added to cell culture medium at the start of the 6-h BrdUrd incubation period. Medium collection was performed over a 24-h period in DMEM, and media were centrifuged to remove particulate matter and concentrated 10x using a Savant DNA Speed Vac (GMI, Albertville, MN).

Immunocytochemistry.
Cells were fixed in 4% paraformaldehyde for 20 min, rinsed in PBS, permeabilized with 0.075% Triton X-100, blocked for 20 min in 2 mg/ml BSA (Sigma, St. Louis, MO) in PBS (BSA/PBS), and incubated primary antibody diluted in 2 mg/ml BSA/PBS for 2 h at room temperature. Antibodies used for study include COOH-terminal APP antibodies [1:500 (Chemicon International) and 1:500 (Sigma)] and an NH2-terminal APP antibody (1:500; Chemicon International). Cells were then rinsed and incubated for 1 h in donkey antirabbit FabCy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Phalloidin colabeling was performed with a 1:1000 dilution of FITC-labeled reagent (Sigma). Coverslipping was performed using Vectashield mounting media with DAPI (Vector Laboratories, Burlingame, CA). For peptide blocking experiments, a final dilution of 1:500 COOH-terminal antibody (Cell Signaling Technology, Beverly, MA) and 100 µg/ml peptide was performed for 1 h in PBS before immunolabeling.

Microscopy.
Slides were imaged with a Zeiss Axioskop epifluorescence microscope equipped with short-arc mercury lamp illumination (Carl Zeiss Inc., Thornwood, NY) and a x100/1.4 NA oil immersion Neofluar lens. Fluorescent images were captured with a cooled charge-coupled device camera (Micro MAX digital camera; Princeton Instruments, Trenton, NJ).

Cell Extracts and Western Blotting.
Confluent cells were extracted into 62.5 mM Tris-HCl (pH 6.8), 2% w/v SDS, 10% glycerol, 50 mM DTT, and 0.01% w/v bromphenol blue; sonicated for three 10-s intervals; and boiled for 5 min. Proteins were fractionated on SDS-polyacrylamide gels containing 12% acrylamide/0.4% bis-acrylamide and transferred to Immobilon-P membranes (Millipore, Bedford, MA) in 25 mM Tris-HCl (pH 8.5), 200 mM glycine, and 20% methanol for 2 h at 100 mA. Blots were blocked with 5% skim milk diluted in 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl containing 0.05% Tween 20, incubated in a 1:1000 dilution of a COOH-terminal APP antibody (Chemicon) or a 1:1000 dilution of NH2-terminal antibody (22C11; Chemicon), and rinsed. Blots were then incubated for 1 h with horseradish peroxidase-conjugated donkey antirabbit or antimouse IgG antibody (1:5000; Amersham Corp.) and visualized using the Enhanced Chemiluminescence Kit (Amersham Corp.). Exposure times ranged from 30 s to 2 min.

Patient Material.
Permission for this study was obtained from the Johns Hopkins Joint Committee for Clinical Investigation. Paraffin-embedded material from a series of 10 pancreatectomies containing pancreatic cancer resected at the Johns Hopkins Hospital (Baltimore, MD) between 1996 and 2001 was used for this study.

Immunohistochemistry.
Sections (3–4 µm) from paraffin-embedded tissue were used for immunohistochemistry. Slides were deparaffinized in fresh xylenes and rehydrated through sequential graded ethanol steps. Antigen retrieval was performed by citrate buffer incubation [18 mM citric acid/8 mM sodium citrate (pH 6)] using a household vegetable steamer (Black and Decker) for 60 min. Slides were incubated for 5 min with 3% hydrogen peroxide; washed in 20 mM Tris, 140 mM NaCl, and 0.1% Tween 20 (pH 7.6); and incubated in appropriate antibody dilution for APP (1:100; COOH-terminal domain; Cell Signaling Technology) for 60 min at room temperature. The avidin-biotin-peroxidase complex method from DAKO (Glostrup, Denmark) was used to visualize antibody binding, and slides were subsequently counterstained with hematoxylin.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
APP and APP Cytosolic Interactors Are Expressed in Pancreatic Cancer Cells.
Using RT-PCR analysis, we examined the expression of APP and its cytosolic interactors JIP1b, Fe65, ShcA, and APPBP1 in the pancreatic cancer cell lines PancI and HS766T and the human ductal epithelium line HPDE. We also examined these cells for the presence of PS-1, a key component in the {gamma}-secretase-dependent cleavage of APP (9) . HPDE cells demonstrated the presence of most components of the APP processing and signaling pathway, except PS-1 (Fig. 1B)Citation ; PancI cells demonstrated increased levels of APP as compared with HPDE cells (Fig. 1BCitation ; GAPDH standardization was performed as a control). All putative cytosolic interactors for APP were present with pancreatic cancer cells (Fig. 1C)Citation .

APP Localization in Pancreatic Cancer Cells.
APP is synthesized at ribosomal sites, inserted into the membrane of the endoplasmic reticulum, and posttranslationally modified in the Golgi network (10) . We performed immunofluorescent localization of APP in BxPC3 and PancI cells using antibodies directed against the NH2 and COOH termini. Immunolabeling of BxPC3 and PancI cells with two antibodies directed against different COOH-terminal epitopes revealed a similar perinuclear distribution pattern of APP (Fig. 2, A and B)Citation , which has been previously described to colocalize with markers of the Golgi complex, such as mannosidase II (5) . Immunolabeling with an NH2-terminal APP antibody revealed a diffuse pattern in BxPC3 cells but an intense, perinuclear localization in PancI cells (Fig. 2, C and D)Citation . The diffuse pattern identified on the surface of BxPC3 cells is reminiscent of that identified with labeled sAPP in multiple cell types (11) . HPDE cells demonstrated perinuclear distribution of APP in only scattered cells (<5%; data not shown).



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  		Fig. 2. Localization of the NH2- and COOH-terminal domains of APP in pancreatic cancer cells in vitro. An antibody against the COOH-terminal domain of APP, which recognizes immature and mature forms of the protein, was used for labeling BxPC3 (A) and PancI (B) cells. Fluorescent overlay reveals labeling for APP (red), phalloidin (green), and DAPI (blue) (magnification, x400). APP NH2-terminal labeling (red) is shown on BxPC3 (C) and PancI (D) cells. Detail of APP COOH-terminal antibody labeling of PancI cells [APP, E; phalloidin, F; DAPI, G; overlay, H]. Magnification, x1000, oil immersion. Antibody incubation with the COOH-terminal antibody (I) or peptide-blocked antibody (J) was performed on BxPC3 cells.

 
Analysis of the COOH-terminal labeling of APP identified a punctate staining pattern on the cell membrane (Fig. 2, E–H)Citation , suggesting that APP may localize to specific complexes on the cell surface. A similar pattern has been described in APP-overexpressing systems in which APP and Fe65 colocalize at dynamic adhesion sites and induce cell migration (12) . Peptide blocking experiments for this antibody demonstrated complete abrogation of antibody signal (Fig. 2, I and J)Citation .

APP Undergoes Variable Levels of Proteolytic Processing in Pancreatic Cancer Cell Lines.
The APP protein exists as multiple isoforms produced through alternative splicing of APP mRNA (1) . Although much of APP remains as full-length protein (Mr 105,000–160,000) at cellular membranes, APP can undergo proteolytic processing to release a soluble Mr ~100,000 extracellular NH2-terminal fragment (sAPP), a soluble Mr 3,000 (p3) or Mr 4,500 (Aß) extracellular fragment, and Mr 10,000–12000 CTFs.

We examined APP protein products in HPDE and the pancreatic cancer cell lines HS766T, PancI, BxPC3, and CFPC1 using a COOH-terminal-directed antibody. Variable levels of proteolytic processing of APP were apparent in each cell line, with full-length APP present in HS766T, PancI, and CFPC1 cells (Fig. 3ACitation , FL) and in BxPC3 cells on prolonged exposure. BxPC3 cells, however, demonstrated high levels of CTF products, indicating that virtually all APP produced in this cell line undergoes complete proteolytic processing (Fig. 3ACitation , CTF). In contrast, CFPC1 cells demonstrated only full-length APP and no identifiable CTFs. Blots were reprobed with an actin antibody to ensure equal protein levels.



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  		Fig. 3. NH2-terminal solubilization of APP influences cell proliferation. A, HPDE cells produce no detectable APP by Western blot analysis using an antibody against the COOH-terminal domain. In contrast, APP is produced by all pancreatic cancer cell types examined (HS766T, PancI, BxPC3, and CFPC1). Full-length APP (FL) is detectable in all cells (BxPC3 with prolonged exposure). In BxPC3 cells, APP appears to undergo virtually complete proteolytic processing into CTFs. Solubilized extracellular products are not detectable by this Western analysis of whole cell extracts. Actin was used for protein standardization. B, whole cell extracts were probed with an antibody directed against sAPP. Full-length APP was identified with this antibody in CFPC1 cells, but not BxPC3 cells, indicating solubilization of this product from the surface of BxPC3 cells. C, recovery of the NH2-terminal fragment of APP from the medium of BxPC3 cells. BxPC3 cells were next incubated with mouse IgG1 at a 1:100 dilution (D) or APP blocking antibody A4/22C11 at a dilution of 1:100 (E) or 1:250 (F) for 24 h. A 6-h BrdUrd labeling pulse demonstrated a reduced number of BrdUrd-positive cells in A4/22C11-treated cultures that was significant by Student’s t test analysis (G, asterisk). Values represent the percentage of IgG1 control cells. Experiments were performed six times; greater variability in BrdUrd labeling was apparent at a 1:500 dilution. H, addition of exogenous sAPP to BxPC3 cultures at either 8 or 16 nM increases cellular proliferation by 15% and 38%, respectively. Student’s t test analysis revealed that 16 nM sAPP produced a significant increase in BrdUrd labeling (asterisk).

 
Because proteolytic processing of APP can release sAPP from the cell surface, we examined the presence of the NH2-terminal APP domain both in cell extracts and harvested medium. Whole cell extracts revealed the presence of the noncleaved NH2-terminal region of APP in CFPC1 cells, but not BxPC3 cells (Fig. 3B)Citation . Furthermore, Western blotting of 10x concentrated medium using an antibody specific for sAPP identified a soluble sAPP product in the medium of BxPC3 cells (Fig. 3C)Citation . These findings suggest that sAPP is cleaved and released from BxPC3 cells and may therefore perform a signaling role in a subset of pancreatic cancer cells.

Incubation of BxPC3 Cells with Blocking Antibody Toward sAPP Reduces BxPC3 Cell Number and Proliferative Index.
The monoclonal antibody A4/22C11 directed against sAPP has previously been demonstrated to bind to sAPP and reduce sAPP695-induced neural stem cell proliferation (11 , 13) . We therefore performed BrdUrd labeling of BxPC3 cells in the presence of control IgG1 antibody or 22C11 at various dilutions to determine a potential role for sAPP in pancreatic cancer cell growth.

BxPC3 cells grow in clusters (see Fig. 2ACitation ), and incubation of these cells with a 1:100 dilution of control antibody did not significantly alter cellular morphology (Fig. 3D)Citation . An identical dilution of A4/22C11, however, decreased the total number of cells (Fig. 3E)Citation , as well as the number of cell clusters present (Fig. 3, E and F)Citation . A 6-h BrdUrd pulse in the presence of a 1:100 dilution of 22C11 antibody significantly reduced proliferation to approximately 60–70% of control (Fig. 3G)Citation . A 1:250 dilution also significantly reduced BrdUrd labeling, albeit to a lesser extent. These findings suggest that sAPP may function in these cells to mediate cell number through effects on proliferative activity.

Addition of Exogenous sAPP Increases BXPC3 Proliferation.
We next incubated cells with exogenous, purified sAPP to determine whether addition of this peptide would increase cell proliferation over baseline levels. Addition of 8 nM sAPP increased proliferation of BxPC3 cells 15% over baseline (P = 0.16436), and addition of 16 nM sAPP significantly increased proliferation by 38% over baseline (P = 0.04485; Fig. 3HCitation ). The concentration of sAPP used in these experiments is similar to that used previously to identify an increase in cell proliferation in basal cells of the epidermis (4) , suggesting a physiological role for sAPP in both normal and disease states.

APP Is Expressed in Human Pancreatic Cancer Specimens.
To determine the expression of APP in human pancreas and pancreatic cancer specimens, we performed immunohistochemical labeling using an antibody directed against the COOH-terminal of APP on 10 resected pancreatic cancer specimens and surrounding normal pancreas. Normal adult human pancreas expressed signal in the majority of pancreatic cells, including acinar and islet cells (Fig. 4)Citation . Small and large ductal epithelium, however, demonstrated no immunoreactivity for APP (Fig. 4Citation A, arrowhead). Pancreatic cancer specimens demonstrated robust immunolabeling for APP (Fig. 4, B and C)Citation , confirming in vitro findings of increased expression in pancreatic cancer cells as compared with normal ductal epithelium. Incubation of antibody with blocking peptide eliminated all staining (data not shown).



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  		Fig. 4. Immunolabeling of pancreatic cancer specimens using an antibody against the COOH-terminal of APP. A, APP is expressed in pancreatic acini, but not ductal epithelium (arrowhead). Magnification, x400. B, pancreatic cancer demonstrates expression of APP, x100. C, inset of box from B, x400.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
APP undergoes proteolytic processing to yield multiple peptide products, including soluble NH2- and COOH-terminal domains, which may function in extracellular or intracellular signaling, respectively. The majority of pancreatic cancer cells demonstrate high levels of proteolytic processing of APP, suggesting that these processed peptides may serve an important role in influencing pancreatic cancer cell growth or migration.

APP expression in the normal adult pancreas appears to be localized to acinar and islet cells, but not ductal epithelium cells. Because ductal epithelium has been proposed to give rise to pancreatic adenocarcinoma through a series of genetic alterations, the lack of detectable expression in these cells suggests an up-regulation of APP expression in pancreatic carcinogenesis. The expression of APP in both pancreatic cancer specimens by immunohistochemistry and in pancreatic cancer cell lines by immunocytochemistry confirms this finding.

To further explore up-regulation of APP transcripts in pancreatic adenocarcinoma, we examined online SAGE libraries of pancreatic cancer and normal ductal epithelium (HX and H126). SAGE profiling indicates tags for APP in the pancreatic cancer cell line CAPAN1 (26 tags/million), but not normal ductal epithelium cells. Furthermore, a large increase in SAGE tag number for PS-1, a necessary component of the {gamma}-secretase complex that cleaves APP, is also evident in pancreatic cancer cells and may explain the high levels of proteolytic processing of APP in these cells. Up-regulation of APP promoter activity and increased APP expression have been described secondary to cytokine and growth factor addition in physiological systems (14 , 15) , and similar effects may occur within a cancerous milieu.

sAPP can be cleaved close to the transmembrane domain of APP through the actions of {alpha}-secretase under normal conditions or ß-secretase under pathological conditions such as Alzheimer’s disease. sAPP has been demonstrated to bind to the cell membrane of thryocytes and fibroblasts (16) . In studies using radiolabeled peptide, sAPP competitively bound to localized patches on FRTL-5 and PC-12 cell membranes, indicating the presence of a specific receptor for sAPP (11) . Furthermore, preferential binding of sAPP occurs at undifferentiated cell membranes, suggesting a putative role for this peptide fragment in cell growth or differentiation (11) .

In our studies, the APP NH2-terminal appeared to be fully cleaved and solubilized in BxPC3 cells. Immunolabeling for sAPP demonstrated a fine punctate membranous pattern in these cells that was not evident in Panc1 cells and may reflect surface-associated sAPP in BxPC3 cells.

Incubation of fibroblasts and thyrocytes with sAPP results in an up-regulation of cell proliferation, and sAPP has been shown to mediate this growth-promoting effect through MAP kinase phosphorylation in FRTL-5 thyroid epithelium cells (16) . In addition to effects on the cell cycle, sAPP has also been demonstrated to alter cell morphology by inducing the extension of neurite processes in neuroblastoma cells (17) . Blockade of sAPP signaling in BxPC3 cells demonstrates a reduction in the overall number of cancer cell clusters and an increase in the number of individually identifiable cells. Furthermore, incubation of these cells with exogenous sAPP demonstrates a significant increase in cancer cell proliferation. This proproliferative effect of sAPP may result from activation of MAP kinase, similar to that described in FRTL-5 cells (14) , or by additional mechanisms such as protein kinase C activation (18) , Akt phosphorylation (19) , or activation or potentiation of growth factor signaling pathways (20 , 21) .

The ability of APP to induce growth-promoting effects and morphological alterations occurs not only through effects of the soluble NH2-terminal fragment of APP but may also occur through effects of the cytosolic domain of APP, whether cleaved or intact. We have identified the presence of multiple cytosolic interactors of APP, including JIP1b, Fe65, ShcA, and APPBP1, many of which may function in a neoplastic system to induce cell proliferation and migration (12 , 22 , 23) .

JIP1b serves as a scaffold protein that regulates the activity of c-Jun-NH2-terminal kinase, a member of the MAP kinase signaling family that couples to microtubules via kinesin (22 , 24) . Fe65 has recently been shown to colocalize with APP, actin, and Mena at sites of dynamic actin rearrangement (focal complexes), rather than at static cell-cell adhesion sites (focal adhesions), and overexpression of APP and Fe65 can induce an increase in Madin-Darby canine kidney cell migration (12) . APPBP1 can mediate the cell cycle by promoting S to M transition (23) . Interaction of Shc/Grb2/SOS has been associated with Ras activation, although in the vast majority of pancreatic cancers, mutations in RAS2 induce constitutive activation of the K-ras gene product (25) , which may reduce the relevance of the Shc/Grb2/SOS pathway in this cancer type.

In conclusion, we have identified an increased expression of APP in pancreatic cancer cells, and these cells appear to undergo, in certain instances, virtually complete processing of APP to yield soluble intra- and extracellular peptides. sAPP signaling appears to increase the level of cellular proliferation in BxPC3 cells, similar to that reported in other noncancerous cell types. We propose that sAPP may serve a proproliferative role in certain pancreatic cancer cell subtypes. Additional studies are necessary to evaluate the role of the COOH-terminal region of APP and its cytosolic interactors in pancreatic cancer intracellular signaling pathways.


   ACKNOWLEDGMENTS
 
We thank Drs. Donald Price and Philip Wong from The Johns Hopkins University School of Medicine, Division of Neuropathology, for invaluable insights into the role of APP in pancreatic cancer and insightful comments in reviewing the manuscript. We also acknowledge Norman J. Barker (Division of Informatics, The Johns Hopkins Medical Institutions) for outstanding technical support in the development and presentation of images used in this publication.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Pathology, The Johns Hopkins Medical Institutions, 720 Rutland Avenue, Ross 632, Baltimore, MD 21205. Phone: (410) 955-3511; Fax: (410) 614-0671; E-mail: dhansel{at}jhmi.edu Back

2 The abbreviations used are: APP, amyloid precursor protein; sAPP, soluble APP (NH2-terminal of APP); CTF, COOH-terminal fragment; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BrdUrd, 5-bromo-2'-deoxyuridine; DAPI, 4',6-diamidino-2-phenylindole; APPBP1, APP-binding protein 1; PS-1, presenilin-1; SAGE, serial analysis of gene expression; MAP, mitogen-activated protein. Back

Received 3/21/03. Revised 7/15/03. Accepted 8/26/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Yoshikai S., Sasaki H., Doh-ura K., Furuya H., Sakaki Y. Genomic organization of the human amyloid ß-protein precursor gene. Gene (Amst.), 87: 257-263, 1990.[Medline]
  2. Esch F. S., Keim P. S., Beattie E. C., Blacher R. W., Culwell A. R., Oltersdorf T., McClure D., Ward P. J. Cleavage of amyloid ß peptide during constitutive processing of its precursor. Science (Wash. DC), 248: 1122-1124, 1990.[Abstract/Free Full Text]
  3. Sisodia S. S., Koo E. H., Beyreuther K., Unterbeck A., Price D. L. Evidence that ß-amyloid protein in Alzheimer’s disease is not derived by normal processing. Science (Wash. DC), 248: 492-495, 1990.[Abstract/Free Full Text]
  4. Hoffmann J., Twiesselmann C., Kummer M. P., Romagnoli P., Herzog V. A possible role for the Alzheimer amyloid precursor protein in the regulation of epidermal basal cell proliferation. Eur. J. Cell Biol., 79: 905-914, 2000.[Medline]
  5. Graebert K. S., Lemansky P., Kehle T., Herzog V. Localization and regulated release of Alzheimer amyloid precursor-like protein in thyrocytes. Lab. Investig., 72: 513-523, 1995.[Medline]
  6. Sandbrink R., Masters C. L., Beyreuther K. ß A4-amyloid protein precursor mRNA isoforms without exon 15 are ubiquitously expressed in rat tissues including brain, but not in neurons. J. Biol. Chem., 269: 1510-1517, 1994.[Abstract/Free Full Text]
  7. Schmitz A., Tikkanen R., Kirfel G., Herzog V. The biological role of the Alzheimer amyloid precursor protein in epithelial cells. Histochem. Cell Biol., 117: 171-180, 2002.[Medline]
  8. Ouyang H., Mou L., Luk C., Liu N., Karaskova J., Squire J., Tsao M. S. Immortal human pancreatic duct epithelial cell lines with near normal genotype and phenotype. Am. J. Pathol., 157: 1623-1631, 2000.[Abstract/Free Full Text]
  9. Esler W. P., Wolfe M. S. A portrait of Alzheimer secretases: new features and familiar faces. Science (Wash. DC), 293: 1449-1454, 2001.[Abstract/Free Full Text]
  10. Weidemann A., Konig G., Bunke D., Fischer P., Salbaum J. M., Masters C. L., Beyreuther K. Identification, biogenesis, and localization of precursors of Alzheimer’s disease A4 amyloid protein. Cell, 57: 115-126, 1989.[Medline]
  11. Hoffmann J., Pietrzik C. U., Kummer M. P., Twiesselmann C., Bauer C., Herzog V. Binding and selective detection of the secretory N-terminal domain of the Alzheimer amyloid precursor protein on cell surfaces. J. Histochem. Cytochem., 47: 373-382, 1999.[Abstract/Free Full Text]
  12. Sabo S. L., Ikin A. F., Buxbaum J. D., Greengard P. The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement. J. Cell Biol., 153: 1403-1414, 2001.[Abstract/Free Full Text]
  13. Ohsawa I., Takamura C., Morimoto T., Ishiguro M., Kohsaka S. Amino-terminal region of secreted form of amyloid precursor protein stimulates proliferation of neural stem cells. Eur. J. Neurosci., 11: 1907-1913, 1999.[Medline]
  14. Nishimura I., Takazaki R., Kuwako K., Enokido Y., Yoshikawa K. Upregulation and antiapoptotic role of endogenous Alzheimer amyloid precursor protein in dorsal root ganglion neurons. Exp. Cell Res., 286: 241-251, 2003.[Medline]
  15. Ge Y. W., Lahiri D. K. Regulation of promoter activity of the APP gene by cytokines and growth factors: implications in Alzheimer’s disease. Ann. N. Y. Acad. Sci., 973: 463-467, 2002.[Abstract/Free Full Text]
  16. Popp G. M., Graebert K. S., Pietrzik C. U., Rosentreter S. M., Lemansky P., Herzog V. Growth regulation of rat thyrocytes (FRTL-5 cells) by the secreted ectodomain of ß-amyloid precursor-like proteins. Endocrinology, 137: 1975-1983, 1996.[Abstract]
  17. Jin L. W., Ninomiya H., Roch J. M., Schubert D., Masliah E., Otero D. A., Saitoh T. Peptides containing the RERMS sequence of amyloid ß/A4 protein precursor bind cell surface and promote neurite extension. J. Neurosci., 14: 5461-5470, 1994.[Abstract]
  18. Ishiguro M., Ohsawa I., Takamura C., Morimoto T., Kohsaka S. Secreted form of ß-amyloid precursor protein activates protein kinase C and phospholipase C{gamma}1 in cultured embryonic rat neocortical cells. Brain Res. Mol. Brain Res., 53: 24-32, 1998.[Medline]
  19. Cheng G., Yu Z., Zhou D., Mattson M. P. Phosphatidylinositol-3-kinase-Akt kinase and p42/p44 mitogen-activated protein kinases mediate neurotrophic and excitoprotective actions of a secreted form of amyloid precursor protein. Exp. Neurol., 175: 407-414, 2002.[Medline]
  20. Luo J. J., Wallace M. S., Hawver D. B., Kusiak J. W., Wallace W. C. Characterization of the neurotrophic interaction between nerve growth factor and secreted {alpha}-amyloid precursor protein. J. Neurosci. Res., 63: 410-420, 2001.[Medline]
  21. Mook-Jung I., Saitoh T. Amyloid precursor protein activates phosphotyrosine signaling pathway. Neurosci. Lett., 235: 1-4, 1997.[Medline]
  22. Taru H., Iijima K., Hase M., Kirino Y., Yagi Y., Suzuki T. Interaction of Alzheimer’s ß-amyloid precursor family proteins with scaffold proteins of the JNK signaling cascade. J. Biol. Chem., 277: 20070-20078, 2002.[Abstract/Free Full Text]
  23. Chen Y., McPhie D. L., Hirschberg J., Neve R. L. The amyloid precursor protein-binding protein APP-BP1 drives the cell cycle through the S-M checkpoint and causes apoptosis in neurons. J. Biol. Chem., 275: 8929-8935, 2000.[Abstract/Free Full Text]
  24. Davis R. J. Signal transduction by the JNK group of MAP kinases. Cell, 103: 239-252, 2000.[Medline]
  25. Hoorens A., Lemoine N. R., McLellan E., Morohoshi T., Kamisawa T., Heitz P. U., Stamm B., Ruschoff J., Wiedenmann B., Kloppel G. Pancreatic acinar cell carcinoma. An analysis of cell lineage markers, p53 expression, and Ki-ras mutation. Am. J. Pathol., 143: 685-698, 1993.[Abstract]



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