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A372 AGA ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4 p16: a Newly Characterized Secretory Protein from the exocrine pancreas. AW Lowe, JEA Braun, and SME Wong. Dept of Medicine and the Digestive Disease Center, Stanford Univ., Stanford, CA 94305-5487 p16 is a newly described pancreatic secretory protein from the rat pancreas, cDNA representing the protein was initially cloned from a Igt11 expression library. The cDNA sequence contained an open reading frame of 168 amino acids that included an appropriate start methionine followed by an amino terminal domain consistent with a signal sequence. The cDNA sequence was used to generate a bacterially expressed fusion protein that was used tO produce a rabbit anti-p16 polyclonal antiserum. Western blots of rat pancreatic subcellular fractions detected a 16 kDa band consistent with he predicted size of the protein. p16 was enriched in the zymogen granules but not in the granule membranes. It is detected in secretions collected from the pancreatic duct. Two-dimensional electrophoresis of p16 revealed a pl of > 9.5. Although not enriched in granule membranes, a significant amount of p16 adhered to the membrane despite sodium carbonate washing at pH 11.5. Northern blots using the p16 cDNA detected a transcript of 880 BP. No transcripts were detected in other tissues including heart, spleen, parotid, brain, kidney, and liver. Analysis of the predicted amino acid sequence revealed 29% identity with rat spermine binding protein (SBP), a secretory protein found in the prostate gland. Amino acid residues #78-145 were 78% similar with that of SBP, suggesting a potential conserved functional domain. Anti-SBP antiserum, however, did not cross react with p16, and anti-p16 antiserum did not react with purified SBP. Although p16 and SBP are exocrine secretory proteins, the function of both are unknown. The amino acid sequence also did not produce any motifs consistent known motifs found in some proteases. Thus, p16 is a newly characterized pancreatic exocrine secretory protein for which a function is not known at this time. Current studies are directed toward potential functions either within the acinar cell or after secretion into the intestine. SOMATOSTATIN BLOCKS ETHANOL-INDUCED RELEASE OF CHOLECYSTOKININ-RELEASING FACTOR (CCK-RP) INTO THE DUODENUM. L Lu. A Saluja, A Kaiser, K Yamanaka, Y Yamaguchi, E Donovan, and M Steer. Dept. of Surgery, Beth Israel Hospital and Harvard Medical School, Boston, MA 02215. We have recently reported that intravenous ethanol administration stimulates in vivo pancreatic amylase secretion and an increase in plasma CCK levels, and that these responses are mediated via the release of CCK-RF into the duodenum. We found that the ethanol-induced increase in plasma CCK levels was completely inhibited by somatostatin. In the current study we have investigated the mechanism of this inhibition. Pentobarbitol anesthetized male Wistar rats (200-300 g) were infused with ethanol to maintain a blood alcohol level of 0.15% for the duration of the experiment. Pancreatic juice was collected by the cannulation of bilio-pancreatic duct just proximal toits entry into the duodenum. In some rats ("donors"), the duodenum was perfused with phosphate buffered saline to collect duodenal juice containing CCK-RF. This duodenal juice was~concentrated by ultrafiltration and instilled into the duodenum of the recipient rats. This allowed for the bioassay of CCK-RF. Results: Intraduodenal administration of concentrated perfusate obtained from the ethanol-infused donor rats, resulted in a significant stimulation of amylase release (3.8 4- 0.5 vs. 118 4- 0.2 f01d increase for control saline-infused donors)and a rise in plasma CCK level (30.5 + 5.4 vs. 10.6 _+.4.8 pM in control saline-infused donors). However, this stimulatory effect of concentrated duodenal perfusate obtained from ethanol-infused rats was abolished when the donors were infused with somatostatin (25 p.g/kg/hr) prior to and during the administration of ethanol. Conclusion: These results indicate that somatostatin inhibits the ethanol-induced rise in plasma CCK levels by inhibiting the release of CCK-releasing factor into the duodenum. Supported by NIH grants DK 46331/31396. EXPRESSION OF SRC-FAMILY KINASES IN HUMAN PANCREATIC CARCINOMA CELL LINES CORRELATES WITH GROWTH INHIBITION BY HERBIMYCIN A M.P.Lutz, I.B.S.Kimmritz, R.Vogelmalm, G.Adler Dept. of Gastroenterology, University of UIm, Germany Tyrosine kinases of the Src family are thought to play important roles in cellular growth control. 5 of the 9 family members are present solely in hemopoetic cells, whereas Src, Lyn, Fyn and Yes have been detected in other tissues. As shown by us before, the prototype of this kinase family - p60-Src - is present in 11/12 human pancreatic carcinoma cell lines, and activated in 10/12. Furthemore, Src is activated in all tumor tissue explants examined as compared to normal pancreatic tissue. To date, expression and activity of other members of this kinase family, as well as their potential role for growth control in pancreatic carcinoma have not been examined. Therefore, kinase activities of Src, Lyn, Fyn and Yes were measured in detergent extracts from human pancreatic carcinoma cell lines after immuno- precipitation of kinase proteins. Protein expression levels were determined by western blotting. TO examine the importance of Src family kinases for growth of pancreatic tumor cell lines, cells were incubated with the mainly?Src-kinase specific tyrosine kinase inhibitor Herbimycin A for 3 days. Growth was measured using the MTT assay and by cell counting. Out of the 12 pancreatic carcinoma cell lines examined, significant amounts of Lyn protein levels (and kinase activities) were detected in 11/12 (10/12), of Fyn in 9/12 (6/12), and of Yes in 7/12 (8/12) cell lines. For any combination of kinases, protein levels or activities did not correlate between individual cell lines. Addition of Herbimycin A inhibited cell growth by 60% to 78% in 4/4 cell lines tested. No change in the fraction of non-viable cells was observed. In three of the cell lines half-maximal inhibition was observed at 15, 40 and 100 ng/ml. This correlated well with the ICso's observed in in vitro Src kinase assays. In the forth cell line kinase activities were barely detectable and the IC5o for cell growth was 500 ng/ml. In summary, Src as well as other members of this kinase family are expressed and active in most panc~:eatic cancer cell lines. Since we could not observe consistent activation patterns of different kinases in individual cell lines, there does not seem to be a common mechanism of activation. Growth inhibition by Herbimycin A may indicate a potential role for these kinases in pancreatic rumor cell growth. Further studies using different experimental approaches will have to confirm this observation. METASTATIC BEHAVIOUR OF PANCREATIC CARCINOMA CELL LINES CORRELATES WITH EXPRESSION OF INTEGRIN SUBTYPES AND ADHESION PROPERTIES _M.P.Lutz, R.Vogelmann, E.D.Kreuser. G.Adler. Dept. of Gastroenterology, University of Ulm and Dept. of Hematology, Freie Universitiit Berlin. Germany Metastasis formation is a multistep process of cellular detachment and adhesion. The attachment of tumor cells on extracellular matrix substrates is mediated by interaction of matrix glycoproteins with the integrin family of dimeric glycoprotein-specific cell surface receptors. To examine the significance of integrin subtype expression and function for exocrine pancreatic tumor dissemination we compared two subclones of a human tumor-derived cell line, 8988s and 8988t. Both are derived from the same original tumor tissue and form solid tumors when injected into nude mice. Interestingly only 8988s is able to metastatize. Integrin expression was analyzed immunochemically by flow cytometry using subunit-specific antibodies. Cell attachment was examined at various time points in cell culture medium (DMEM without FCS) on 96well plates coated with different matrix proteins (10-15 mg/cm2). The fraction of attached cells was estimated after thorough washing by staining the +with sulphorhodamine B. lntegrin subunits ~1,a2, m6and I]4 were more porminent in the metastatic cell line 8988s. whereas ~5 was measured at higher levels in 898gt cells. Expression of subunits ~3, ~4 and ~v. as well as 13~_ 3 and 135 was similar in both cell lines. Adhesion properties did correlate with the patterns of matrix receptor expi'ession: When basal membrane proteins were used as substrates, 55% of 8988s cells and 24% of 8988t cells did attach to collagen IV (mediated by integrins ~131, t*2130, with similar but less pronounced effects using laminin (~613~, ~6134,nl.313~). Adhesion to the interstitial matrix attachment factor fibronectin Ia513~, ~t41~)was 25% and 48%. Using the matrix constituent collagen I (~2131, ~13~ ~313~) maximal adhesion was 46% and 34%. respectively. Adhesion kinetics were similar with all substrates: Attachment was halfmaximal after 20rain and completed within 60min. In summary~ the metastatic cell line 8988s. as compared to the non-metastatic cell line 8988t. expresses higher levels of adhesion receptors specific for basal membrane proteins: and lower levels of receptors for the interstitial attachment factor fibronectin. This was reflected by the adhesion properties observed. These observations may explain at least in part the differences in metastatic behaviour of the two cell lines. Future studies will have to confirm the relevance of these observations for the in vivo metastatic behaviour of these cell lines.

p16: a newly characterized secretory protein from the exocrine pancreas

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Page 1: p16: a newly characterized secretory protein from the exocrine pancreas

A372 AGA ABSTRACTS GASTROENTEROLOGY, Vol. 108, No. 4

• p16: a Newly Characterized Secretory Protein from the exocrine pancreas. AW Lowe, JEA Braun, and SME Wong. Dept of Medicine and the Digestive Disease Center, Stanford Univ., Stanford, CA 94305-5487

p16 is a newly described pancreatic secretory protein from the rat pancreas, cDNA representing the protein was initially cloned from a Igt11 expression library. The cDNA sequence contained an open reading frame of 168 amino acids that included an appropriate start methionine followed by an amino terminal domain consistent with a signal sequence. The cDNA sequence was used to generate a bacterially expressed fusion protein that was used tO produce a rabbit anti-p16 polyclonal antiserum. Western blots of rat pancreatic subcellular fractions detected a 16 kDa band consistent with he predicted size of the protein. p16 was enriched in the zymogen granules but not in the granule membranes. It is detected in secretions collected from the pancreatic duct. Two-dimensional electrophoresis of p16 revealed a pl of > 9.5. Although not enriched in granule membranes, a significant amount of p16 adhered to the membrane despite sodium carbonate washing at pH 11.5. Northern blots using the p16 cDNA detected a transcript of 880 BP. No transcripts were detected in other tissues including heart, spleen, parotid, brain, kidney, and liver. Analysis of the predicted amino acid sequence revealed 29% identity with rat spermine binding protein (SBP), a secretory protein found in the prostate gland. Amino acid residues #78-145 were 78% similar with that of SBP, suggesting a potential conserved functional domain. Anti-SBP antiserum, however, did not cross react with p16, and anti-p16 antiserum did not react with purified SBP. Although p16 and SBP are exocrine secretory proteins, the function of both are unknown. The amino acid sequence also did not produce any motifs consistent known motifs found in some proteases. Thus, p16 is a newly characterized pancreatic exocrine secretory protein for which a function is not known at this time. Current studies are directed toward potential functions either within the acinar cell or after secretion into the intestine.

• SOMATOSTATIN BLOCKS ETHANOL-INDUCED RELEASE OF C H O L E C Y S T O K I N I N - R E L E A S I N G F A C T O R (CCK-RP) INTO THE D U O D E N U M . L Lu. A Saluja, A Kaiser, K Yamanaka , Y Yamaguchi , E Donovan, and M Steer. Dept. of Surgery, Beth Israel Hospital and Harvard Medical School, Boston, MA 02215.

W e h a v e r e c e n t l y r e p o r t e d tha t i n t r a v e n o u s e t h a n o l adminis t ra t ion s t imulates in v ivo pancreatic amylase secretion and an increase in p l a sma CCK levels , and that these responses are med ia ted v ia the re lease of C C K - R F into the duodenum. W e found that the e thanol- induced increase in p lasma CCK levels was comple t e ly inh ib i ted by somatosta t in . In the current s tudy we have inves t iga ted the m e c h a n i s m of this inhibit ion. Pentobarbi tol anes the t ized m a l e W i s t a r ra ts (200-300 g) were infused wi th ethanol to mainta in a blood alcohol level of 0.15% for the duration of the expe r imen t . Pancrea t i c j u i c e was co l l ec t ed by the cannulat ion of bi l io-pancreat ic duct jus t p rox imal t o i t s entry into the duodenum. In some rats ("donors") , the d u o d e n u m was perfused wi th phosphate buffered sal ine to co l lec t duodenal ju ice conta in ing CCK-RF. This duodena l j u i c e was~concent ra ted by ultrafi l trat ion and inst i l led into the duodenum of the recipient rats. This a l lowed for the bioassay of CCK-RF. Results: In t raduodenal adminis t ra t ion of concentra ted perfusate ob ta ined f r o m the e thano l - in fused donor rats, resu l ted in a s igni f icant s t imula t ion of amylase release (3.8 4- 0.5 vs. 118 4- 0.2 f01d increase for control saline-infused donor s )and a r ise in p lasma CCK level (30.5 + 5.4 vs. 10.6 _+. 4.8 pM in control sal ine-infused donors) . H o w e v e r , this s t imu la to ry e f fec t o f concen t r a t ed duodena l p e r f u s a t e ob t a ined f r o m e thano l - i n fu sed r a t s was abol ished when the donors were infused wi th somatos ta t in (25 p.g/kg/hr) pr ior to and during the administrat ion of ethanol. C o n c l u s i o n : These results indica te that somatosta t in inhibi ts the ethanol- induced rise in p lasma CCK levels by inhibit ing the release of CCK-releas ing factor into the duodenum. Supported by NIH grants DK 46331/31396.

• EXPRESSION OF SRC-FAMILY KINASES IN HUMAN PANCREATIC CARCINOMA CELL LINES CORRELATES WITH GROWTH INHIBITION BY HERBIMYCIN A M.P.Lutz, I.B.S.Kimmritz, R.Vogelmalm, G.Adler Dept. of Gastroenterology, University of UIm, Germany

Tyrosine kinases of the Src family are thought to play important roles in cellular growth control. 5 of the 9 family members are present solely in hemopoetic cells, whereas Src, Lyn, Fyn and Yes have been detected in other tissues. As shown by us before, the prototype of this kinase family - p60-Src - is present in 11/12 human pancreatic carcinoma cell lines, and activated in 10/12. Furthemore, Src is activated in all tumor tissue explants examined as compared to normal pancreatic tissue. To date, expression and activity of other members of this kinase family, as well as their potential role for growth control in pancreatic carcinoma have not been examined.

Therefore, kinase activities of Src, Lyn, Fyn and Yes were measured in detergent extracts from huma n pancreatic carcinoma cell lines after immuno- precipitation of kinase proteins. Protein expression levels were determined by western blotting. TO examine the importance of Src family kinases for growth of pancreatic tumor cell lines, cells were incubated with the mainly?Src-kinase specific tyrosine kinase inhibitor Herbimycin A for 3 days. Growth was measured using the MTT assay and by cell counting.

Out of the 12 pancreatic carcinoma cell lines examined, significant amounts of Lyn protein levels (and kinase activities) were detected in 11/12 (10/12), of Fyn in 9/12 (6/12), and of Yes in 7/12 (8/12) cell lines. For any combination of kinases, protein levels or activities did not correlate between individual cell lines. Addition of Herbimycin A inhibited cell growth by 60% to 78% in 4/4 cell lines tested. No change in the fraction of non-viable cells was observed. In three of the cell lines half-maximal inhibition was observed at 15, 40 and 100 ng/ml. This correlated well with the ICso's observed in in vitro Src kinase assays. In the forth cell line kinase activities were barely detectable and the IC5o for cell growth was 500 ng/ml.

In summary, Src as well as other members of this kinase family are expressed and active in most panc~:eatic cancer cell lines. Since we could not observe consistent activation patterns of different kinases in individual cell lines, there does not seem to be a common mechanism of activation. Growth inhibition by Herbimycin A may indicate a potential role for these kinases in pancreatic rumor cell growth. Further studies using different experimental approaches will have to confirm this observation.

METASTATIC BEHAVIOUR OF PANCREATIC CARCINOMA CELL LINES CORRELATES WITH EXPRESSION OF INTEGRIN SUBTYPES AND ADHESION PROPERTIES _M.P.Lutz, R.Vogelmann, E.D.Kreuser. G.Adler. Dept. of Gastroenterology, University of Ulm and Dept. of Hematology, Freie Universitiit Berlin. Germany

Metastasis formation is a multistep process of cellular detachment and adhesion. The attachment of tumor cells on extracellular matrix substrates is mediated by interaction of matrix glycoproteins with the integrin family of dimeric glycoprotein-specific cell surface receptors. To examine the significance of integrin subtype expression and function for exocrine pancreatic tumor dissemination we compared two subclones of a human tumor-derived cell line, 8988s and 8988t. Both are derived from the same original tumor tissue and form solid tumors when injected into nude mice. Interestingly only 8988s is able to metastatize.

Integrin expression was analyzed immunochemically by flow cytometry using subunit-specific antibodies. Cell attachment was examined at various time points in cell culture medium (DMEM without FCS) on 96well plates coated with different matrix proteins (10-15 mg/cm2). The fraction of attached cells was estimated after thorough washing by staining the +with sulphorhodamine B.

lntegrin subunits ~1,a2, m6 and I]4 were more porminent in the metastatic cell line 8988s. whereas ~5 was measured at higher levels in 898gt cells. Expression of subunits ~3, ~4 and ~v. as well as 13~_ 3 and 135 was similar in both cell lines. Adhesion properties did correlate with the patterns of matrix receptor expi'ession: When basal membrane proteins were used as substrates, 55% of 8988s cells and 24% of 8988t cells did attach to collagen IV (mediated by integrins ~131, t*2130, with similar but less pronounced effects using laminin (~613~, ~6134, nl.313~). Adhesion to the interstitial matrix attachment factor fibronectin Ia513~, ~t41~) was 25% and 48%. Using the matrix constituent collagen I (~2131, ~13~ ~313~) maximal adhesion was 46% and 34%. respectively. Adhesion kinetics were similar with all substrates: Attachment was halfmaximal after 20rain and completed within 60min.

In summary~ the metastatic cell line 8988s. as compared to the non-metastatic cell line 8988t. expresses higher levels of adhesion receptors specific for basal membrane proteins: and lower levels of receptors for the interstitial attachment factor fibronectin. This was reflected by the adhesion properties observed. These observations may explain at least in part the differences in metastatic behaviour of the two cell lines. Future studies will have to confirm the relevance of these observations for the in vivo metastatic behaviour of these cell lines.