Molecular Cloning of the Oncofetal Isoform of the Human Pancreatic Bile Salt-dependent Lipase
1998; Elsevier BV; Volume: 273; Issue: 43 Linguagem: Inglês
10.1074/jbc.273.43.28208
ISSN1083-351X
AutoresEric Pasqualini, Nathalie Caillol, Laurence Panicot‐Dubois, Eric Mas, Roland Lloubès, Dominique Lombardo,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoSpecific transcripts for bile salt-dependent lipase (BSDL), a 100-kDa glycoprotein secreted by the human pancreas, were immunodetected in BxPC-3 and SOJ-6 pancreatic tumoral cell lines. Sequencing of fragments, obtained by mRNA reverse transcription and amplification, confirmed the presence of BSDL transcripts in these cancer cells. The protein was detected in lysates of pancreatic tumoral cells, where it was mainly associated with membranes. Only a minute amount of the enzyme was detected in the culture media. Immunofluorescence studies demonstrated that in SOJ-6 cells, BSDL colocates with the p58 Golgi protein and suggested that the protein may be sequestrated within the Golgi compartment. These results demonstrated that BSDL is expressed in human pancreatic tumoral cells and cannot be secreted (or for the least very poorly). Subsequently, a cDNA covering the entire sequence of BSDL was obtained by reverse transcription-polymerase chain reaction. The sequence of this cDNA indicated that the N-terminal domain encoded by exons 1–10 was identical to that of BSDL expressed by the human normal pancreas. However, the sequence corresponding to exon 11, which should code for the 16 tandem-repeated identical mucin-like sequences of BSDL, was deleted by 330 base pairs (bp) and encoded only 6 of these repeated sequences. We conclude that this truncated variant of BSDL would be its oncofetal form, referred to as feto-acinar pancreatic protein. We then investigated whether the deletion of 330 bp affected the secretion of the protein. For this purpose, the cDNA corresponding to the mature form of the BSDL variant expressed in SOJ-6 cells was cloned into an expression/secretion vector and transfected into CHO-K1 cells. Results indicated that the variant of BSDL isolated from SOJ-6 cells was expressed and secreted by transfected cells. However, the level of BSDL secreted by these transfected CHO-K1 cells was significantly higher than that observed for SOJ-6 cells. Consequently, the retention of the oncofetal variant of BSDL observed in human pancreatic tumoral cells might not result from inherent properties of the protein. Specific transcripts for bile salt-dependent lipase (BSDL), a 100-kDa glycoprotein secreted by the human pancreas, were immunodetected in BxPC-3 and SOJ-6 pancreatic tumoral cell lines. Sequencing of fragments, obtained by mRNA reverse transcription and amplification, confirmed the presence of BSDL transcripts in these cancer cells. The protein was detected in lysates of pancreatic tumoral cells, where it was mainly associated with membranes. Only a minute amount of the enzyme was detected in the culture media. Immunofluorescence studies demonstrated that in SOJ-6 cells, BSDL colocates with the p58 Golgi protein and suggested that the protein may be sequestrated within the Golgi compartment. These results demonstrated that BSDL is expressed in human pancreatic tumoral cells and cannot be secreted (or for the least very poorly). Subsequently, a cDNA covering the entire sequence of BSDL was obtained by reverse transcription-polymerase chain reaction. The sequence of this cDNA indicated that the N-terminal domain encoded by exons 1–10 was identical to that of BSDL expressed by the human normal pancreas. However, the sequence corresponding to exon 11, which should code for the 16 tandem-repeated identical mucin-like sequences of BSDL, was deleted by 330 base pairs (bp) and encoded only 6 of these repeated sequences. We conclude that this truncated variant of BSDL would be its oncofetal form, referred to as feto-acinar pancreatic protein. We then investigated whether the deletion of 330 bp affected the secretion of the protein. For this purpose, the cDNA corresponding to the mature form of the BSDL variant expressed in SOJ-6 cells was cloned into an expression/secretion vector and transfected into CHO-K1 cells. Results indicated that the variant of BSDL isolated from SOJ-6 cells was expressed and secreted by transfected cells. However, the level of BSDL secreted by these transfected CHO-K1 cells was significantly higher than that observed for SOJ-6 cells. Consequently, the retention of the oncofetal variant of BSDL observed in human pancreatic tumoral cells might not result from inherent properties of the protein. bile salt-dependent lipase feto-acinar pancreatic protein Chinese hamster ovary 4-nitrophenyl hexanoate base pair(s) endoplasmic reticulum fluorescein isothiocyanate tetrarhodamine isothiocyanate phosphate-buffered saline lactate dehydrogenase polymerase chain reaction polyacrylamide gel electrophoresis reverse transcription kilobase(s). The bile salt-dependent lipase (BSDL,1 EC 3.1.1) is a 100-kDa glycoprotein secreted by the pancreas into the duodenum, where it is thought to play an important role in cholesterol and lipid-soluble vitamin ester hydrolysis and absorption (1Lombardo D. Fauvel J. Guy O. Biochim. Biophys. Acta. 1980; 611: 136-146Crossref PubMed Scopus (115) Google Scholar, 2Lombardo D. Guy O. Biochim. Biophys. Acta. 1980; 611: 147-155Crossref PubMed Scopus (172) Google Scholar). The cDNA for pancreatic BSDL has been isolated and sequenced (3Reue K. Zambaux J. Wong H. Lee G. Leete H. Ronk M. Shively J.E. Sternby B. Borgström B. Ameis D. Schotz M. J. Lipid Res. 1991; 32: 267-276Abstract Full Text PDF PubMed Google Scholar). The amino acid sequence is rich in proline (12%), the majority of these residues (68%) being located within 16 C-terminal tandemly repeated sequences (repeats numbered 1 to 16) (3Reue K. Zambaux J. Wong H. Lee G. Leete H. Ronk M. Shively J.E. Sternby B. Borgström B. Ameis D. Schotz M. J. Lipid Res. 1991; 32: 267-276Abstract Full Text PDF PubMed Google Scholar, 4Baba T. Downs D. Jackson K.W. Tang J. Wang C-S. Biochemistry. 1991; 30: 500-510Crossref PubMed Scopus (92) Google Scholar). Further studies have shown that exon 11 encoded these tandem repeats (5Kumar B.V. Aleman-Gomez J.A. Colwell N. Lopez-Candales A. Bosner M-S. Spilburg C.A. Lowe M. Lange L.G. Biochemistry. 1992; 31: 6077-6081Crossref PubMed Scopus (35) Google Scholar), the size of which varied by species (5Kumar B.V. Aleman-Gomez J.A. Colwell N. Lopez-Candales A. Bosner M-S. Spilburg C.A. Lowe M. Lange L.G. Biochemistry. 1992; 31: 6077-6081Crossref PubMed Scopus (35) Google Scholar, 6Fontaine R.N. Carter C.P. Hui D.Y. Biochemistry. 1991; 30: 7008-7014Crossref PubMed Scopus (35) Google Scholar). This accounts, in part, for the previously observed species variation in BSDL size and amino acid composition (7Abouakil N. Rogalska E. Bonicel J. Lombardo D. Biochim. Biophys. Acta. 1988; 961: 299-308Crossref PubMed Scopus (69) Google Scholar). In contrast to the other secretory pancreatic enzymes, BSDL is associated with membranes during its intracellular processing (8Bruneau N. Lechêne de la Porte P. Sbarra V. Lombardo D. Eur. J. Biochem. 1995; 233: 209-218Crossref PubMed Scopus (26) Google Scholar, 9Bruneau N. Lombardo D. J. Biol. Chem. 1995; 270: 13524-13533Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This association involves a multimeric folding complex including p94, a protein immunologically related to the glucose-regulated protein of 94 kDa (Grp94) and two other proteins of 56 and 46 kDa (9Bruneau N. Lombardo D. J. Biol. Chem. 1995; 270: 13524-13533Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). It has been suggested that the interaction of BSDL with the Grp94-related p94 protein is essential for the O-glycosylation of C-terminal tandem-repeated sequences (9Bruneau N. Lombardo D. J. Biol. Chem. 1995; 270: 13524-13533Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). These repeated sequences contain PEST regions, which are signals for rapid degradation (10Rogers S. Wells R. Rechsteiner M. Science. 1986; 234: 364-369Crossref PubMed Scopus (1964) Google Scholar). It is therefore possible that glycosylation of these PEST regions may contribute to the removal of BSDL from a possible degradation route (11Bruneau N. Nganga A. Fisher E.A. Lombardo D. J. Biol. Chem. 1997; 272: 27353-27361Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Once fully glycosylated, the enzyme is phosphorylated and released from membranes either in or after the trans-Golgi compartment (12Pasqualini E. Caillol N. Mas E. Bruneau N. Lexa D. Lombardo D. Biochem. J. 1997; 327: 527-535Crossref PubMed Scopus (17) Google Scholar). It is then aggregated in the trans-Golgi network (8Bruneau N. Lechêne de la Porte P. Sbarra V. Lombardo D. Eur. J. Biochem. 1995; 233: 209-218Crossref PubMed Scopus (26) Google Scholar, 9Bruneau N. Lombardo D. J. Biol. Chem. 1995; 270: 13524-13533Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) with other digestive enzymes and enters the regulated secretion pathway. In a previous study, we have isolated an oncofetal variant of BSDL (13Mas E. Abouakil N. Roudani S. Miralles F. Guy-Crotte O. Figarella C. Escribano M.J. Lombardo D. Biochem. J. 1993; 289: 609-615Crossref PubMed Scopus (50) Google Scholar), later identified as the feto-acinar pancreatic protein (FAPP) (14Escribano M.J. Imperial S. J. Biol. Chem. 1989; 264: 21865-21871Abstract Full Text PDF PubMed Google Scholar). FAPP was first characterized by the monoclonal antibody J28(mAb J28) (14Escribano M.J. Imperial S. J. Biol. Chem. 1989; 264: 21865-21871Abstract Full Text PDF PubMed Google Scholar). FAPP is expressed in human embryonic and fetal pancreas. The earliest expression of this protein was seen in undifferentiated mesenchymal cells and in nascent acini at the beginning of the morphological differentiation of the pancreas (15Albers G.H.R. Escribano M.J. Gonzalez M. Mulliez N. Nap N. Differentiation. 1987; 34: 210-215Crossref PubMed Scopus (29) Google Scholar). Maximal synthesis of FAPP occurs at the time of intense proliferation of acinar cells and declines progressively thereafter (15Albers G.H.R. Escribano M.J. Gonzalez M. Mulliez N. Nap N. Differentiation. 1987; 34: 210-215Crossref PubMed Scopus (29) Google Scholar) to reach a very low level of expression in normal adult pancreas (16Imperial S. Escribano M.J. Biochim. Biophys. Acta. 1988; 967: 25-33Crossref PubMed Scopus (6) Google Scholar). FAPP concentration is elevated in the blood of patients suffering from pancreatitis and pancreatic cancer, suggesting an enhanced synthesis in cases of pancreatic pathologies. This finding corroborates the increased level of FAPP in pathological pancreatic juices (17Shimizu H. Fujii Y. Sekiguchi M. Sugawara I. Escribano M.J. Oncologia. 1990; 23: 83-91Google Scholar). FAPP was shown to be different from BSDL in many aspects: (a) FAPP is much less active than BSDL; (b) theN-linked glycosylation of FAPP seems to be a high-mannose type, whereas that of BSDL is complex (18Mas E. Franc J.L. Lecestre D. Crotte C. Lombardo D. Sadoulet M.O. Eur. J. Biochem. 1997; 243: 299-305Crossref PubMed Scopus (6) Google Scholar); (c) the amount of O-linked structures is largely decreased in FAPP (18Mas E. Franc J.L. Lecestre D. Crotte C. Lombardo D. Sadoulet M.O. Eur. J. Biochem. 1997; 243: 299-305Crossref PubMed Scopus (6) Google Scholar); (d) carbohydrate accounts for 47% of the FAPP mass (14Escribano M.J. Imperial S. J. Biol. Chem. 1989; 264: 21865-21871Abstract Full Text PDF PubMed Google Scholar) instead of 20% for BSDL (19Guy O. Lombardo D. Brahms J.G. Eur. J. Biochem. 1981; 117: 457-460Crossref PubMed Scopus (35) Google Scholar); and (e) the amino acid composition of FAPP differs from that of BSDL (13Mas E. Abouakil N. Roudani S. Miralles F. Guy-Crotte O. Figarella C. Escribano M.J. Lombardo D. Biochem. J. 1993; 289: 609-615Crossref PubMed Scopus (50) Google Scholar), although their N-terminal sequences are identical (20Mas E. Crotte C. Lecestre D. Michalski J.C. Escribano M.J. Lombardo D. Sadoulet M.O. Glycobiology. 1997; 7: 745-752Crossref PubMed Scopus (21) Google Scholar). Data suggest that the two proteins may have different C-terminal tails (18Mas E. Franc J.L. Lecestre D. Crotte C. Lombardo D. Sadoulet M.O. Eur. J. Biochem. 1997; 243: 299-305Crossref PubMed Scopus (6) Google Scholar, 20Mas E. Crotte C. Lecestre D. Michalski J.C. Escribano M.J. Lombardo D. Sadoulet M.O. Glycobiology. 1997; 7: 745-752Crossref PubMed Scopus (21) Google Scholar). Finally, FAPP migration on SDS-PAGE is diffuse and lower than that of BSDL (13Mas E. Abouakil N. Roudani S. Miralles F. Guy-Crotte O. Figarella C. Escribano M.J. Lombardo D. Biochem. J. 1993; 289: 609-615Crossref PubMed Scopus (50) Google Scholar). Other studies have shown that FAPP is expressed in pancreatic tumor cells; however, the secretion of the protein was not detected (21Miralles F. Langa F. Mazo A. Escribano M.J. Eur. J. Cell Biol. 1993; 60: 115-121PubMed Google Scholar). Miralles et al. (21Miralles F. Langa F. Mazo A. Escribano M.J. Eur. J. Cell Biol. 1993; 60: 115-121PubMed Google Scholar) have postulated that the absence of secretion was due to the retention of the protein within the endoplasmic reticulum (ER) and suggested that FAPP would remain associated to ER resident protein(s) as a consequence of its improper folding. Recent findings suggested that glycosylation of the C-terminal region of BSDL regulates the secretion of the protein (11Bruneau N. Nganga A. Fisher E.A. Lombardo D. J. Biol. Chem. 1997; 272: 27353-27361Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The aim of this study was 2-fold: first, to investigate the molecular properties of BSDL expressed by human pancreatic tumoral cells; second, to determine whether the retention of the variant expressed by tumoral cells is due to inherent properties of the protein. We showed that part of C-terminal tandem repeated sequences were deleted in the BSDL variant expressed by pancreatic tumoral cells, suggesting that we are dealing with FAPP. Moreover, CHO-K1 cells transfected with the truncated cDNA secreted the protein. This result indicates that the retention of FAPP within tumor cells cannot be due to the truncation of the C-terminal region of the protein. Glutamine, penicillin, trypsin-EDTA, and streptomycin were from Life Technologies, Inc. Fetal calf serum was from Dutscher (Brumath, France). Phenylmethylsulfonyl fluoride, benzamidine, and β-phenyl propionate were from Fluka (Buchs, Switzerland). Mouse antibodies specific for the p58 Golgi protein, FITC-conjugated anti-rabbit goat IgG, TRITC-conjugated anti-mouse goat IgG, saponin, 4-nitrophenyl hexanoate, and soybean trypsin inhibitor were from Sigma. [α-32P]dCTP and125I-labeled protein A were from NEN Life Science Products. [35S]Methionine (Tran35S-label) was from ICN biochemicals (Costa Mesa, CA). BxPC-3 cells, obtained from the American Type Culture Collection (ATCC, Rockville, MD), were cultured in RPMI 1640 (Life Technologies, Inc.). SOJ-6 cells were kindly provided by Dr. M. J. Escribano (INSERM U 260, Marseille, France) and cultured in RPMI 1640. Media were supplemented with 10% fetal calf serum, glutamine (1%), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were maintained at 37 °C in a 5% CO2, 95% air atmosphere. Cell culture medium was removed after the required incubation time, collected, and stored frozen until use. CHO-K1 cells were also obtained from ATCC and cultured under the same conditions in Ham's F12 nutrient mixture (Life Technologies, Inc.) supplemented with 10% fetal calf serum. Normal human pancreatic tissues came from three donors (two females and one male, aged 60–65), and cancer tissue was from a 63-year-old female suffering with an adenocarcinoma of the pancreas. Diagnosis was confirmed by histology examination performed during the time course of surgery. All tissues were generous gifts from Prof. J. R. Delpéro (Institut Paoli-Calmettes, Marseilles, France). Tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C until used. Cells grown to confluence were rinsed twice with incomplete PBS buffer (10 mm sodium phosphate buffer, pH 7.4, with 0.15 m NaCl and no Ca2+ and no Mg2+ ions) and harvested with 0.25% trypsin-EDTA. Cells were suspended in culture medium and centrifuged at 400 × g for 3 min. Pellets were washed twice with PBS and suspended in PBS (500 μl) containing 100 μg/ml soybean trypsin inhibitor, 2 mm phenylmethylsulfonyl fluoride, 2 mm β-phenyl propionate, and 2 mmbenzamidine and sonicated for 10 s. The cell membrane fraction was separated from the soluble fraction by centrifugation at 12,000 ×g for 20 min at 4 °C (22Roudani S. Pasqualini E. Margotat A. Gastaldi M. Sbarra V. Malezet-Desmoulin C. Lombardo D. Eur. J. Cell Biol. 1994; 65: 132-144PubMed Google Scholar). The pellet containing membrane proteins was solubilized in 0.125 mm Tris/HCl, pH 7.0, buffer (3% SDS) (21Miralles F. Langa F. Mazo A. Escribano M.J. Eur. J. Cell Biol. 1993; 60: 115-121PubMed Google Scholar). Proteins were quantitated using the bicinchoninic acid test from Pierce using bovine serum albumin as standard. α-Amylase and lactate dehydrogenase (LDH) activities were determined using adequate methods (23Rauscher E. Von Buelow S. Neumann U. Schaich E. Ber. Osterr. Ges. Klin. Chem. 1981; 4: 150-155Google Scholar, 24Goldberg E. J. Biol. Chem. 1972; 247: 2044-2048Abstract Full Text PDF PubMed Google Scholar). BSDL was assayed on 4-nitrophenyl hexanoate (4-NPH) in the presence of 4 mm sodium taurocholate as specific activator of the enzyme (25Gjellesvik D.R. Lombardo D. Walther B.J. Biochim. Biophys. Acta. 1992; 1124: 123-134Crossref PubMed Scopus (214) Google Scholar). In BxPC-3 and SOJ-6 tumoral pancreatic cells, more than 90% of the activity on 4-NPH can be immunoprecipitated with antibodies specific for BSDL (22Roudani S. Pasqualini E. Margotat A. Gastaldi M. Sbarra V. Malezet-Desmoulin C. Lombardo D. Eur. J. Cell Biol. 1994; 65: 132-144PubMed Google Scholar). The 4-NPH activity determined in CHO-K1 cells transfected with PCR products in pSecTag (see below) was corrected for the activity found in control cells (i.e. transfected with the empty vector). Note that the 4-NPH activity detected in control CHO-K1 cells represented less than 5% of that found in CHO-K1 cells transfected with PCR products. BSDL was purified from human pancreatic juice devoid of free proteolytic activity (a gift of Prof. R. Laugier, Hôpital de la Conception, Marseille, France) (13Mas E. Abouakil N. Roudani S. Miralles F. Guy-Crotte O. Figarella C. Escribano M.J. Lombardo D. Biochem. J. 1993; 289: 609-615Crossref PubMed Scopus (50) Google Scholar, 26Lombardo D. Guy O. Figarella C. Biochim. Biophys. Acta. 1978; 257: 142-149Crossref Scopus (145) Google Scholar) as judged from SDS-PAGE (13Mas E. Abouakil N. Roudani S. Miralles F. Guy-Crotte O. Figarella C. Escribano M.J. Lombardo D. Biochem. J. 1993; 289: 609-615Crossref PubMed Scopus (50) Google Scholar). α-Amylase was also purified to homogeneity (27Guy O. Lombardo D. Bartelt D.C. Amic J. Figarella C. Biochemistry. 1978; 17: 1669-1672Crossref PubMed Scopus (126) Google Scholar). Antibodies against pure antigens were raised in rabbit as already described (28Lechêne de la Porte P. Lafont H. Lombardo D. Histochemistry. 1986; 86: 211-214Crossref PubMed Scopus (16) Google Scholar). Antibodies directed against α-amylase revealed, in human pancreatic microsomes, one band at 55 kDa (8Bruneau N. Lechêne de la Porte P. Sbarra V. Lombardo D. Eur. J. Biochem. 1995; 233: 209-218Crossref PubMed Scopus (26) Google Scholar), a migration compatible with that of α-amylase (27Guy O. Lombardo D. Bartelt D.C. Amic J. Figarella C. Biochemistry. 1978; 17: 1669-1672Crossref PubMed Scopus (126) Google Scholar). Antibodies directed against BSDL (referred to as pAbL64) detected, in human pancreatic juice, one band associated with anM r ≈100 kDa (13Mas E. Abouakil N. Roudani S. Miralles F. Guy-Crotte O. Figarella C. Escribano M.J. Lombardo D. Biochem. J. 1993; 289: 609-615Crossref PubMed Scopus (50) Google Scholar). pAbL64 also reacted with the oncofetal variant of BSDL (i.e. FAPP) (13Mas E. Abouakil N. Roudani S. Miralles F. Guy-Crotte O. Figarella C. Escribano M.J. Lombardo D. Biochem. J. 1993; 289: 609-615Crossref PubMed Scopus (50) Google Scholar). Polyacrylamide gel electrophoreses (10% acrylamide, 0.1% sodium dodecyl sulfate, SDS-PAGE) were performed according to Laemmli (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Proteins were electrophoretically transferred to nitrocellulose membranes (30Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5927) Google Scholar) and probed using polyclonal antibodies specific for pancreatic proteins as primary antibody. The antigen-antibody complexes were then detected by125I-protein A (0.25 μCi/ml) overlay. After extensive washing, replicas were autoradiographed. Total RNA was extracted from pellets of human pancreatic cells or tissues using the method of Chirgwin et al. (31Chirgwin J.M. Pryzbyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16652) Google Scholar). RNA were reverse-transcribed as described by Roudaniet al. (32Roudani S. Miralles F. Margotat A. Escribano M.J. Lombardo D. Biochim. Biophys. Acta. 1995; 1264: 141-150Crossref PubMed Scopus (25) Google Scholar). The cDNA(−) pool obtained was amplified by performing a polymerase chain reaction using a pair of primers designed to cover the entire sequence of secreted BSDL (3Reue K. Zambaux J. Wong H. Lee G. Leete H. Ronk M. Shively J.E. Sternby B. Borgström B. Ameis D. Schotz M. J. Lipid Res. 1991; 32: 267-276Abstract Full Text PDF PubMed Google Scholar, 4Baba T. Downs D. Jackson K.W. Tang J. Wang C-S. Biochemistry. 1991; 30: 500-510Crossref PubMed Scopus (92) Google Scholar) (BSDL-5′,5′-TTCGTaagcttGCGAAGCTGGGCGCCGTGTACAGAA; BSDL-3′,5′-TTTGTgaattcACGCTAAACCTAATGACTGCAGGCATCTG) and using the GC-rich PCR kit from CLONTECH. These two primers include HindIII and EcoRI restriction sequences (lowercase letters), which were used for the subsequent cloning of transcripts. Bases were randomly added in 5′ of these primers to allow restrictive cleavage of PCR transcripts. The DNA was amplified using a 35 reaction cycles program as follows: denaturation (94 °C, 1 min), annealing (64 °C, 1 min), and extension (68 °C, 4 min). The reaction was terminated by an incubation at 68 °C for 10 min. PCR products were then analyzed on 1% agarose gel. After purification, transcripts were subcloned into pCR 2.1 TOPO vector (Invitrogen) and sequenced using M13 forward and reverse or T7 primers. Alternatively, RNA was quantitated by dot-blot dilution. For this purpose, RNA was blotted after cascade half-dilutions on nitrocellulose membrane. Prehybridization and hybridization using specific cDNA probes for BSDL (22Roudani S. Pasqualini E. Margotat A. Gastaldi M. Sbarra V. Malezet-Desmoulin C. Lombardo D. Eur. J. Cell Biol. 1994; 65: 132-144PubMed Google Scholar), α-amylase (a gift of Prof. J-C Chaix, Univ. Aix-Marseille III), and actin (a gift of Dr. R. Planells, INSERM-U38, Marseille) were performed essentially as described (32Roudani S. Miralles F. Margotat A. Escribano M.J. Lombardo D. Biochim. Biophys. Acta. 1995; 1264: 141-150Crossref PubMed Scopus (25) Google Scholar). Before hybridization, probes were32P-labeled by random priming (Life Technologies, Inc.) using [α-32P]dCTP at a specific radioactivity of 4·108 cpm/μg DNA probe. In vitrotranslations of RNA were performed using the rabbit reticulocyte lysate system from Promega in accordance with the manufacturer's instruction. 10 μg of RNA were incubated with the reticulocyte lysate and [35S]methionine (0.4 mCi/ml) in a final volume of 50 μl. After 2 h of incubation at 30 °C, the translated products were immunoprecipitated with pAbL64 and analyzed by SDS-PAGE followed by autoradiography. Transcripts obtained by RT-PCR were digested by HindIII and EcoRI and ligated into pSecTag expression vector (Invitrogen). Stable transfection of CHO-K1 cells was performed with the pSecTag vector comprising RT-PCR transcripts and using the LipofectAMINE-mediated transfection procedure according to the manufacturer (Life Technologies, Inc.). The selection of stable clones was performed for 6 weeks in Ham's F12 medium with zeocin (500 μg/ml, Invitrogen). Control cells, transfected with the empty pSecTag vector, were cloned under the same conditions. Cells grown to confluence on microscope slides were washed three times with incomplete PBS buffer and fixed with 3% (v/v) paraformaldehyde for 20 min. The excess paraformaldehyde was eliminated by washing slides in 50 mmNH4Cl, and cells were permeabilized with 0.05% saponin in PBS buffer. Nonspecific sites were saturated by cell incubation for 30 min in 10% calf serum. The cells were then incubated for the same time with polyclonal antibodies specific for BSDL (pAbL64) and for the p58 Golgi protein. Slides were then exhaustively rinsed with 0.05% saponin in incomplete PBS and incubated for 20 min with FITC-conjugated antibodies directed against rabbit immunoglobulins (10 μg/ml) and with TRITC-conjugated antibodies directed against mouse immunoglobulins (10 μg/ml). Slides were washed with 0.05% saponin in incomplete PBS and mounted in 25 mm Tris/HCl, pH 8.0, buffer, 75% glycerol, and 0.1% p-phenylenediamine illuminated using adequate filters of a fluorescence microscope and photographed. The presence of a BSDL messenger has been detected byin situ hybridization performed on SOJ-6 cells (22Roudani S. Pasqualini E. Margotat A. Gastaldi M. Sbarra V. Malezet-Desmoulin C. Lombardo D. Eur. J. Cell Biol. 1994; 65: 132-144PubMed Google Scholar). Therefore, the presence of mRNA encoding pancreatic enzymes was studied in BxPC-3 and SOJ-6 pancreatic tumoral cell lines. Radiolabeled probes specific for BSDL and α-amylase were used to detect transcripts of respective enzymes. A negative reaction was found when RNA was hybridized with the α-amylase probe, whereas that of BSDL detected a specific transcript in tumoral cell lines (Fig. 1). BSDL and α-amylase transcripts were detected in normal adult pancreas, whereas transcript encoding BSDL was only detected in tumoral pancreas; values were similar to those found with cancer cells. These data suggested that BSDL mRNA was expressed in tumoral tissue and cancer cells, whereas that of α-amylase was not. When normalized to actin mRNA transcript, it appeared that tumoral tissue and cells expressed 20-fold less BSDL than normal pancreatic tissue. The secretion of BSDL was first investigated by Western blotting using polyclonal antibodies specific for BSDL and 125I-protein A overlay. This very sensitive assay allowed us to detect BSDL in cell culture medium of BxPC-3 and SOJ-6 cell lines (Fig. 2). The migration of BSDL secreted by these tumoral cells, although diffuse, appeared lower (arrow, 110–125 kDa, p125) than that of the protein (arrow, 100 kDa, p100) present in normal human pancreatic juice (Fig. 2, HPJ). This could be related to highM r glycoforms of BSDL also referred to as concanavalin A reactive forms (13Mas E. Abouakil N. Roudani S. Miralles F. Guy-Crotte O. Figarella C. Escribano M.J. Lombardo D. Biochem. J. 1993; 289: 609-615Crossref PubMed Scopus (50) Google Scholar, 18Mas E. Franc J.L. Lecestre D. Crotte C. Lombardo D. Sadoulet M.O. Eur. J. Biochem. 1997; 243: 299-305Crossref PubMed Scopus (6) Google Scholar), which preferentially display the J28 epitope. Two immunoreactive forms of BSDL were detected in cell culture of SOJ-6 cells, the lowerM r form having the same electrophoretic migration than that of the protein detected in normal pancreatic juice (100 kDa). Second, BxPC-3 and SOJ-6 cells were incubated for 6 h in fresh medium, and BSDL activity was then recorded on 4-NPH. This activity was found in cell culture medium of SOJ-6 cells, whereas no activity could be detected in that of BxPC-3. BSDL activity represented approximately 12% of the total esterolytic activity detected in SOJ-6 cell line (Table I). The presence of BSDL in the extracellular medium did not correlate with that of the cytoplasmic marker LDH, the activity of which never exceeded 5% of its intracellular activity. This indicated that cell lysis may not be responsible for the presence of extracellular BSDL activity. Because BSDL activity represented at least 85% of the total esterolytic activity associated with tumoral cells (22Roudani S. Pasqualini E. Margotat A. Gastaldi M. Sbarra V. Malezet-Desmoulin C. Lombardo D. Eur. J. Cell Biol. 1994; 65: 132-144PubMed Google Scholar), it seems that only a minute amount of BSDL activity was secreted by SOJ-6 cells. Attempts to detect α-amylase in the cell culture medium by Western blot or by recording activity were unsuccessful.Table ISecretion of BSDL by cell lines and transfected CHO cellsCellsActivity (10−3units)Extracellular activity (% Total activity)IntracellularExtracellularSOJ-629.5 ± 1.34.1 ± 0.712.2 ± 2.4BxPC-345.1 ± 4.10 0CHO-pSecFAPP2.8 ± 0.73.6 ± 0.456.3 ± 4.7CHO-pSecBSDL11.2 ± 2.0130.7 ± 4.692.1 ± 1.4Cells were cultured in adequate fresh medium for 6h. Then, BSDL activity was recorded in cell-free medium (extracellular activity), and cells were harvested and lysed before determination of BSDL activity (intracellular activity). Open table in a new tab Cells were cultured in adequate fresh medium for 6h. Then, BSDL activity was recorded in cell-free medium (extracellular activity), and cells were harvested and lysed before determination of BSDL activity (intracellular activity). We next attempted to determine whether the BSDL expressed by tumoral cells was associated with membranes, as found in normal pancreatic tissue (8Bruneau N. Lechêne de la Porte P. Sbarra V. Lombardo D. Eur. J. Biochem. 1995; 233: 209-218Crossref PubMed Scopus (26) Google Scholar). For this purpose, cells were grown to 80% confluence and lysed, and the lysate was clarified as described previously (2
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