Differential expression of RAGE in human pancreatic carcinoma cells

BACKGROUND/AIMS: Amphoterin is a key protein in normal neurite outgrowth and that its receptor on the cell surface, RAGE, is an important molecule for invasion. RAGE induces neurite outgrowth and regulate gene expression through NF-kappa B. Because the pancreatic cancer shows the constitutively activated NF-kappa B, the involvement of RAGE could be possible as a determinant for the metastatic ability. METHODOLOGY: To see the involvement of RAGE in pancreatic cancer, three representative human pancreatic carcinoma cells were rendered for the study which have different metastatic potential, PANC-1 and MIA PaCa-2 as the cells with high ability, BxPC-3 as those with low. The expression of RAGE (receptor for advanced glycation end-products) was examined by Western blot. RESULTS: The expression of RAGe was strong in MIA PaCa-2 and PANC-1 that have high metastatic ability. On the contrary, RAGE is expressed little in BxPC-3 whose ability is low. CONCLUSIONS: RAGE is expressed in concordant to the metastatic ability of the human pancreatic cancer cells. Control of this molecule could be a key to regulate the metastatic ability of pancreatic cancer.     

Human pancreatic adenocarcinomas express parathyroid hormone-related protein

PTH-related protein (PTHrP) is expressed in many common malignancies such as breast and prostate cancer and can regulate their growth. Little is known, however, about the role of PTHrP in pancreatic adenocarcinoma. To study PTHrP in pancreatic exocrine cancer, we studied its expression in pancreatic cancer cell lines and surgical specimens. Eight human pancreatic adenocarcinoma cell lines were evaluated: AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, PANC-1, PANC-28, and PANC-48. Murine monoclonal antibodies to the amino-terminal (1-34), mid-region (38-64), and carboxyl-terminal peptides (109-141) of PTHrP were used to identify cellular PTHrP and secreted PTHrP, including Western blotting and immunocytochemical staining for PTHrP from each cell line. Cellular PTHrP was detected in all cell line extracts by both Western blotting and immunoassay. CFPAC-1, derived from a pancreatic liver metastasis, had the highest concentration of PTHrP, and MIA PaCa-2, derived from primary pancreatic adenocarcinoma, had the lowest. PTHrP was localized by immunocytochemical staining in the cytoplasm in all but one cell line, and both nuclear and cytoplasmic immunostaining were observed in the MIA PaCa-2 and PANC-1 cells. Secretion of PTHrP into cell medium was also observed for each cell line and paralleled intracellular PTHrP levels. Evidence for differential processing of PTHrP expression was provided by studies demonstrating different patterns of PTHrP among the cell lines when assessed by PTHrP immunoassays directed against different PTHrP peptides. In specific, PTHrP secretion measured by a PTHrP-(38-64) assay was highest for BxPC-3, whereas the highest levels of secreted PTHrP-(109-141) occurred in CFPAC-1 and PANC-1. Growth of AsPC-1 cells was stimulated in a dose-dependent manner by PTHrP-(1-34). Immunostaining from archival tissue of patients with pancreatic adenocarcinoma revealed strong PTHrP expression in all 14 specimens. All patients were eucalcemic preoperatively. These results demonstrate that PTHrP is commonly expressed in pancreatic cancer. Our data suggest that PTHrP may have growth-regulating properties in pancreatic adenocarcinoma cells, but further studies are required.     

Alteration of integrins by interleukin-1alpha in human pancreatic cancer cells

INTRODUCTION: Adhesion of tumor cells to extracellular matrix (ECM) proteins plays an important role in tumor invasion and metastasis. AIMS: To investigate the expression of integrins in human pancreatic cancer cell lines and its alteration by interleukin (IL)-1alpha to examine the mechanism of adhesion of metastatic human pancreatic cancer cells to ECM proteins. METHODOLOGY: The expression of integrin subunits and their alteration by IL-1alpha were examined by flow-cytometric analysis and cellular enzyme-linked immunosorbent assay in three metastatic human pancreatic cancer cell lines (AsPC-1, BxPC-3, and SW1990) and two nonmetastatic cancer cell lines (PaCa-2 and PANC-1). In addition, assays of cancer cell adhesion to ECM proteins were performed to investigate if increased integrin expression actually affected the adhesive interaction between cancer cells and the putative integrin ECM ligands. RESULTS: The alpha(6) subunit expressed in metastatic cancer cells was enhanced by IL-1alpha. Metastatic cancer cells also showed preferential adherence to laminin compared with nonmetastatic cancer cells, and this was enhanced by IL-1alpha. CONCLUSION: In pancreatic cancer, the enhancement of alpha(6)beta(1) integrin by IL-1alpha through IL-1 receptor type I, as well as the expression of alpha(6)beta(1) integrin, plays an important role in metastasis formation.     

Suppression of human pancreatic carcinoma cell growth and invasion by epigallocatechin-3-gallate

INTRODUCTION: The consumption of green tea is associated with a lower risk of several types of human carcinomas. A number of studies have focused on the possible mechanisms of cancer prevention by tea extracts, especially polyphenols such as epigallocatechin-3-gallate (EGCG). AIMS AND METHODOLOGY: Green tea-derived EGCG was tested in human pancreatic carcinoma cells. The cells (PANC-1, MIA PaCa-2, and BxPC-3) were treated with different doses of EGCG (0, 25, 50, 100, and 200 micromol/L) for 48 hours in culture medium. Proliferation of pancreatic carcinoma cells was measured by means of the WST-1 colorimetric assay. For the study of cell invasion, the cells were incubated with 100 micromol/L EGCG for 2 hours. Then, the cells were added into the cell insert, coated with Matrigel basement membrane matrix. After incubation at 37 degrees C for 24 hours, the cells that had invaded through the Matrigel were counted visually under the microscope. RESULTS: The growth of all three pancreatic carcinoma cells was significantly suppressed by EGCG treatment in a dose-dependent manner. EGCG treatment caused significant suppression of the invasive ability of pancreatic carcinoma cells PANC-1, MIA PaCa-2, and BxPC-3 but did not affect the cell cycle protein cyclin D1. CONCLUSION: EGCG may be a potent biologic inhibitor of human pancreatic carcinomas, reducing their proliferative and invasive activities.     

Claudin 7 as a possible novel molecular target for the treatment of pancreatic cancer

Background/objectives

Pancreatic cancer consists of various subpopulations of cells, some of which have aggressive proliferative properties. The molecules responsible for the aggressive proliferation of pancreatic cancer may become molecular targets for the therapies against pancreatic cancer.

Cholecystokinin analog,JMV-180, stimulates growth of human pancreatic cancer

The gastrointestinal peptide CCK has been shown to stimulate growth of normal and malignant pancreatic tissue. The CCK receptor possesses several different binding sites for CCK. By using the CCK analog JMV-180, which is a functional agonist at CCK high-and low-affinity receptors and an antagonist at very low affinity receptors, and carbachol, which down-regulates binding to CCK high-affinity receptors, we evaluated which receptor is involved in growth of human pancreatic cancer cells. PANC-1 and MIA PaCa-2 human pancreatic cancer cell lines were grown for four to six days in the presence or absence of JMV-180 (10^–10–10^–6 M) alone or in combination with carbachol (10 mM). Growth was evaluated by counting cells and by [³H]thymidine incorporation. JMV-180 increased cell number in PANC-1 and MIA PaCa-2 cells 123% and 86%, respectively, over controls (P=0.004). DNA synthesis by [³H]thymidine uptake was increased 64% and 40% in PANC-1 and MIA PaCa-2 cells, respectively, over controls (P<0.001). The trophic effect of JMV-180 was not inhibited by the addition of carbachol. Since JMV-180 stimulated the growth and since the effect was not inhibited by carbachol, we suggest that the growth effects of CCK in pancreatic cancer cells are mediated by the low-affinity receptor.     

Comparative analysis of a human pancreatic undifferentiated cell line (MIA PaCa-2) to acinar and ductal cells

Pancreatic ductal cell secretion has not been well characterized due to the difficulty in obtaining sufficient quantities of purified ductal cells. To determine if the MIA PaCa-2 cell line would provide a useful model for in vitro studies of pancreatic ductal cell secretion, the present study was designed to characterize these cells in greater detail. In this investigation, the human pancreatic undifferentiated cell line, MIA PaCa-2, was compared with PANC-1 cells (a human ductal cell line previously characterized), isolated rat and human ducts, acinar cells, and nonpancreatic cell lines. The results indicate that while the morphology of the MIA PaCa-2 cell line is nonpolarized and generally atypical of either ductal or acinar cells, the cell line has retained certain biochemical similarities to ductal cells. Additional morphological studies indicated (a) the presence of intermediate filaments characteristic of epithelial cells, (b) the absence of zymogen granules, and (c) an apparent basolateral plasma membrane localization of Na+, K+-ATPase. Similar to ductal cells, biochemical analyses indicated (a) the presence of Na+, K+-ATPase based on [3H]-ouabain binding assays, (b) high levels of carbonic anhydrase, (c) low levels of gamma-glutamyl transpeptidase, (d) nondetectable levels of amylase, and (e) protein composition and protein synthetic patterns comparable to PANC-1 cells. Finally, as with PANC-1 cells and isolated rat and human ducts, the major sulfated secretory product of MIA PaCa-2 cells was a protein with a molecular weight of approximately 660,000 to 1 million.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trophic effects of unsulfated cholecystokinin on mouse pancreas and human pancreatic cancer.

The effect of unsulfated cholecystokinin on pancreatic growth was evaluated in two experimental models in vivo and in vitro. Mice were injected with sulfated cholecystokinin (CCKs) or unsulfated cholecystokinin (CCKu) (10 or 20 micrograms/kg) or vehicle twice daily for 15 days. Animals were then killed and pancreatic weights, protein, amylase, and DNA content were evaluated. In vitro, growth was evaluated by DNA synthesis and viable cell counts. MIA PaCa-2 and BxPC-3 human pancreatic cancer cells were treated with CCKs or CCKu (10(-12) to 10(-9) M) for 48 or 72 h in the presence of [3H]thymidine to evaluate DNA synthesis. Viable cell counts were performed on both cell lines grown in the presence or absence of unsulfated CCK (10(-12) to 10(-9) M) for 96 h. Pancreatic weight, protein, amylase, and DNA were significantly increased in animals treated with either CCKs or CCKu. However, pancreatic weight, protein, and amylase were significantly higher in mice treated with CCKs compared to CCKu (p less than 0.005). DNA content and index of hyperplasia were the same whether mice were treated with CCKs or CCKu. CCKu was as potent a stimulus for DNA synthesis as CCKs in MIA PaCa-2 and BxPC-3 cells. Finally, CCKu increased cell counts in both pancreatic cancer cell lines. These data suggest that the mechanisms responsible for CCK-induced growth of normal pancreas and pancreatic cancer may differ from those that regulate secretion.     

Different capabilities of morphological pattern formation and its association with the expression of differentiation markers in a xenograft model of human pancreatic cancer cell lines.

AIMS: New concepts of tumorigenesis favor an unregulated process recapitulating different stages of embryogenic development with dysregulation of transition states. The aim of our study was to investigate the possibility of differentiation pathways of human pancreatic cancer cell lines in vivo. MATERIAL AND METHODS: Different human pancreatic cancer cell lines (YAPC, DAN-G, CAPAN-1, PANC-1 and MIA PaCa-2) were implanted subcutaneously (3 x 10(6) cells) for 28 days in nude mice. Xenotransplants were characterized with histochemistry (HE, PAS), immunohistochemistry (cytokeratin (CK)7, CK8, CK18, CK19, CK20, vimentin, chromogranin A (Chr-A), alpha1-antichymotrypsin (alpha1-chym), beta-catenin, laminin-5, pancreatic and duodenal homeobox gene 1 (pdx-1), sonic hedgehog protein (shh), Patched (ptc)), Western blotting and real-time PCR (CK7, CK8, CK20, Chr-A, pdx-1, shh, ptc). RESULTS: Depending on three major morphologic phenotypes of tumor cell xenotransplants (ductal (YAPC), ductal/solid (DAN-G, CAPAN-1), solid (PANC-1, MIA PaCa-2)), a decrease of CK7/CK19 was found, accompanied by an increase of CK8/18 and vimentin. Predominantly the CK7-positive ductal phenotype (YAPC and DAN-G) was associated with pdx-1 expression, whereas the CK8-positive solid phenotype was associated with shh/ptc expression on protein and mRNA level. Additionally, CK-20 expression was mainly linked to the ductal phenotype, co-localized with nuclear beta-catenin. The endocrine-exocrine transdifferentiation, as assessed by Chr-A and alpha1-chym, was on a constant low to moderate level in all xenotransplants. Finally, an intensive epithelial-mesenchymal interaction was observed by overexpression of laminin-5 at the invasion front. CONCLUSION: The observed patterns of morphology and molecular differentiation in human pancreatic cancer xenografts indicate that these cancer cell lines have different capabilities of pattern formation in vivo associated with molecular differentiation markers, especially of embryonic pancreatic development.     

Peroxisome proliferator-activated receptor gamma ligand inhibits cell growth and invasion of human pancreatic cancer cells

Summary Background. Peroxisome proliferator-activated receptor γ (PPARγ) is expressed in certain human cancers; ligand-induced PPARγ activation can result in growth inhibition and differentiation in these cells. However, the precise mechanism for the antiproliferative effect of PPARγ ligands is not entirely known. Aim of Study. The purpose of this study was to examine the effect of PPARγ ligands on pancreatic cancer cell growth and invasiveness. Methods. The effect of two PPARγligands, 15 deoxy-Δ^12,14prostaglandin J₂ (15d-PGJ₂) and ciglitazone, on the growth of four human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, Panc-1, and L3.6) was assessed. Expression of cell-cycle and apoptotic-related proteins was measured. Finally, the effect of 15d-PGJ₂ on pancreatic cancer cell invasiveness and matrix metalloproteinase expression was determined. Results. Both 15d-PGJ₂ and ciglitazone inhibited the growth of all four pancreatic cancer cell lines in a dose- and time-dependent fashion. Treatment of BxPC-3 cells with 15d-PGJ₂ resulted in a time-dependent decrease in cyclin D1 expression associated with a concomitant induction of p21^waf1 and p27^kip1. In addition, 15d-PGJ₂ treatment induced apoptosis through activation of caspase-8, -9, and -3. Moreover, pancreatic cancer cell invasiveness was significantly suppressed after treatment with a nontoxic dose of 15d-PGJ₂, which was associated with a reduction of MMP-2 and MMP-9 protein levels and activity. Conclusion. These results demonstrate that PPARγ ligands have the dual advantage of inhibiting pancreatic cancer cell growth while reducing the invasiveness of the tumor cells, suggesting a potential role for these agents in the adjuvant treatment of pancreatic cancer.     

Expression of intercellular adhesion molecule (ICAM)-1 or ICAM-2 is critical in determining sensitivity of pancreatic cancer cells to cytolysis by human γδ-T cells: Implications in the design of γδ-T-cell-based immunotherapies for pancreatic cancer

Background and Aims:  γδ-T cells can recognize and kill malignant cells, particularly those of epithelial origin, through mechanisms which do not require the recognition of tumor-specific antigens (innate immune response). This natural ability of γδ-T cells to kill tumor cells in a tumor antigen-independent manner provides a strong rationale for developing clinical trials designed to exploit the innate antitumor properties of γδ-T cells.
Methods:  In vitro studies were carried out to asses the sensitivity of pancreatic cancer cells (MIA PaCa2, BxPC-3, PANC-1) to killing by ex vivo expanded human γδ-T cells.
Results:  The capacity of γδ-T cells to bind to as well as to kill pancreatic cancer cells correlated with the degree of surface expression of key intercellular adhesion molecules (ICAM) present on pancreatic cancer cells. Moreover, pancreatic cancer cells expressing neither ICAM-1 nor ICAM-2 were bound poorly by γδ-T cells and were found to be resistant to γδ-T-cell killing. However, upon transfection of resistant cells with ICAM-1 or ICAM-2, γδ-T cells were then able to bind to and subsequently kill these cells.
Conclusion:  In vitro , the expression of ICAM-1 or ICAM-2 on human pancreatic cancer cells is critically important in determining the extent to which these cells are sensitive to killing by human γδ-T cells. Accordingly, in ongoing and future clinical studies using γδ-T cells for the treatment of a variety of epithelial-derived solid tumors—including pancreatic cancer—interventions intended to modulate ICAM expression on tumor cells may become important adjuncts to γδ-T-cell-based immunotherapies.     

Fascin promotes the motility and invasiveness of pancreatic cancer cells

AIM:To explore the role of actin-bundling protein, fascin during the progression of pancreatic cancer. METHODS:The plasmid expressing human fascin-1 was stably transfected into the pancreatic cancer cell line MIA PaCa-2. The proliferation, cell cycle, motility, scattering, invasiveness and organization of the actin filament system in fascin-transfected MIA PaCa-2 cells and control non-transfected cells were determined. RESULTS:Heterogeneous overexpression of fascin markedly enhanced the motility, scattering...     

Binding and biological effects of tumor necrosis factor and gamma interferon in human pancreatic carcinoma cells

The cytotoxic/cytostatic effects of recombinant human tumor necrosis factor alpha (rhTNF) and gamma interferon (rhIFN-gamma) were studied in five human pancreatic tumor cell lines. During a 48-h incubation, MIA PaCa-2 cells were most sensitive to rhTNF (56% cytotoxicity, 500 U/ml), T3M4 cells were most sensitive to rhIFN-gamma (54% cytostasis, 250 U/ml), and ASPC-1 and COLO 357 cells were most sensitive to the combination of rhTNF and rhIFN-gamma (56 and 55% cytotoxicity, respectively, 250 U/ml of each cytokine). The PANC-1 cells were relatively insensitive to either the individual or the combined effects of these cytokines. All five cell lines exhibited specific, high-affinity receptors for 125I-labeled rhTNF (480-8,610 sites/cell) and rhIFN-gamma (2,050-6,280 sites/cell). The MIA PaCa-2 cells, which were the most sensitive to the inhibitory effects of rhTNF, also possessed the largest number of 125I rhTNF receptors; all other cell lines had a relatively low number of binding sites and low sensitivity. In contrast, no direct correlation could be made between the number of IFN-gamma binding sites and inhibitory sensitivity in any of the cell lines. Incubation of COLO 357 cells at 37 degrees C with either 125I rhTNF or 125I rhINF-gamma led to internalization of the respective 125I-labeled ligand. Our findings document the presence of cytokine receptors in human pancreatic carcinoma cells and suggest that postreceptor events rather than differences in receptor number or affinity more likely govern the responsiveness of pancreatic cancer cells to TNF and IFN-gamma.     

KAI1 reverses the epithelial-mesenchymal transition in human pancreatic cancer cells

Background: Epithelial-mesenchymal transition(EMT) plays an important role in pancreatic cancer(PC). In the present study, we investigated the effects of KAI1 gene overexpression on the EMT of human PC cell lines, MIA Pa Ca-2 and PACN-1. Methods: Plasmids overexpressing KAI1 and p CMV were transfected into MIA Pa Ca-2 and PACN-1 cells, respectively. After selection of differently transfected cells by G418, KAI1 protein levels were examined by Western blotting, and transfected cells were renamed as MIA Pa Ca-2-K, MIA Pa Ca-2-p, PACN-1-K and PACN-1-p. Wound healing and Transwell migration assays were then performed comparing the two groups of cells. EMT-related markers were analyzed by Western blotting. Results: The percentage of wound closure significantly decreased in MIA Pa Ca-2-K cells compared with MIA Pa Ca-2-p and MIA Pa Ca-2 cells after 24, 48 and 72 h( P 0.05). In PACN-1-K cells, the percentage of wound closure significantly decreased as well( P 0.05). Numbers of invading MIA Pa Ca-2, MIA Pa Ca-2-p and MIA Pa Ca-2-K cells were determined as 48.0 ± 15.4, 50.0 ± 12.4, and 12.0 ± 3.8, respectively. The corresponding numbers of invading PACN-1, PACN-1-p and PACN-1-K cells were 29.0 ± 10.6, 31.0 ± 11.4, and 8.0 ± 4.2, respectively. KAI1 overexpression induced a significant upregulation of E-cadherin and also significant downregulation of Snail, vimentin, matrix metalloproteinase 2(MMP2) and MMP9(all P 0.05) in PC cells. Conclusions: KAI1 reversed EMT-related marker expression and inhibited migration and invasion of PC cells. Thus, KAI1 might represent a novel potential therapeutic target for PC.     

Production of transforming growth factor alpha in human pancreatic cancer cells: evidence for a superagonist autocrine cycle.

Previous work showed that cultured human pancreatic cancer cells overexpress the epidermal growth factor (EGF) receptor. In the present study, we sought to determine whether some of these cell lines produce transforming growth factor alpha (TGF-alpha). Utilizing a radiolabeled TGF-alpha cDNA in hybridization experiments, we determined that ASPC-1, T3M4, PANC-1, COLO-357, and MIA PaCa-2 cell lines expressed TGF-alpha mRNA. Serum-free medium conditioned by T3M4 and ASPC-1 cells contained significant amounts of TGF-alpha protein. Although unlabeled TGF-alpha readily competed with 125I-labeled EGF for binding, each cell line exhibited lower surface binding and internalization of 125I-labeled TGF-alpha as compared to 125I-labeled EGF. Both TGF-alpha and EGF significantly enhanced the anchorage-independent growth of PANC-1, T3M4, and ASPC-1 cells. However, TGF-alpha was 10- to 100-fold more potent than EGF. These findings suggest that the concomitant overexpression of EGF receptors and production of TGF-alpha may represent an efficient mechanism for certain cancer cells to obtain a growth advantage.     

The expression of growth hormone-releasing hormone (GHRH) and splice variants of its receptor in human gastroenteropancreatic carcinomas

Splice variants (SVs) of receptors for growth hormone-releasing hormone (GHRH) have been found in primary human prostate cancers and diverse human cancer cell lines. GHRH antagonists inhibit growth of various experimental human cancers, including pancreatic and colorectal, xenografted into nude mice or cultured in vitro, and their antiproliferative action could be mediated in part through SVs of GHRH receptors. In this study we examined the expression of mRNA for GHRH and for SVs of its receptors in tumors of human pancreatic, colorectal, and gastric cancer cell lines grown in nude mice. mRNA for both GHRH and SV(1) isoform of GHRH receptors was expressed in tumors of pancreatic (SW1990, PANC-1, MIA PaCa-2, Capan-1, Capan-2, and CFPAC1), colonic (COLO 320DM and HT-29), and gastric (NCI-N87, HS746T, and AGS) cancer cell lines; mRNA for SV(2) was also present in Capan-1, Capan-2, CFPAC1, HT-29, and NCI-N87 tumors. In proliferation studies in vitro, the growth of pancreatic, colonic, and gastric cancer cells was stimulated by GHRH(1-29)NH(2) and inhibited by GHRH antagonist JV-1-38. The stimulation of some gastroenteropancreatic cancer cells by GHRH was followed by an increase in cAMP production, and GHRH antagonist JV-1-38 competitively inhibited this effect. Our study indicates the presence of an autocrine/paracrine stimulatory loop based on GHRH and SV(1) of GHRH receptors in human pancreatic, colorectal, and gastric cancers. The finding of SV(1) receptor in human cancers provides an approach to an antitumor therapy based on the blockade of this receptor by specific GHRH antagonists.     

Proteinase-activated receptor-2-mediated signaling and inhibition of DNA synthesis in human pancreatic cancer cells

Summary Conclusion Proteinase-activated receptor-2 (PAR-2)-mediated effects contribute to the intracellular signaling network in pancreatic tumor cells. A role of PAR-2 as negative regulator in human pancreatic tumor growth might be implied. Background Using the human pancreatic tumor cell line MIA PaCa-2, we evaluated cellular effects of trypsin and the PAR-2-activating peptide SLIGRL on [Ca²⁺]_i mobilization, Ins(1,4,5)P₃ level, and protein kinase (PKC) activation. Furthermore, PAR-2 involvement in the regulation of cell proliferation has been estimated by measurement of [³H]thymidine incorporation in MIA PaCa-2 cells. Results Trypsin and the PAR-2 synthetic peptide agonist SLIGRL induced [Ca²⁺]_i mobilization, transient increase in inositol (1,4,5) triphosphate level, and PKC translocation in MIA PaCa-2 cells. In addition, SLIGRL induced a decrease in DNA synthesis in MIA PaCa-2 cells.     

Local secretion of complement C3 in the exocrine pancreas: Ductal epithelial cells as a possible biosynthetic site

BACKGROUND & AIMS: The complement system participates in the local immune system in various tissues. In this study, we investigated the local secretion of complement C3 into the pancreatic fluid and attempted to determine a possible biosynthetic site. METHODS: C3 protein in human pancreatic fluid was analyzed by, immunoblotting. The C3 messenger RNA (mRNA) expression in several pancreatic carcinoma cell lines was analyzed by the polymerase chain reaction and/or Northern blotting. The secretion of C3 by these pancreatic carcinoma cells was assessed by metabolic labeling and immunoprecipitation experiments. RESULTS: In five samples of human pancreatic fluid, C3 was detected as a molecule composed of alpha and beta chains. C3 mRNA expression was observed in the ductal cell carcinoma lines (PANC-1 and MIA PaCa-2) but not in the acinar cell line (HPC-YO and AR-42J). C3 production in these cells was enhanced by interleukin 1 beta and tumor necrosis factor alpha at both the protein and the mRNA levels. CONCLUSIONS: (1) Complement C3 is secreted into the exocrine fluids of the pancreas. (2) Ductal epithelial cells are possible biosynthetic sites for C3. (3) The proinflammatory cytokines, interleukin 1 beta and tumor necrosis factor alpha are effective stimulators of local C3 production in the pancreas. (Gastroenterology 1996 Jun;110(6):1919-25)