Etienne Hollande

Gastric M1 mucin, an early oncofetal marker of colon carcinogenesis, is encoded by the MUC5AC gene

Gastric M1 mucin and the MUC5AC gene show a similar oncofetal expression in the colon. Our aim was to determine whether M1 mucin is the product of the MUC5AC gene. A recombinant baculovirus encoding the C‐terminal portion of the MUC5AC gene as a fusion protein was isolated and the immunoreactivity of the recombinant mucin (rM) toward M1 antibodies studied. Chicken antibodies also were raised against purified rM. Besides its reactivity with L56/C, a serum recognizing the bacterially expressed MUC5AC gene product, rM was endowed with M1 immunoreactivity: (i) rM‐expressing cells were stained specifically with anti‐M1 serum and with the monoclonal antibody (MAb) 21M1, defining the M1‐f epitope; (ii) both L56/C and anti‐M1 antibodies recognized the same bands in immunoblots of rM‐containing cell extracts; (iii) the 21M1 antibody reacted with rM in an immunoradiometric assay. Among the 7 M1 epitopes, M1‐f was the only one encoded by the 3′ portion of the MUC5AC gene. It was the only epitope detected in a native mucin M1‐derived 170 kDa bromelain proteolytic fragment. Furthermore, the staining patterns of human tissues obtained with either anti‐rM chicken antibodies or anti‐M1 antibodies were identical. We conclude that M1 immunoreactivity is encoded at least in part by the MUC5AC gene. Int. J. Cancer 75:767–773, 1998.© 1998 Wiley‐Liss, Inc.

Possible regulation of CFTR‐chloride channels by membrane‐bound phosphatases in pancreatic duct cells

We have studied CFTR‐Cl− channels in non‐CF CAPAN‐1 and in CFTR‐transfected CFPAC‐PLJ‐CFTR‐6 epithelial cells from human pancreas. Theophylline and IBMX induced the opening of cell‐attached CFTR‐Cl− channels. Theophylline, IBMX and the alkaline phosphatase (AP) inhibitor levamisole enhanced the activity of excised channels and reduced by 70–75% the apical membrane‐associated APs activity. Okadaic acid had no effect on APs and channel activities. A polyclonal anti‐alkaline phosphatase antibody (which detected apical APs) reduced APs activity and activated quiescent excised chloride channels. These results suggest that CFTR channels may be regulated by membrane‐bound phosphatases.

Anion channels in a human pancreatic cancer cell line (Capan-1) of ductal origin

Transepithelial solute transport and bicarbonate secretion are major functions of pancreatic duct cells, and both functions are thought to involve the presence of chloride channels in the apical membrane of the cell. After being isolated from a human pancreatic adenocarcinoma, the Capan-1 cell line conserves most of the properties of ductal cells and thus constitutes a useful system for investigating the role of chloride channels. Using patch-clamp techniques, we identified three different chloride-selective channels in the apical membrane of confluent Capan-1 cells. Two were non-rectifying chloride channels with low (50 pS) and high (350 pS) unitary conductances. Both channels were active in cell-attached recordings, and they were consistently located together in the same patch. Maxi Cl− channels displayed multiple subconductance states, and were reversibly inactivated by either positive or negative voltage changes, which indicates that they were optimally opened at the cell resting potential. The third was an outwardly rectifying chloride channel with a unitary conductance of 38 pS and 70 pS at negative and positive potentials respectively. Rectifying Cl− channels were clustered in discrete loci. They were silent in situ, but became active after patch excision. In inside-out excised patches, the three channels displayed a high selectivity for Cl− over monovalent cations (Na+ and K+) and gluconate. They were blocked by 20–200 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and were insensitive to changes in the Ca2+ concentration. Our results show that the apical membrane of Capan-1 cells contains a high density of chloride channels; these channels may provide pathways for transepithelial solute transport as well as for bicarbonate secretion.

Differential Regulation of Fibroblast Growth Factor (FGF) Receptor-1 mRNA and Protein by Two Molecular Forms of Basic FGF MODULATION OF FGFR-1 mRNA STABILITY

To evaluate possible functional differences between basic fibroblast growth factor (FGF) 2 isoforms we analyzed the effects of the 18-kDa FGF-2 which mainly localizes in the cytosol and that of the nuclear-targeted 22.5-kDa form on FGF receptors (FGFR) expression. These peptides were expressed at low amounts through a retroviral-infection system. Point mutated FGF-2 cDNAs under the control of the β-actin promoter were used to infect a pancreatic cell line (AR4-2J) which does not produce FGF-2. Saturation and competition binding studies with I-FGF-2 revealed a 3-fold increase in both high and low affinity receptors in cells expressing the 22.5-kDa form and a 2-fold increase only in the high affinity receptors in cells producing the 18-kDa form. K values and molecular weights of the high affinity receptors were unaffected. Increasing cell densities or cell treatment with exogenous FGF-2 resulted in FGFR down-regulation as in control cells. Neutralizing anti-FGF-2 antibodies and suramin did not affect receptor density in control and in cells producing the 22.5-kDa form but further increased by 60 and 80%, respectively, the receptor level in cells synthesizing the 18-kDa form. These data suggest the involvement of the intracellular stored FGF-2 in FGFR up-regulation. Although all cells expressed FGFR-1, −2, and −3 mRNA only the FGFR-1 transcript was found increased, 6-fold in 22.5-kDa expressing cells and 3-fold in cell producing the shortest secreted isoform. The increase in FGFR-1 mRNA levels in the 22.5-kDa expressing cells was due to enhanced stability of the transcript. Confocal microscopy detected the presence of FGFR-1 at the cell surface whereas secretory isoforms of the receptor were not observed. Reverse transcriptase-polymerase chain reaction did not reveal significant differences in the expression of FGFR-1 variants. In the 22.5-kDa expressing cells exogenous FGF-2 evoked a stronger translocation of the calcium-phospholipid-dependent PKC. These results indicate that the transfected FGF-2 isoforms up-regulated FGFR-1 mRNA and protein. The 22.5-kDa form acted by increasing FGFR-1 mRNA stability enhancing cell responses to exogenous FGF-2.

The expression of carbonic anhydrases II and IV in the human pancreatic cancer cell line (Capan 1) is associated with bicarbonate ion channels

Summary— Human pancreatic ductal cells of the Capan 1 cell line differentiate progressively during growth. After the exponential growth phase, the cells elongate and become polarized with their apical poles covered by microvilli and separated from the basolateral pole by tight junctions. In this stationary phase, they form domes, which are thought to result from the exchange of water and electrolytes. In this study, we demonstrated, using patch‐clamp techniques, that HCO3− ions exit via the g350 high conductance anionic channel we observed recently at the Capan 1 cell surface. This g350 channel was thought to be either a Cl−/HCO3− antiport or a simple HCO3− channel. The stilbene derivatives 4‐acetamido‐4 isothiocyano‐2‐2′‐disulfonic acid (SITS) and 4,4′ diisothiocyano stilbene‐2,2′ disulfonic acid (DIDS) reduced both the number of domes and the Cl− and HCO3− flux through the g350 channel. Moreover, using histochemical, immunocytochemical and biochemical methods we showed that Capan 1 cells express a specific pattern of carbonic anhydrases (CA). Two types of CA were detected: the CA II isozyme mainly localized in the cytoplasm, but also found beneath the inner leaflet of the apical plasma membrane, and the CA IV isozyme localized on the outer leaflet of the apical plasma membrane and microvilli. Their molecular masses were 30 (CA II) and 55 kDa (CA IV), respectively. They were expressed continuously during the exponential growth phase, although their activity increased greatly during the stationary phase. Inhibition of dome formation by acetazolamide indicated the existence of a direct relationship between dome formation and CA. Characteristic structures with a central electron‐dense core surrounded by a light halo were observed on the surface of cell membranes using histochemical and immunocytochemical methods. These structures were thought to represent a channel, corresponding possibly to CA IV. Our observations suggest that Capan 1 cells, despite their neoplasic transformation, produce HCO3− ions in the same way as normal human pancreatic ductal cells. Capan 1 cells in culture may therefore represent a suitable model for studying pancreatic duct HCO3− secretion at the cellular and molecular levels.

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl− channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 μmol/l), dibutyryl-adenosine 3′,5′-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3′,5′-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 μmol/l) and forskolin (10 μmol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl−>I−≫>HCO3−>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg2+). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl− channel was 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS, 100 μmol/l) and 4-acetamido-4′-isothiocyanatostilbene-2, 2′-disulphonic acid (SITS, 100 μmol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 μmol/l). These results demonstrate that the apical low-conductance Cl− channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel.

Noninvasive fluorine-19 NMR study of fluoropyrimidine metabolism in cell cultures of human pancreatic and colon adenocarcinoma.

SummaryFluorine-19 NMR spectrometry was used to monitor the metabolism of two antineoplastic fluoropyrimidines, 5-fluorouracil (5FU) and 5′-deoxy-5-fluorouridine (5′dFUrd), in cell cultures of human pancreatic (Capan-1) and colon (HT-29) adenocarcinoma. The preliminary results showed, for the two tumor cell lines treated with 5FU, the presence in nonperfused cells of three signals corresponding to intracellular metabolites: 5FU, F-nucleotides and F-nucleosides. When the cells were perfused only the signals of F-nucleotides and 5FU were present. The F-nucleosides observed during the analysis of the nonperfused cells came from the conversion of F-nucleotides. During the NMR recording of Capan-1 cells at 37 °C the first metabolite of the catabolic pathway of 5FU, 5,6-dihydro-5-fluorouracil, occurred. At the beginning of the NMR recording of Capan-1 cells treated with 5′dFUrd, two signals corresponding to F-nucleotides and F-nucleosides (consistent with 5′dFUrd) were observed; during the analysis, a supplementary signal corresponding to 5FU appeared. Even after pretreatment with methotrexate the signal of 5FU incorporated into RNA was not detected. Our experiments, performed in attempts to observe the signal of the ternary complex between thymidylate synthetase (TS), 5-fluoro-2′-deoxyuridine-5′-monophosphate (FdUMP) and 5,10-methylene-tetrahydrofolate (5,10-CH2FH4), allowed detection in some cases of a broad signal, whose chemical shift was similar to that reported in the literature following incubation of TS with FdUMP and 5,10-CH2FH4, but our results were not always reproducible.

MORPHOGENESIS OF "DUCT-LIKE" STRUCTURES IN THREE-DIMENSIONAL CULTURES OF HUMAN CANCEROUS PANCREATIC DUCT CELLS (CAPAN-1)

SummaryPancreatic duct cells secrete water and ions, bicarbonate in particular. The study of these secretion processes is hindered by the unavailability of human pancreatic tissue. In this study, pancreatic human cells of the Capan-1 cell line were employed to investigate secretion in vitro. These cells are of ductal origin because in standard culture they polarize spontaneously forming domes in the culture dishes, indicating the existence of transepithelial exchange of water and electrolytes.In culture in suspension, Capan-1 cells form hollow spheroids bounded by a cell monolayer in a radial organization. These three-dimensional structures could be maintained in culture for more than 140 days. In young cultures, the cells of these spheroids grew rapidly (mitotic index=9.2% on Day 2). Their cytologic features were analyzed by immunocytochemical, cytoenzymatic methods, and by electron microscopy. We showed that they are : a) polarized with an apical pole facing the culture medium; b) organized in a monolayer; c) bound by tight junctions and desmosomes; d) characterized by a particular distribution of enzyme systems known to play a role in ion exchanges, with placental-type alkaline phosphatases and carbonic anhydrases IV on their apical membranes and Ca2+-ATPases on their basolateral membranes. Crystalline structures were detected histochemically in the closed cavities and in the intercellular spaces of the spheroids. X-ray emission spectroscopy and electron diffraction showed that they consisted of calcium phosphate in an apatite structure. They were assumed to derive from a raised concentration of Ca2+ and phosphate ions under the impermeable monolayer of the spheroids. In addition, numerous cells secreted M1 gastric-type mucins, and acquired the ability to produce colonic-type M3 mucins. These hollow spheroids swelled during the culture period. Taken together these results suggest that the Capan-1 cells organized in these hollow spheroids exchange ions. Their three-dimensional structure resembles that of human pancreatic ducts, and they may therefore represent a useful model system for investigation of Cl− and HCO3− ion exchange processes in the human pancreas.

TARGETING OF CFTR PROTEIN IS LINKED TO THE POLARIZATION OF HUMAN PANCREATIC DUCT CELLS IN CULTURE

A relationship between targeting of the protein CFTR (Cystic Fibrosis Transmembrane conductance Regulator) and cellular polarization has been observed in various types of epithelial cells. However, there are no reports on this in human exocrine pancreatic cells, which are functionally altered in patients with cystic fibrosis. The expression of CFTR and its targeting to apical plasma membranes was investigated during growth and polarization of human ductal pancreatic cancerous Capan-1 cells. Despite their neoplastic origin, the cancerous pancreatic duct cells of the Capan-1 line secrete Cl- and HCO3- ions. We showed by electron microscopy, impregnation of cells with tannin and freeze-fracture that these cells become polarized during growth in culture, and are joined by tight junctions. The expression of CFTR and the various stages in its anchorage to membranes was followed using a specific polyclonal antibody, ECL-885, directed against a synthetic peptide mimicking one of the extracellular loops of CFTR. Qualitative and quantitative confocal microscopic studies showed that: (i) the expression of CFTR was constant during growth, irrespective of cellular conformation, (ii) the number of cells presenting CFTR anchored to membranes increased with time in culture, (iii) the rise in membrane-bound CFTR-immunoreactivity accompanied the polarization of the cells, (iv) CFTR anchored to plasma membranes was distributed regularly over the surface of non-polarized cells, but was localized only at the apical membranes of the polarized cells. Moreover, patch-clamp studies indicated the presence of few Cl- cAMP-dependent conductance CFTR channels on unpolarized cells, and a larger number of CFTR channels on the apical plasma membranes of polarized cells. These results indicated that the anchorage of a functional CFTR to the plasma membrane is progressive and occurs in step with polarization of these human pancreatic duct cells in culture. We suggest that the targeting of CFTR to the apical membranes is directly linked to the process of cellular polarization.

FGF-2 isoforms of 18 and 22.5 kDa differentially modulate t-PA and PAI-1 expressions on the pancreatic carcinoma cells AR4–2J: Consequences on cell spreading and invasion

Pancreatic tumors overexpress FGF‐2 and t‐PA, but the implication of the growth factor in t‐PA synthesis and t‐PA‐dependent tumor invasion remains unknown. FGF‐2 is present in different isoforms: The 18 kDa FGF‐2 is secreted, while the 22.5 kDa one is nuclearized and exerts intracrine regulations bypassing cell‐surface FGF receptors. Rat pancreatic carcinoma AR4–2J cells producing either the 18 or the 22.5 kDa FGF‐2 after transfection with FGF‐2 cDNAs have been used to analyze the role of FGF‐2 in t‐PA expression and t‐PA‐related cell spreading. The 22.5 kDa FGF‐2 reduced t‐PA and PAI‐1 synthesis 2‐fold. Addition of recombinant 18 kDa FGF‐2 (rFGF‐2) to cell cultures resulted in increased t‐PA and decreased PAI‐1 expression. By contrast, rFGF‐2 did not significantly modify t‐PA synthesis in cells producing the 22.5 kDa FGF‐2. Cell spreading was t‐PA‐dependent. Furthermore, cells producing the 22.5 kDa FGF‐2 migrated less than control cells and cells producing the 18 kDa FGF‐2. Overall, our data show that secretory FGF‐2 is involved in t‐PA synthesis by pancreatic cancer cells and facilitates cell spreading. The 22.5 kDa FGF‐2 exerts opposite effects by decreasing t‐PA expression in basal conditions and during rFGF‐2 stimulation. Since the expression of the 22.5 kDa FGF‐2 is under specific controls, its up‐regulation might have the potential to reduce spreading of pancreatic cancer cells. Int. J. Cancer 85:555–562, 2000.