JOURNAL OF CELLULAR PHYSIOLOGY 147:333343 (1991)
Evidence for a Common Cell of Origin for Primitive Epithelial Cells Isolated From Rat Liver and Pancreas HANNE CATHRINE BISCAARD AND SNORRI S. THORCEIRSSON* laboratory of Experimental Carcinogcnecis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 The appearance of differentiated hepatocytes in the adult rat pancreas as well as pancreatic-type tissue in the adult rat liver can be experimentally induced (Reddy et al.: 1. Cell Biol., 98:2082-2090, 1984; Rao et al., 1. Histochem. Cytochem., 34:197-201, 1986). These observations suggest a lineage relationship between cell compartments present in rat liver and pancreas. The present data demonstrate that epithelial cell lines with almost identical phenotypes can be established from adult rat liver and pancreas. The established cell lines showed similar morphologies as established by light- and electron-microscopic studies. The cell lines showed a unique expression pattern of intermediate filament proteins. Vimentin, actin, and P-tubulin were present in all cell lines. In addition, simple epithelial type I 1 cytokeratins 7 and 8 were found to be coexpressed with the type I cytokeratin 14 in several of the cell lines. Neither the type I cytokeratins 18 and 19, which are the normal partners for cytokeratins 8 and 7 in filament formation, nor the type I I cytokeratin 5 could be detected despite the fact that filaments were formed by both cytokeratins 8 and 14. This suggests that cytokeratin 14 acts as an indiscriminate type I cytokeratin in filament formation in the established cell lines. The cell lines expressed the same sets of LDH and aldolase isoenzymes and identical sets of glutathione transferase subunits. In addition, the epithelial cell lines from liver and pancreas were equally sensitive to the growth-inhibitory effects of TGF-Pl. No expression of tissue- or cell-specific proteins such as wfetoprotein, albumin, amylase, elastase, or y-glutamyl transpeptidase were detected. The almost identical phenotypes of the hepatic and pancreatic cell lines suggest that they may be derived from a common primitive epithelial cell type present in both rat liver and pancreas. In contrast to parenchymal cells, these cells have an extended capacity for proliferation in vitro and may represent a progeny from a “precursor“ or “stem” cell compartment in vivo.
The rat liver and pancreas have a common embryonic origin, both being derived from almost identical regions of the primitive endoderm. These organs are comprised of predominantly epithelial cells, which, based on their expression of cytokeratins, have been characterized as simple epithelia (Moll et al., 1982). In the early fetal period hepatic epithelial cells express predominantly cytokeratins 8, 14, and 18 (Germain et al., 1988a,b; Marceau, 1990). However, as cell differentiation in the developing liver proceeds along the hepatocytic or biliary epithelial lineages a more complex pattern of cytokeratin expression arises. While hepatocytes express primarily cytokeratins 8 and 18, biliary ductular cells express primarily cytokeratins 7, 8, 18, and 19 (Marceau, 1990). Interestingly, the cytokeratin expression in the adult liver parallels that of the adult pancreas. Pancreatic acini cells express cytokeratins 8 and 18, while pancreatic ductal cells express cytokeratins 7, 8, 18, and 19 (Marceau, 1990). Extensive research in he atocarcinogenesis has revealed that one of the earyiest cellular responses to chemical hepatocarcinogens in the rat involves the 0 1991 WILEY-LISS, INC.
proliferation of cells located around the terminal bile ductules followed by the appearance of a rapidly growing population of small, distinct epithelial cells characterized by oval nuclei and dense cytoplasm commonly referred to as “oval cells” (Farber, 1984; Sell, 1990). During the early stages of oval cell proliferation, a majority of the small epithelial cells seems to express cytokeratins similar to that of biliary ductular cells (Germain et al., 1985; Dunsford et al., 1989; Dunsford and Sell, 1989; Evarts et al., 1990). Once induced, oval cells may undergo necrosis or differentiate into hepatocytes (Farber, 1956; Rubin, 1964; Inaoka, 1967; Grisham and Porta, 1964; Evarts et al., 1987, 1989, 1990). Additionally, oval cells can undergo intestinal Received November 20, 1990; accepted February 8, 1991. *To whom reprint requestsicorrespondence should be addressed at the National Cancer Institute, Building 37, Room 3C28, Bethesda, MD 20892. H.C. Bisgaard is on leave from the Institute of Toxicology, National Food Agency, Ministry of Health, Copenhagen, Denmark.
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metaplasia (Tatematsu et al., 1985) and may also differentiate into pancreatic tissue in rat liver (Kimbrough et al., 1972; Rao et al., 1986). Presence of both “oval” cells and exocrine pancreatic tissue similar to that seen in rat liver has recently been observed in human livers (Wolf et al., 1990; Hsia, C.-H., and Thorgeirsson, S.S., unpublished data). Hepatocytes have been shown to appear in the pancreas of rats durin aging and after feeding a diet deficient in copper ( hiu, 1987; Reddy et al., 1984; Rao et al., 1989). The pancreatic hepatocytes in Cu’+deficient animals seem to arise from periductular or ductular cells in a manner very similar, if not identical, to the oval-cell-derived hepatocytes in livers of animals exposed to chemical hepatocarcinogens (Farber, 1984; Evarts et al., 1987; Rao et al., 1989). Thus, it has been hypothesized that a “stem” cell compartment of common embryonic origin is present in both the liver and pancreas (Sell, 1990). Liver-derived diploid or near-diploid epithelial cell lines from normal as well as hepatocarcinogen-treated rats have been established by several investigators (reviewed by Fausto et al., 1987). Studies of the biochemical and ultrastructural characteristics of newly isolated oval cells, cultured oval cells, bile duct cells, and long-term cultures of rat-liver-derived epithelial cells have shown a common pattern of expression of many cellular markers including cytokeratins (Germain et al., 1988a,b; Hayner et al., 1984; Marceau et al., 1986; Tsao et al., 1984). The suggestion that oval cells are descendants from a putative liver “stem” cell (Sell, 1990) raises the possibility that rat liver epithelial cells because of their phenotypic similarities to oval cells are also progeny from such a “stem” cell compartment. Epithelial cell lines from adult pancreas have previously been established by Tsao and Duguid (1987).The almost identical ultrastructural characteristics of pancreatic cells, liver epithelial cells, and oval cells in primary culture as revealed by light- and electronmicroscopic studies (Hayner et al., 1984; Tsao and Duguid, 1987) led us t o hypothesize that epithelial cell lines established from liver and pancreas are derived from equivalent compartments of cells with an extended capacity for proliferation in vitro. Using a series of cell lineage markers, structural as well as functional, we have demonstrated a close lineage relationship between apparently normal epithelial cell lines established from adult rat liver and pancreas.
Rockville, MD) supplemented with 20 mM L-glutamine (Whittaker M.A. Bioproducts, Walkersville, MD), 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), and 50 mgiliter (1) gentamicin sulfate (50 mgie) (GIBCO, Grand Island, NY). The primary cell cultures were incubated at 37°C in a humidified atmosphere containing 5% C 0 2 for a period of 15 days during which the medium was changed every second day. At the end of the incubation period, cell clones with a typical a pearance of epithelial cells were isolated from the f?asks by use of filter paper pieces (3 MM, Whatman Int. Ltd., UK) dipped in a solution containing 0.1% trypsin. The filter paper to which detached cells adhered was transferred to a culture dish of appropriate size and the cells were propagated in Ham’s F12 complete medium. At a cell number of approximately 1 x lo5 cells the cultures were single-cell cloned by the limiting dilution method. Clonal lines were used in the characterization studies between passages 9 and 19 in vitro. RPEAM3 and RPEAM4. The clonal lines RPEAM3 and RPEAM4 (Rat Pancreas Epithelial Adult Male clones #3 and #4)were established from pancreas of a 9 week-old male Fischer rat. The gastric and splenic lobes of the pancreas were isolated and placed in ice-cold Ca’+-Mg”-free Hank‘s Balanced Salt Solution (HBSS, GIBCO Lab.). The peripancreatic tissue was dissected away and the pancreas was minced. The tissue fragments were washed twice with ice-cold HBSS and transferred to Ham’s F12 supplemented with 20 mM L-glutamine, gentamicin sulfate (50 mgil), and collagenase (1 gil) (Sigma Chemical Co., St. Louis, MO). The tissue fragment suspension was incubated with intermittent agitation at 37°C for 30 min. Then trypsin at a final concentration of 0.25%was added and the suspension incubated for an additional 10 min. The suspension was filtered (80 pm mesh nylon filter), thc cells were sedimented at 150g for 5 min, and the cell pellet was washed twice in Ham’s F12 supplemented with glutamine, gentamicin sulfate, and 10% fetal bovine serum. The cell pellet was resuspended in Ham’s F12 medium supplemented as above and cells were plated at a final density of 1 x lo3 cellsicm’ (AccelP’, Costar, Cambridge, MA). The primary cell cultures were incubated at 37°C in a humidified atmosphere containing 5% COzfor a period of 3 weeks during which the medium was changed every third day. At the end of the incubation period, cells with a typical appearance of epithelial cells were isolated and the cells were propagated and single-cell cloned. In the present study, the MATERIALS AND METHODS clonal lines were generally used between the ninth and Isolation of epithelial cell lines 18th passage in vitro. RLEAM7 and RLEAM12. The clonal lines RLESF13. The clonal line RLESFl3 (Rat Liver RLEAM7 and RLEAMl2 (Rat Liver Epithelial Adult Epithelial Suckling Female clone #13) was established Male clones #7 and #12) were established from a 9 from a 10 day-old female Fischer rat as reviously week-old male Fischer rat by a novel method involving described (McMahon et al., 1986). The cel line was perfusion of the liver in situ. Liver cells were isolated routinely maintained as monolayer cultures in Ham’s by the two-step collagenase perfusion technique as F-12 medium supplemented with 20 mM L-glutamine, described by Seglen (1979).The initial cell preparation 10%fetal bovine serum and 50 mgil gentamicin sulfate. was enriched for epithelial cells by sedimentation The RLESF13 cell line was generally used after 18 through a Percoll gradient (Kraemer et al., 1986). The passages in vitro. purified liver cell suspension was plated at a density of In some experiments the epithelial cell lines were 5 x lo4 cells/cm2(AccelPtissue culture flasks, Costar, utilized between passages 35 and 39 in vitro. Cambridge, MA) in Ham’s F-12 medium (Biofluids Inc., FR. The cell line FR (Epidermis, Germ-free fetal rat)
8
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EPITHELIAL CELL LINES FROM LIVER AND PANCREAS
was obtained from the American Type Tissue Culture Collection (Rockville, MD). The cell line was propagated in Ham's F12 supplemented with 20 mM Lglutamine, gentamicin sulfate (50 mg/l), and 10%fetal bovine serum. The cells were used between passages 12 and 16 in vitro. Electron microscopy Ultrastructural studies were performed on cultured cells attached as monolayers in tissue culture flasks. The cell monolayers were grown to approximately 70% confluence, washed twice with Dulbecco's phosphatebuffered saline, and fixed in a phosphate-buffered solution (100 mM, pH 7.2) containing 1%glutaraldehyde. The cell monolayers were postfixed in osmium tetroxide and the samples routinely processed for transmission electron microscopy. Cell proliferation analysis Doubling times. Cells were plated at a density of 5 x l o 4 cellsiwell in six-well culture dishes (Costar, Cambridge, MA). Cells were detached by trypsination at different time points (typically every 24 hr) and counted in a Coulter counter (Coulter Electronics Inc., Hialeah, FL). Doubling times were calculated from growth curves generated from several experiments. TGF-P1 inhibition. The inhibition of cell proliferation by TGF-P1 was assessed by examining the rate of DNA synthesis normalized against cell number by using the procedure of Richards et al. (1985) and modified as described by Chapekar et al. (1989).
Cytoskeletal extraction Cellular cytoskeletons were metabolically labeled by using [35S]methionine (Amersham Corp., Arlington Heights, IL). Cellular monolayers were incubated with complete medium containing 100 pCi/ml ["'Slmethionine for 4 hr. Cytoskeletal proteins were prepared according to Bowden et al. (1984). Briefly, cell monolayers, prewashed in ice-cold phosphate-buffered saline (PBS), were extracted with buffered 1%Triton X-100 (10 mM TrisiHCl pH 7.4, 2 mM DTE, 140 mM NaC1). The insoluble residue was extracted with buffered 0.5% Triton X-100 (10 mMTridHC1 pH 7.4,0.5 mM DTE, 1.5 M KC1) and then washed with buffered 0.1%)Triton X-100 (5mM TrisiHCl pH 7.4, 0.5 mM DTE, 70 mM NaC1). All buffers were supplemented with the following components to prevent protein breakdown by proteases: 5 mM EDTA, 5 mM EGTA, 200 pM PMSF, 1pM leupeptin, and 1 pM pepstatin (all Boehringer-Mannheim, IN). Cytoskeletal pellets were then lysed in SDS sample preparation buffer (50 mM TrisiHCl, pH 6.8; 1 mM MgC1,; 1%SDS; 2% 2-mercaptoethanol), and the samples were sedimented at 100,OOOg for 10 min at room temperature. Cytosolic fractionation Cell monolayers were washed twice with PBS and harvested by scraping, and the cell pellets were homogenized by sonication for 15 s in ice-cold 10 mM Tris/ HC1, pH 7.2, containing the different protease inhibitors. For fractionation of tissues, the excised tissue (approximately 1.5g) from brain, liver, kidney, and pancreas was minced and homogenized in 10 mM
335
TrisiHCl, pH 7.2, containing the different protease inhibitors. Homogenates were centrifuged at 100,OOOg for 30 rnin at 4°C to obtain clear supernatants. Analysis of cytoskeletal proteins Analysis of [35S]methionine-labelled cytoskeletal proteins by two-dimensional gel electrophoresis was performed as described by Hochstrasser et al. (1988); 500,000 dpm of extracted material was loaded on the first-dimension isoelectric focusing gel. Ampholytes (LKB Instruments, Rockville, MD) were used at concentrations of 1.6%, pH range 5 to 8, and 0.4%, pH range 3.5-10. SDS-PAGE in the second dimension was performed by using 1mm-thick 7.5%to 17.5%gradient gels. Polypeptides were detected by direct autoradiography following an exposure of 3 days.
Analysis of glutathione transferase subunits Analysis of glutathione transferase subunits by electrophoresis in one dimension was performed by SDSPAGE according to Laemmli (1970). Samples (10 pg of protein) were loaded onto 10%polyacrylamide mini els (10 x 10 cm, 0.75 mm thick) and electrophoresef at constant voltage (200 V). The relative amounts of the polypeptides were visualized by transferring the polypeptides from the gels to nitrocellulose membranes (Schleicher and Schuell, Keene, NHj in a transblot apparatus (Hoefer Scientific Instruments, San Francisco, CA) at 100 V, 0.2 amp at 4°C for 90 min by using a transfer buffer containing 20 mM TrisiHCl, 150 mM glycine, and 20% (viv) methanol. The nitrocellulose blot was then washed for 5 min in 50 mM Tris/HCl and 100 mM NaC1, pH 7.2 (Tris-buffered saline (TBS)),and blocked overnight in 0.5%nonfat dry milk (Carnation, USA) in TBS at 4°C. The blots were washed with TBS for 5 min and incubated for 2 hr with the rimary antibody diluted in TBS containing 0.5% non at dry milk. After two washes, each for a period of 10 min in TBS supplemented with 0.05% Tween 20, the blots were incubated for 1hr in TBS supplemented with 0.5% nonfat dry milk and the secondary antibody conjugated to alkaline phosphatase as the detection enzyme (Goat antirabbit IgG (H + L) (Bio-Rad Lab., Richmond, CAj). The blots were washed twice in TBS supplemented with 0.05% Tween 20 (10 rnin for each wash) and once in 0.1 M Tris; pH 9.5 ( 5 rnin). The blots were developed by using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitro blue tetrazolium salt (NBT) (Bio-Rad Lab.) as the substrate for alkaline phosphatase. Blots were developed until clear purple bands appeared. For immunoblot analysis, the polyclonal antibody against the glutathione transferase subunit Y (kindly provided by Dr. K. Satoh) was used at a difution of 1:1,000. The polyclonal antibodies against the glutathione transferase subunits Y,, Yb2,and Y, (Medlabs, Dublin, Ireland) were used at dilutions of 1:250.
P
Lactate dehydrogenase LDH isozymes were separated electrophoretically on cellulose acetate membranes by using a standard isoenzyme kit (Sigma Chemical Co., St. Louis, MO); 10 pg of cytosolic protein was loaded on a cellulose acetate membrane (Super Sepraphore cellulose acetate mem-
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brane (Gelman Sciences, MI). Electrophoresis was carried out at constant voltage (250 V) for 25 min in a resolution buffer containing 50 mM Tris, 12 mM citrate, 25 mM barbital. The isoenzyme pattern was visualized by staining with LDH Agaro-Stain@Reagent (Sigma).
Aldolase Cytosolic protein (25 pg) was subjected to electrophoresis on cellulose acetate membranes (Gelman Super Sepraphore) for 60 min at 250 V, in 60 mM high-resolution buffer (Gelman Sciences) containing 10 mM P-mercapto-ethanol. Isozymes were visualized by reactions coupled to the reduction of p-nitrotetrazolium blue as described by Penhoet et al. (1966). Immunofluorescence Indirect immunofluorescence was performed on ethanol-fixed monolayer cultures grown on glass slides (Miles Lab., Naperville, IL). Following rehydration in PBS for 30 min, the slides were incubated with the primary antibody diluted in PBS supplemented with 12% bovine serum albumin for 12 hr at 4°C. The monoclonal antibody against cytokeratin 8 (Amersham) was used at a dilution of 1:lOO. The monoclonal antibody against cytokeratin 14 (Sigma) was used at a dilution of 1:250. The slides were washed twice for 10 min in PBS followed by incubation with a 1:lOO dilution of biotinylated horse anti-mouse IgG (Vector Lab,, Burlingham, CA) in PBS supplemented with 12% albumin for 30 min at room temperature. The slides were washed as before and incubated with streptavidinconjugated Texas Red (Life Technologies Inc., Gaithersburg, MD) diluted 1:400 in PBS supplemented with 12%albumin for 30 min. After washing, the slides were mounted and examined on a Nikon fluorescent microscope. RESULTS Cell morphology Phase-contrast light microscopy of the newly established clonal lines RLEAM7, RLEAM12, RPEAM3, and RPEAM4 showed small cuboidal-shaped cells of relative uniform size (Fig. la,b,d,e). Typical electron micrographs representing the clonal lines RLEAMl2 and RPEAM4 are shown in Figure l c and If. The micrographs revealed cells with a large, rounded nucleus, a small amount of cytoplasm, little rough endoplasmic reticulum, and relatively few mitochondria, small in size. It has previously been reported that a typical feature of small epithelial cells isolated from liver and pancreas is their irregular, indented nucleus (Hayner et al., 1984; Tsao and Duguid, 1987). However, electron micrographs of small epithelial cells as bile duct cells, hepatic oval cells, and pancreatic duct cells in vivo show large, rounded nuclei (Hayner et al., 1984; Tsao and Duguid, 1987). The previous studies of isolated epithelial cells were done on cells fixed in suspension. In contrast, our studies were done on fixed monolayer cultures. Therefore, the irregular, indented nucleus can be attributed to an artifact of the fixation method used. The electron micrographs of the established hepatic and pancreatic cell lines all showed the presence of
tight junctions and desmosomes, supporting the epithelial origin of the cell lines.
Karyotyping The karyotypes of the clonal epithelial cell lines showed that all cell lines were rat-derived diploids with 42 chromosomes. The lines RLEAM7, RLEAM12, RPEAM3, and RPEAM4 were characterized as diploid male (XY) rat while RLESFl3 was characterized as a diploid female (XX) rat. No marker or unassignable chromosomes were present in the lines RLEAM7, RLEAM12, RPEAM3, and RPEAM4, typing these cell lines as apparent normal. A single marker chromosome (chromosome 1)was found in RLESF13. Growth characteristics The doubling times at early passage numbers varied among the epithelial cell lines. While RLESF13 had a doubling time of 18.3 +- 1.3 hr (n = 31, the two lines isolated from adult liver, RLEAM7 and RLEAM12, had doubling times of 31.2 2 2.9 hr (n = 3) and 23.5 2 1.0 hr (n = 3 ) . respectively. The cell lines isolated from adult pancreas, RPEAM3 and RPEAM4, proliferated with doubling times of 30 k 2.7 hr (n = 3) and 34.2 t 1.4 hr (n = 3), respectively. Proliferation of all the epithelial cell lines were inhibited to the same extent by the potent growth inhibitor TGF-P1. The concentration of TGF-P1 causing 50% inhibition of the DNA synthesis (IDs0) was 50-75 pgiml. The proliferation of the FR line established from rat epidermis was not inhibited by TGF-P1 at concentrations up to 1,000 pgiml. Expression of cytoskeletal proteins The two-dimensional " eel natterns of 35S-methioninelabelled intermediate fdaments extracted from RLEAM7, RLEAM12, RPEAM3. RPEAM4. RLESF13. and FR are shown in Figure 2. Relatively few interme: diate filament proteins were present in the cell lines. For identification, the polypeptides were transferred to nitrocellulose and their location was visualized by immunostaining with specific antibodies (data not shown). All the cell lines expressed vimentin, actin, and p-tubulin although the expression varied between the lines. In addition, the cell lines RLEAM12, RPEAM3, and RPEAM4 synthesized several other intermediate filament subunits. Using immunoblotting analysis of cytoskeletal extracts from RPEAM3, three cytokeratins were identified. In addition to the rat equivalents of human cytokeratins 7 and 8 an equivalent of human cytokeratin 14 was expressed. The normal type I partners for cytokeratins 8 and 7, cytokeratins 18 and 19, respectively, as well as the type I1 partner for cytokeratin 14, cytokeratin 5, could not be detected in any of the cell lines by immunoblotting analysis. The two-dimensional gel analysis indicated that cells from the different epithelial cell lines synthesized different quantities of cytokeratins. Using the more sensitive immunofluorescence analysis of cell monolayers with antibodies against cytokeratins 8 and 14, cells showing typical patterns of cytokeratin filaments were found to be present in all the liver and pancreatic cell lines although in varying numbers (Fig. 3). The overall
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Fig. 1. Phase-contrast micrographs of cell lines isolated from rat liver (a) RLEAM7 passage 8, (b) RLEAMl2 passage 7, and rat pancreas (d)RPEAM3 passage 12, (el RPEAM4 passage 11. x 250. Electron microscopic photographs of ( c )RLEAM12 assage 12 isolated from liver and (0 RPEAM4 passage 10 isolated %om rat pancreas. X 3,500.
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Fig. 2. Two-dimensional electrophoretic patterns of ["'Slmethioninelabelled cytoskeletal proteins extracted (a) RLEAM7 passage 13, (b) RLEAMl2 passage 11, (c) RPEAM3 passage 9, and (d) RPEAM4 passage 9, (e) RLESFl3 passage 18, (0 FR. Abscissa, pH range; ordinate, molecular weight x lo3. The abbreviations used are 7, cytokeratin 7 ; 8, cytokeratin 8; 14, cytokeratin 14; a, actin; v, vimentin; t, P-tubulin.
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Fig. 3. Immunofluorescent staining of epithelial cell lines at early passage number by cytokeratins 8 (a, c, e, $, i) and 14 (b, d, f, h, j), RLEAM7 passage 18 (a,b), RLEAMl2 pasage 19 (c,d). RpEAM3 passage 18 (e,D,RPEAM4 passage 18 (g,hi, RLESF13 passage 18 (i,jl.
number of cytokeratin-expressing cells correlated well with the amount of labelled protein detected on the two-dimensional els. Relatively few cells (< 10%) from the liver ce 1 lines RLEAM7, RLEAM12, and RLESF13 were found to express cytokeratins in contrast to a larger number of cells (> 40%) in the pancreatic cell lines RPEAM3 and RPEAM4. However, prolonged passage in vitro (> 35 passages) resulted in a dramatic increase in the number of cells showing filament formation in the liver cell line RLEAMl2 (> 90%) and in the pancreatic line RPEAM4 (> 75%) (Fig. 4c,d,g,h).Cells staining for cytokeratin 5,18, or 19 could not be detected by immunofluorescence in any of the epithelial cell lines at early passage numbers or after at least 35 passages in vitro. No cells stained in monolayers of the epidermal cell line FR.
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Fig. 4. Immunofluorescent staining of epithelial cell lines after prolonged Passage in vitro by cytokeratin 8 (a, c, e, g, i) and 1 4 (b, d, f~ h, j). RLEAM7 Passage 35 (ah), RLEAM12 Passage 39 (cjd)t RPEAM3 passage 33 (e,fl, RPEAM4 passage 37 (g,h), RLESF13 passage 35 (i,j).
Isozyme expression Lactate dehydrogenase. The relative distribution of LDH isoenzymes varies among both different organs and cell types and can therefore be used as a cell lineage marker (Hayner et al., 1984). Five different isoenzymes, LDH,, LDH4, LDH,, LDH2, and LDH1, were detected in the cytosolic fraction of whole homogenates from brain, kidney, and pancreas, while four isozymes-LDH,, LDH4, LDH,, and LDH,-were observed in whole liver homogenates (Fig. 5A). While the different isoenzymes were expressed in almost equal amounts in brain and kidney, the liver and pancreas expressed predominantly LDH,. All of the epithelial cell lines showed a similar expression pattern of LDH
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Fig. 6. Cellulose acetate electrophoresis of aldolase isozymes in supernatants from whole organ homogenates (A) and homogenates from epithelial cell lines isolated from liver and pancreas (B).
isoenzymes (Fig, 5B). The predominant isozymes expressed were LDH5, LDH,, and LDH,, with a lesser expression of LDH2. Only two isozymes, LDH5 and LDH,, were expressed in the cell line FR established from rat epidermis. Aldolase. Three types of aldolase subunits representing three different gene products designated A, B, and C are known to exist in mammals (Penhoet et al., 1966).Aldolase is a tetrameric enzyme and hybrids are readily formed in cells expressing more than one subunit gene (Penhoet et al., 1966; Hayner et al., 1984).In liver homogenates B, was the predominant isozyme, but A4 and AB, isozymes were also present (Fig. 6A). In homogenates from rat pancreas the hybrids AB,, A,B2, A,B, and A, were found. A and C subunits in various hybrid combinations were expressed in homogenates from whole rat brain. The only hybrid expressed in homogenates from all the cell lines including the FR line was A4 (Fig. 6B).
Glutathione S-transferase. Similar to LDH and aldolase, a complex mixture of different GST isoenzymes composed of different subunits is present in different organs and cell types (Abramovitz et al., 1989; Evarts et al., 1990).As shown in Figure 7, the subunits Y, and Y, were expressed in the adult rat liver and kidney but not in pancreas. The subunit Y,, was expressed in liver and pancreas but not in the kidney. The placental form Y, could not be detected in either organ by the method used in the present study. Newly isolated adult rat hepatocytes expressed Y,, Y,, and Ybz while Y, was absent. The cell line isolated from rat epidermis with a morphological appearance of fibroblasts showed a very low expression of the Y b 2 subunit and no expression of the other three subunits. All the epithelial cell lines showed an almost identical expression pattern of GST subunits with a strong expression of Y, and Y,, a lower expression of Yb2, and a very low expression of Y,.
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Expression of proteins related to cell-specific functions In the adult rat liver specific functions such as albumin production and secretion are features of the differentiated hepatocyte while differentiated bile duct cells express y-glutamyl transpeptidase. In the adult pancreas, elastase and amylase are produced specifically in the exocrine acinar cells while epithelial cells of ductal structures express y-glutamyl transpeptidase. None of the e ithelial cell lines showed expression of albumin, amy ase, elastase, or y-glutamyl transpeptidase, indicating that none of the cell lines expressed tissue- or cell-specific functions. The fetal liver protein a-fetoprotein was not detected in any of the cell lines.
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DISCUSSION In the present study, epithelial cell lines having extended capacity to proliferate in vitro were established from adult rat liver and pancreas. The cell lines exhibited almost identical phenotypes as judged by both morphological appearance and biochemical characterization, suggesting a common cell or origin for primitive epithelial cells isolated from rat liver and pancreas. Studies of the cytokeratin expression in the hepatic and pancreatic epithelial cell lines revealed a unique coexpression of the “simple” epithelial type I1
cytokeratins 7 and 8 with type I cytokeratin 14. The data suggest that cytokeratin 14 can act as an indiscriminate cytokeratin partner in filament formation with cytokeratin 8. In an attempt to investigate the possibility of a common cell of origin for the established hepatic and pancreatic epithelial cell lines a series of morphologic as well as biochemical parameters were used as cell lineage markers. The newly established clonal hepatic and pancreatic cell lines appeared similar with respect to both morphology and growth characteristics. All the cell lines had apparent normal diploid karyotypes and displayed a morphology similar t o what has been reported for other hepatic and pancreatic derived epithelial cells in culture (Hayner et al., 1984; Tsao et al., 1984; Tsao and Duguid, 1987). The epithelial nature of the cell lines was indicated by their cuboidal appearance and the presence of desmosomes and gap junctions. The cell lines showed similar proliferation rates and all were equally sensitive to the growth-inhibitory effects of TGF-P1. TGF-P1 has previously been shown to be a potent inhibitor of normal epithelial liver cell proliferation (McMahon et al., 1986; Nagy et al., 1989). The present data suggest that TGF-P1 is an equally potent inhibitor of normal epithelial pancreatic cell proliferation. The use of intermediate filament proteins as epithe-
EPITHELIAL CELL LINES FROM LIVER AND PANCREAS
lial cell lineage markers is well established (Moll et al., 1982; Osborn and Weber, 1982). Among this family of proteins, cytokeratins are of special interest since they are specifically and differentially expressed in different epithelial cell types (Moll et al., 1982).The cytokeratins are frequently expressed in distinct pairs consisting of one acidic (type I) and one basic (type III cytokeratin. In the rat liver two cytokeratins equivalent to the human cytokeratins 8 (type 11) and 18 (type I) are present in both mature hepatocytes and bile duct cells, while two cytokeratins equivalent to human cytokeratins 7 (type 11)and 19 (type I) are expressed specifically in bile duct and oval cells (Marceau, 1990).A similar distribution of simple cytokeratins seems to exist within pancreas where cytokeratins 8 and 18 are expressed in exocrine cells and cytokeratins 7 and 19 in addition to 8 and 18 in epithelial cells of the pancreatic ducts (Marceau, 1990). The two-dimensional gel analysis performed on our established cell lines showed a unique expression of cytokeratins 7, 8, and 14 in several of the cell lines. Immunofluorescent localization of cytokeratins 8 and 14 revealed formation of filament bundles expanding from the nucleus to the membrane. Filament formation of cytokeratins normally requires expression of distinct pairs of type I and type I1 cytokeratins, the normal counterpart for cytokeratin 8 being cytokeratin 18 (Hatzfeld and Franke, 1985). However, we were unable to detect an equivalent of cytokeratin 18 as well as 19 in the cell lines at early as well as late passage number in vitro. Cytokeratin 8 has been shown to be indiscriminate when forming filaments in reconstituted systems in vitro (Hatzfeld and Franke, 1985). In addition, it has been suggested to act as an indiscriminate type I1 cytokeratin in filament formation with other type I cytokeratins such as 19 in intestinal epithelia and MCF-7 cells (Moll et al., 1982). In view of the coexpression of cytokeratins 8 and 14 observed in the RLEAMl2 after prolonged passage in vitro and our inability t o detect cytokeratins 18,19, and 5, it seems reasonable to suggest that cytokeratin 8 acts as the type I1 partner in filament formation with cytokeratin 14 in all our epithelial cell lines. Whether cytokeratin 7 can act as an indiscriminate cytokeratin in filament formation in the epithelial cell lines is presently under investigation. All the established cell lines expressed a similar set of epithelia-specific intermediate filaments although to varying degrees (Fig. 2). In two of the cell lines, RLEAMl2 and RPEAM4, the extent of the cytokeratin expression seems to be related to the passage number of the cells (Fig. 3, 4). In both these cell lines prolonged passage in vitro resulted in an increased expression of cytokeratins 8 and 14. The unique phenotype of the established hepatic and pancreatic cell lines regarding the cytokeratin expression is supported by data provided on rat epithelial cell lines established from other tissues. Lobach and co-workers (1987) found that a newly established thymic epithelial cell line (IT26R21) expressed cytokeratins 5, 7, 8, and 14 and upon prolonged passage also 6 and 16. A rat kidney epithelial cell line was shown to express exclusively cytokeratins 8 and 18 (Storey et al., 1988). Moreover, in vitro transition of pulmonary type I1 epithelial cells to type I cells is accompanied by an induction of cytokeratins 8
341
and 18, and cytokeratins 5 , 7, 14, and 19 were not detected during this transition (Woodcock-Mitchell et al., 1989). In the present study the FR cell line was chosen as a control cell line because of its derivation from rat epidermis and possible relationship to keratinocytes. However, the morphological appearance and the expression of cytoskeletal proteins similar to a rat fibroblast cell line characterized by Garrels and Franza (1989)indicate that this cell line is of mesenchymal and not epithelial origin. Although a cytokeratin with a molecular weight identical to the rat cytokeratin 14 identified in the present study has been found in cells of early embryonic rat liver a definitive identification still is not complete (Germain et al., 1988a,b). To date the expression of cytokeratin 14 has not been reported in adult rat liver or pancreas. However, preliminary observations from our laboratory indicate that cytokeratin 14 is expressed during early stages of oval cell proliferation (Bisgaard, H.C., and Evarts, R.P., unpublished data). Whether expression of this unique set of keratins in the cell lines is merely related to the in vitro culture conditions or reflects origin from as-yet-undefined “stem” cell populations in vivo is presently under investigation. Similar to that observed with the cytoskeletal proteins, a complex mixture of different GST isoenzymes is also present in mammalian organs due to variable expression of some forms and the complete absence of other forms (for review see Boyer, 1989).Recent data on the expression of mRNA coding for the placental subunit of glutathione transferase (Y,) during early stages of hepatocarcinogenesis (Evarts et al., 1990) and data on the differential expression of mRNA coding for other subunits in primary hepatocytes (Abramovitz et al., 1989) indicate that GST can be used as a cell lineage marker in rat liver. Primary adult rat hepatocytes show transcripts for the Y,, Y,, Y b l , Yb2, and Yb3 subunits but not for Y,. However, during early stages of hepatocarcinogenesis Y, transcripts are found in bile ducts, ductal structures, oval cells, and basophilic foci of small hepatocytes but not in normal mature hepatocytes, endothelial cells, or the mesothelial cells of the Glisson capsula (Evarts et al., 1990).In addition, the Y, protein has been detected in human fetal liver (Guthenberg et al., 1986). Our data using immunoblotting analysis with antibodies specific for the Y,, Y,, Yb2, and Y, subunits confirmed that freshly isolated rat hepatocytes express the Y,, Y,, and Yb2subunits while Y, is absent. However, the different pattern of GST subunit expression in the liver-derived epithelial cell lines characterized by high expression of the Y, and Y, subunits and low expression of Y, and Yb2 indicates that these cell lines might be derived from cell populations located in the biliary structures of the normal rat liver. The lack of expression of Y, in endothelial and mesothelial cells of rat liver (Evarts et al., 1990) indicates that the established cell lines are not derived from these cell compartments. Rat liver epithelial cells in long-term culture have phenotypic traits almost identical to those of oval cells (Tsao et al., 1984; Grisham et al., 1974). However, the isoenzyme pattern of both LDH and aldolase present in the liver-derived cell lines differs significantly from the pattern previously found in isolated oval cells and bile
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duct cells (Hayner et al., 1984). While LDH,, LDH,, and LDH, are the main isoforms detected in our established cell lines, LDH4,LDH3, and LDHz were the main isoforms found in both oval and bile duct cells. Our data therefore support the suggestion that the propagable liver epithelial cell lines may originate from a compartment of cells located in the ductular structures (Grisham, 1980) but with phenotypic features that differ somewhat from oval cells and terminal bile duct cells. Our study was designed to investigate a possible relationship between epithelial cell lines isolated from liver and pancreas. During embryogenesis both the liver and pancreas develop from a common origin, the endoderm. Recent findings in rats have shown that a unique transdifferentiation of epithelial cells in pancreas into fully differentiated hepatocytes occurs after treatment with a copper-deficient diet (Reddy et al., 1984; Rao et al., 1989). The fact that populations of small epithelial cells with common morphological phenotypes arise after extensive hepatic and pancreatic injury led us to hypothesize that identical compartments of primitive epithelial cells capable of proliferating in vitro are present in these organs. The present findings that the phenotypic traits characteristic of epithelial cell lines isolated from pancreas closely resemble those observed in epithelial cells isolated from liver suggest that these cell lines may originate from a common rimitive cell population that resides in both organs. It as recently been shown that chemical transformation of rat liver epithelial cell lines results in the generation of a wide range of tumors including hepatocellular carcinomas, cholangiocarcinomas, and hepatoblastomas as well as sarcomas and mixed epithelial-mesenchymal tumors (Tsao and Grisham, 1987; Lee et al., 1989). Furthermore, we have recently shown that similar tumor types are generated when rat-liver-derived epithelial cells are transformed by selective oncogenes (Garfield et al., 1988). These data strongly indicate the blastic multipotent nature of the liver-derived epithelial cell lines. We are currently investigating whether transformation of the epithelial cell lines from pancreas results in formation of tumorigenic cells reflecting bhe same blastic multipotent nature.
K
ACKNOWLEDGMENTS P.T. Ton is acknowledged for skillful technical assistance. We wish to thank Dr. P.J. Wirth and Dr. B.S. Warren for many helpful discussions and for critical reading of the manuscript. H.C. Bisgaard was supported by a grant from the Danish Research Academy.
LITERATURE CITED Abramovitz, M.M., Ishigaki, S., and Listowsky, I. (1989) Differential expression of glutathione S-transferase in cultured hepatocytes. Hepatology, 92235-239. Bowden, P.E., Quinlan, R., Breitkreutz, D., and Feusenig, N.E. (1984) Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis. Eur. J. Biochem., 142:29-36. Boyer, T.D. (1989) The glutathione S-transferases: An update. Hepatblogy, 9:486-497. Chapekar, M.S., Iluggett, A.C., and Thorgeirsson, S.S. (1989)Growth modulatory effects of a liver-derived growth inhibitor, transforming
growth factor p1, and recombinant tumor necrosis factor a, in normal and neoplastic cells. Exp. Cell Res., 185.247-257. Chiu, T. 11987) Focal eosinophilic hypertrophic cells in the rat pancreas. Toxicol. Pathol., 15:331-333. Dunsford? H.A., Karnasuta, C., Hunt, J.M., and Sell, S. (1989) Different lineages of chemically induced hepatocellular carcinoma in rats defined by monoclonal antibodies. Cancer Res., 49:48944900. Dunsford, H.A., and Sell, S. (19891Production of monoclonal antibodies to preneoplastic liver cell populations induced by chemical carcinogens in rats and t o transplantable Morris hepatomas. Cancer Res.; 49:48874893. Evarts, R.P., Nagy, P., Marsden, E., and Thorgeirsson, S.S. (1987) A precursor-product relationship exists b e h e e n oval cells and hepatocytes in rat liver. Carcinogenesis, 8:1737-1740. Evarts, R.P., Nagy, P., Nakatsukasa. H., Marsden, E., and Thorgeirsson? S.S. (1989) In vivo differentiation of rat liver oval cells into hepatocytes. Cancer Res., 49:1541-1547. Evarts, R.P., Nakatsukasa, H., Marsden, E., Hsia, C.-C., Dunsford, H.A., and Thorgeirsson, S.S. (1990) Cellular and molecular changes in the early stages of chemical hepatocarcinogenesis in the rat. Cancer Res., 50:3439-3444. Farber, E. 11956) Similarities in the sequence of early histological changes induced in the liver by ethionine, 2-acetylaminofluorene and 3'-methyl-4-dimethylaminoazobenzene. Cancer Res., 16:142148. Farber, E.(1984)The multistep nature of cancer development. Cancer Res., 44:42174223. Fausto, N., Thompson, N.L., and Braun, L. (1987) Purification and culture of oval cells from rat liver. In: Cell Separation: Methods and Selected Applications, vol. 4. T.G. Pretlow, and T.P.P. Pretlow, ed. Academic Press Inc., New York, pp. 45-77. Garfield, S., Huber, B.E., Nagy, P., Cordingley, M.G., and Thorgeirsson, S.S. (1988) Neoplastic transformation and lineage switching of rat liver epithelial cells by retrovirus-associated oncogenes. Mol. Carcinog., 1:189-195. Garrels, J.I., and Franza, B.R. (1989) Transformation-sensitive and growth-related changes of protein synthesis in REF52 cells. J. Biol. Chem., 26452994312. Germain, L., Blouin. M.-J.,and Marceau, N. (1988a) Biliary epithelial and hepatocytic cell lineage relationships in embryonic rat liver as determined by the differential expression of cytokeratins, a-fetoprotein, albumin, and cell surface-exposedcomponents. Cancer Res., 48:49094918. Germain, L.,Goyette, R., and Marceau, N. (1985) Differential cytokeratin and a-fetoprotein expression in morphologically distinct cells emerging at early stages of rat hepatocarcinogenesis. Cancer Res., 45:673-681. Germain, L.,Noel, M., Gourdeau, H., and Marceau, N. (198813) Promotion of growth and differentiation of ductular oval cells in primary culture. Cancer Res., 48:368-378. Grisham, J.W. (1980) Cell types in long term propagable cultures of rat liver. Ann. NY Acad. Sci., 349:128-137. Grisham, J.W., and Porta, E.A. (1964) Origin and fate of proliferated ductul cells in the rat: electron microscopic and autoradiographic studies. Exp. Mol. Patbol., 3:242-261. Grisham, J.W., Thal, S.B., and Nagel, A. (1974) Cellular derivation of continuously cultured epithelial cells from normal rat liver. In: Gene Expression and Carcinogenesis in Cultured Liver. L.E. Gerschenson and E. Thompson, eds. Academic Press, New York. Guthenberg, C., Warholm, M., Rane, A,, and Mannervik, B. (1986) Two distinct forms of glutathione transferase from human fetal liver. Biochem. J . , 235:741-745. Hatzfeld, M., and Franke, W.W. (1985) Pair formation and promiscuity of cytokeratins: Formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J. Cell Biol., 101r1826-1842. Hayner, N.T., Braun, L.? Yaswen, P., Brooks, M., and Fausto, N. (1984) Isoenzymes of oval cells, parenchymal cells and biliary cells isolated by centrifugal elution from normal and preneoplastic livers. Cancer Res., 44:332-338. Hochstrasser, D.F., Harrington, M.G., Hochstrasser, A.C., Miller, M.J., and Merril, C.R. (1988) Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal. Biochem., 173:424-135. Inaoka, Y. (1967) Significance of the so-called oval cell proliferation during azo-dye hepatocarcinogenesis. GANN, 68:355-366. Kimbrough, R.D., Linder, R.E., and Gaines, T.B. (1972)Morphological
EPITHELIAL CELL LINES FROM LIVER AND PANCREAS chances in liver of rats fed polychlorinated biphenyls. Arch. Environ. Health, 253354. Kraemer, B.L., Staecker, J.L., Sawada, N., Sattler, G.L., Hsia, M.T.S., and Pitot, H.C. (1986) Use of low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell. Dev. Biol.. 22:201-211. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 2273680-685. Lee, L.W., Tsao, M.S., Grisham, J.W., and Smith, G.J. 11989) Emergence of neoplastic transformants spontaneously or after exposure to N-methyl-N’-nitro-N-nitrosoguanidine in populations of rat liver epithelial cells cultured under selective or non-selective conditions. Am. J. Pathol., 135:63-71. Lobach, D.F., Itoh, T., Singer, K.H., and Haynes, B.F. (1987) The thymic microenvironment. Characterization of in vitro differentiation of the IT26R21 rat thymic epithelial cell line. Differentiation, 32~50-59. Marceau, N. (19901Biology of diseases: cell lineages and differentiation urotrrams in euidermal. urothelial and heuatic tissues and their neo&asms. Lab. &vest., 63.4-20. Marceau. N., Germain, L., Goyette, R., Noel. M., and Gourdeau, H. 11986) Cell of orinin of distinct cultured rat liver euithelial cells. as typed by cytokeitin and surface component selective expression. Biochem. Cell Biol., 64:788-802. McMahon. J.B.. Richards. D.W.. del Camuo, A.A.. Song. M.-K.H.. and Thorgeirsson; S.S.(1986) Differential effects of transforming growth factor$ on proliferation of normal and malignant rat liver epithelial cells in culture. Cancer Res., 46346674671. Moll, R., Pranke, W.W., Schiller, D.C., Geiger, B., and Krepler, R. (1982) The catalog of human cytokeratin: Patterns of expression in normal epithelia, tumors and cultured cell. Cell, 31:ll-24. N a g , P., Evarts, R.P., McMahon, J.B., and Thorgeirsson, S.S. (1989) Role of TGF-PI in normal differentiation and oncogenesis in rat liver. Mol. Carcinog., 2 3 4 5 3 5 4 . Osborn, M., and Weber, K. 11982) Intermediate filaments: celltype-specific markers in differentiation and pathology. Cell, 31:303-306. Penhoet. E.. Raikumar. T.. and Rutter. W.J. (19661 Multiple forms of fructose diphisphate aldolase in mammalian tissues. Proc. Natl. Acad. Sci. U S A . , 56:1275-1282. Rao, M.S., Bendayan, R.D., Kimbrough, R.D., and Reddy, J.K. (1986) Characterization of uancreatic-tvue tissue in the liver of rat induced by polychlorinated *biphenyls. j: Histochem. Cytochem., 34~197on1
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Rao; M.S.: Dwiwedi, R.S., Yeldandi, A.V., Subbarao, V., Tan, X., Usman, M.I., Thangada, S., Nemali, M.R., Kumar, S., Scarpelli, D.G., and Reddy, J.K. (1989) Role of periductal and ductular epithelial cells of the adult rat pancreas in pancreatic hepatocyte lineage: A change in the differentiation commitment. Am. J. Pathol., 134:1069-1086. Reddy, J.K., Rao, M.S., Qureshai, S.A., Reddy, M.K., Scarpelli, D.G., and Lalwani, N.D. (1984) Induction and origin of hepatocytes in pancreas. J . Cell Biol., 982082-2090. Richards, W.L., Song, M.K., Krutzsch, H., Evarts, R.P., Marsden, E., and Thorgeirsson, S.S. (1985) Measurement of cell proliferation in microculture using Hoechst 33342 for rapid semiautomated microfluorimetric determination of chromatin. Exp. Cell Res., 159235246. Rubin, E. 11964)The origin and fate of proliferated bile ductular cells. Exp. Mol. Pathol., 3279-286. Seglen, P.O. (1979) Separation approaches for liver and other cell sources. Cell Popul. Methodological Surv., 8326-46. Sell, S. !19901 Is there a liver stem cell? Cancer Res.?50:38113815. Storey, A.?Pim, D., Murray, A.; Osborn, K., Banks, L., and Crawford, L. (1988) Comparison of the in vitro transforming activities of human papillomavirus types. EMBO J.: 7~1815-1820. Tatematsu, M., Thohru, K.. Medline, A., and Farber, E. (1985) Intestinal metaplasia as a common option of oval cells in relation to cholangiofibrosis in the livers of rats exposed to Z-acetylaminofluorene. Lab. Invest., 52:354. Tsao, M.A., and Duguid, W.P. (1987) Establishment of propagable epithelial cell lines from normal adult rat pancreas. Exp. Cell Res., 168:365-375. Tsao, M.-S., and Grisham, J.W. 11987) Hepatocarcinomas, cholangiocarcinomas and hepatoblastomas produced by chemically transformed cultured rat liver epithelial cells: A light and electron microscopic analysis. Am. J. Pathol., 127:16%181. Tsao, M.S., Smith, J.D., Nelson, K.G., and Grisham, J.W. (1984) A diploid epithelial cell line from normal adult liver with phenotypic properties of “oval” cells. Exp. Cell Res., 154:38-52. Wolf, H.K., Burchette, J.L., Garcia, J.A., and Michalopoulos,G. 11990) Exocrine pancreatic tissue in human liver: a metaplastic process? Am. J. Surg. Pathol., 14:590-595. Woodcock-Mitchell, J.?Rannels, S.R., Mitchell, J., Rannels, D.E., and Low, R.B. (1989) Modulation of keratin expression in type 11 pneumocytes by the extracellular matrix. Am. J . Respir. Dis., 1393343-351.
Evidence for a Common Cell of Origin for Primitive Epithelial Cells Isolated From Rat Liver and Pancreas HANNE CATHRINE BISCAARD AND SNORRI S. THORCEIRSSON* laboratory of Experimental Carcinogcnecis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 The appearance of differentiated hepatocytes in the adult rat pancreas as well as pancreatic-type tissue in the adult rat liver can be experimentally induced (Reddy et al.: 1. Cell Biol., 98:2082-2090, 1984; Rao et al., 1. Histochem. Cytochem., 34:197-201, 1986). These observations suggest a lineage relationship between cell compartments present in rat liver and pancreas. The present data demonstrate that epithelial cell lines with almost identical phenotypes can be established from adult rat liver and pancreas. The established cell lines showed similar morphologies as established by light- and electron-microscopic studies. The cell lines showed a unique expression pattern of intermediate filament proteins. Vimentin, actin, and P-tubulin were present in all cell lines. In addition, simple epithelial type I 1 cytokeratins 7 and 8 were found to be coexpressed with the type I cytokeratin 14 in several of the cell lines. Neither the type I cytokeratins 18 and 19, which are the normal partners for cytokeratins 8 and 7 in filament formation, nor the type I I cytokeratin 5 could be detected despite the fact that filaments were formed by both cytokeratins 8 and 14. This suggests that cytokeratin 14 acts as an indiscriminate type I cytokeratin in filament formation in the established cell lines. The cell lines expressed the same sets of LDH and aldolase isoenzymes and identical sets of glutathione transferase subunits. In addition, the epithelial cell lines from liver and pancreas were equally sensitive to the growth-inhibitory effects of TGF-Pl. No expression of tissue- or cell-specific proteins such as wfetoprotein, albumin, amylase, elastase, or y-glutamyl transpeptidase were detected. The almost identical phenotypes of the hepatic and pancreatic cell lines suggest that they may be derived from a common primitive epithelial cell type present in both rat liver and pancreas. In contrast to parenchymal cells, these cells have an extended capacity for proliferation in vitro and may represent a progeny from a “precursor“ or “stem” cell compartment in vivo.
The rat liver and pancreas have a common embryonic origin, both being derived from almost identical regions of the primitive endoderm. These organs are comprised of predominantly epithelial cells, which, based on their expression of cytokeratins, have been characterized as simple epithelia (Moll et al., 1982). In the early fetal period hepatic epithelial cells express predominantly cytokeratins 8, 14, and 18 (Germain et al., 1988a,b; Marceau, 1990). However, as cell differentiation in the developing liver proceeds along the hepatocytic or biliary epithelial lineages a more complex pattern of cytokeratin expression arises. While hepatocytes express primarily cytokeratins 8 and 18, biliary ductular cells express primarily cytokeratins 7, 8, 18, and 19 (Marceau, 1990). Interestingly, the cytokeratin expression in the adult liver parallels that of the adult pancreas. Pancreatic acini cells express cytokeratins 8 and 18, while pancreatic ductal cells express cytokeratins 7, 8, 18, and 19 (Marceau, 1990). Extensive research in he atocarcinogenesis has revealed that one of the earyiest cellular responses to chemical hepatocarcinogens in the rat involves the 0 1991 WILEY-LISS, INC.
proliferation of cells located around the terminal bile ductules followed by the appearance of a rapidly growing population of small, distinct epithelial cells characterized by oval nuclei and dense cytoplasm commonly referred to as “oval cells” (Farber, 1984; Sell, 1990). During the early stages of oval cell proliferation, a majority of the small epithelial cells seems to express cytokeratins similar to that of biliary ductular cells (Germain et al., 1985; Dunsford et al., 1989; Dunsford and Sell, 1989; Evarts et al., 1990). Once induced, oval cells may undergo necrosis or differentiate into hepatocytes (Farber, 1956; Rubin, 1964; Inaoka, 1967; Grisham and Porta, 1964; Evarts et al., 1987, 1989, 1990). Additionally, oval cells can undergo intestinal Received November 20, 1990; accepted February 8, 1991. *To whom reprint requestsicorrespondence should be addressed at the National Cancer Institute, Building 37, Room 3C28, Bethesda, MD 20892. H.C. Bisgaard is on leave from the Institute of Toxicology, National Food Agency, Ministry of Health, Copenhagen, Denmark.
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metaplasia (Tatematsu et al., 1985) and may also differentiate into pancreatic tissue in rat liver (Kimbrough et al., 1972; Rao et al., 1986). Presence of both “oval” cells and exocrine pancreatic tissue similar to that seen in rat liver has recently been observed in human livers (Wolf et al., 1990; Hsia, C.-H., and Thorgeirsson, S.S., unpublished data). Hepatocytes have been shown to appear in the pancreas of rats durin aging and after feeding a diet deficient in copper ( hiu, 1987; Reddy et al., 1984; Rao et al., 1989). The pancreatic hepatocytes in Cu’+deficient animals seem to arise from periductular or ductular cells in a manner very similar, if not identical, to the oval-cell-derived hepatocytes in livers of animals exposed to chemical hepatocarcinogens (Farber, 1984; Evarts et al., 1987; Rao et al., 1989). Thus, it has been hypothesized that a “stem” cell compartment of common embryonic origin is present in both the liver and pancreas (Sell, 1990). Liver-derived diploid or near-diploid epithelial cell lines from normal as well as hepatocarcinogen-treated rats have been established by several investigators (reviewed by Fausto et al., 1987). Studies of the biochemical and ultrastructural characteristics of newly isolated oval cells, cultured oval cells, bile duct cells, and long-term cultures of rat-liver-derived epithelial cells have shown a common pattern of expression of many cellular markers including cytokeratins (Germain et al., 1988a,b; Hayner et al., 1984; Marceau et al., 1986; Tsao et al., 1984). The suggestion that oval cells are descendants from a putative liver “stem” cell (Sell, 1990) raises the possibility that rat liver epithelial cells because of their phenotypic similarities to oval cells are also progeny from such a “stem” cell compartment. Epithelial cell lines from adult pancreas have previously been established by Tsao and Duguid (1987).The almost identical ultrastructural characteristics of pancreatic cells, liver epithelial cells, and oval cells in primary culture as revealed by light- and electronmicroscopic studies (Hayner et al., 1984; Tsao and Duguid, 1987) led us t o hypothesize that epithelial cell lines established from liver and pancreas are derived from equivalent compartments of cells with an extended capacity for proliferation in vitro. Using a series of cell lineage markers, structural as well as functional, we have demonstrated a close lineage relationship between apparently normal epithelial cell lines established from adult rat liver and pancreas.
Rockville, MD) supplemented with 20 mM L-glutamine (Whittaker M.A. Bioproducts, Walkersville, MD), 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), and 50 mgiliter (1) gentamicin sulfate (50 mgie) (GIBCO, Grand Island, NY). The primary cell cultures were incubated at 37°C in a humidified atmosphere containing 5% C 0 2 for a period of 15 days during which the medium was changed every second day. At the end of the incubation period, cell clones with a typical a pearance of epithelial cells were isolated from the f?asks by use of filter paper pieces (3 MM, Whatman Int. Ltd., UK) dipped in a solution containing 0.1% trypsin. The filter paper to which detached cells adhered was transferred to a culture dish of appropriate size and the cells were propagated in Ham’s F12 complete medium. At a cell number of approximately 1 x lo5 cells the cultures were single-cell cloned by the limiting dilution method. Clonal lines were used in the characterization studies between passages 9 and 19 in vitro. RPEAM3 and RPEAM4. The clonal lines RPEAM3 and RPEAM4 (Rat Pancreas Epithelial Adult Male clones #3 and #4)were established from pancreas of a 9 week-old male Fischer rat. The gastric and splenic lobes of the pancreas were isolated and placed in ice-cold Ca’+-Mg”-free Hank‘s Balanced Salt Solution (HBSS, GIBCO Lab.). The peripancreatic tissue was dissected away and the pancreas was minced. The tissue fragments were washed twice with ice-cold HBSS and transferred to Ham’s F12 supplemented with 20 mM L-glutamine, gentamicin sulfate (50 mgil), and collagenase (1 gil) (Sigma Chemical Co., St. Louis, MO). The tissue fragment suspension was incubated with intermittent agitation at 37°C for 30 min. Then trypsin at a final concentration of 0.25%was added and the suspension incubated for an additional 10 min. The suspension was filtered (80 pm mesh nylon filter), thc cells were sedimented at 150g for 5 min, and the cell pellet was washed twice in Ham’s F12 supplemented with glutamine, gentamicin sulfate, and 10% fetal bovine serum. The cell pellet was resuspended in Ham’s F12 medium supplemented as above and cells were plated at a final density of 1 x lo3 cellsicm’ (AccelP’, Costar, Cambridge, MA). The primary cell cultures were incubated at 37°C in a humidified atmosphere containing 5% COzfor a period of 3 weeks during which the medium was changed every third day. At the end of the incubation period, cells with a typical appearance of epithelial cells were isolated and the cells were propagated and single-cell cloned. In the present study, the MATERIALS AND METHODS clonal lines were generally used between the ninth and Isolation of epithelial cell lines 18th passage in vitro. RLEAM7 and RLEAM12. The clonal lines RLESF13. The clonal line RLESFl3 (Rat Liver RLEAM7 and RLEAMl2 (Rat Liver Epithelial Adult Epithelial Suckling Female clone #13) was established Male clones #7 and #12) were established from a 9 from a 10 day-old female Fischer rat as reviously week-old male Fischer rat by a novel method involving described (McMahon et al., 1986). The cel line was perfusion of the liver in situ. Liver cells were isolated routinely maintained as monolayer cultures in Ham’s by the two-step collagenase perfusion technique as F-12 medium supplemented with 20 mM L-glutamine, described by Seglen (1979).The initial cell preparation 10%fetal bovine serum and 50 mgil gentamicin sulfate. was enriched for epithelial cells by sedimentation The RLESF13 cell line was generally used after 18 through a Percoll gradient (Kraemer et al., 1986). The passages in vitro. purified liver cell suspension was plated at a density of In some experiments the epithelial cell lines were 5 x lo4 cells/cm2(AccelPtissue culture flasks, Costar, utilized between passages 35 and 39 in vitro. Cambridge, MA) in Ham’s F-12 medium (Biofluids Inc., FR. The cell line FR (Epidermis, Germ-free fetal rat)
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EPITHELIAL CELL LINES FROM LIVER AND PANCREAS
was obtained from the American Type Tissue Culture Collection (Rockville, MD). The cell line was propagated in Ham's F12 supplemented with 20 mM Lglutamine, gentamicin sulfate (50 mg/l), and 10%fetal bovine serum. The cells were used between passages 12 and 16 in vitro. Electron microscopy Ultrastructural studies were performed on cultured cells attached as monolayers in tissue culture flasks. The cell monolayers were grown to approximately 70% confluence, washed twice with Dulbecco's phosphatebuffered saline, and fixed in a phosphate-buffered solution (100 mM, pH 7.2) containing 1%glutaraldehyde. The cell monolayers were postfixed in osmium tetroxide and the samples routinely processed for transmission electron microscopy. Cell proliferation analysis Doubling times. Cells were plated at a density of 5 x l o 4 cellsiwell in six-well culture dishes (Costar, Cambridge, MA). Cells were detached by trypsination at different time points (typically every 24 hr) and counted in a Coulter counter (Coulter Electronics Inc., Hialeah, FL). Doubling times were calculated from growth curves generated from several experiments. TGF-P1 inhibition. The inhibition of cell proliferation by TGF-P1 was assessed by examining the rate of DNA synthesis normalized against cell number by using the procedure of Richards et al. (1985) and modified as described by Chapekar et al. (1989).
Cytoskeletal extraction Cellular cytoskeletons were metabolically labeled by using [35S]methionine (Amersham Corp., Arlington Heights, IL). Cellular monolayers were incubated with complete medium containing 100 pCi/ml ["'Slmethionine for 4 hr. Cytoskeletal proteins were prepared according to Bowden et al. (1984). Briefly, cell monolayers, prewashed in ice-cold phosphate-buffered saline (PBS), were extracted with buffered 1%Triton X-100 (10 mM TrisiHCl pH 7.4, 2 mM DTE, 140 mM NaC1). The insoluble residue was extracted with buffered 0.5% Triton X-100 (10 mMTridHC1 pH 7.4,0.5 mM DTE, 1.5 M KC1) and then washed with buffered 0.1%)Triton X-100 (5mM TrisiHCl pH 7.4, 0.5 mM DTE, 70 mM NaC1). All buffers were supplemented with the following components to prevent protein breakdown by proteases: 5 mM EDTA, 5 mM EGTA, 200 pM PMSF, 1pM leupeptin, and 1 pM pepstatin (all Boehringer-Mannheim, IN). Cytoskeletal pellets were then lysed in SDS sample preparation buffer (50 mM TrisiHCl, pH 6.8; 1 mM MgC1,; 1%SDS; 2% 2-mercaptoethanol), and the samples were sedimented at 100,OOOg for 10 min at room temperature. Cytosolic fractionation Cell monolayers were washed twice with PBS and harvested by scraping, and the cell pellets were homogenized by sonication for 15 s in ice-cold 10 mM Tris/ HC1, pH 7.2, containing the different protease inhibitors. For fractionation of tissues, the excised tissue (approximately 1.5g) from brain, liver, kidney, and pancreas was minced and homogenized in 10 mM
335
TrisiHCl, pH 7.2, containing the different protease inhibitors. Homogenates were centrifuged at 100,OOOg for 30 rnin at 4°C to obtain clear supernatants. Analysis of cytoskeletal proteins Analysis of [35S]methionine-labelled cytoskeletal proteins by two-dimensional gel electrophoresis was performed as described by Hochstrasser et al. (1988); 500,000 dpm of extracted material was loaded on the first-dimension isoelectric focusing gel. Ampholytes (LKB Instruments, Rockville, MD) were used at concentrations of 1.6%, pH range 5 to 8, and 0.4%, pH range 3.5-10. SDS-PAGE in the second dimension was performed by using 1mm-thick 7.5%to 17.5%gradient gels. Polypeptides were detected by direct autoradiography following an exposure of 3 days.
Analysis of glutathione transferase subunits Analysis of glutathione transferase subunits by electrophoresis in one dimension was performed by SDSPAGE according to Laemmli (1970). Samples (10 pg of protein) were loaded onto 10%polyacrylamide mini els (10 x 10 cm, 0.75 mm thick) and electrophoresef at constant voltage (200 V). The relative amounts of the polypeptides were visualized by transferring the polypeptides from the gels to nitrocellulose membranes (Schleicher and Schuell, Keene, NHj in a transblot apparatus (Hoefer Scientific Instruments, San Francisco, CA) at 100 V, 0.2 amp at 4°C for 90 min by using a transfer buffer containing 20 mM TrisiHCl, 150 mM glycine, and 20% (viv) methanol. The nitrocellulose blot was then washed for 5 min in 50 mM Tris/HCl and 100 mM NaC1, pH 7.2 (Tris-buffered saline (TBS)),and blocked overnight in 0.5%nonfat dry milk (Carnation, USA) in TBS at 4°C. The blots were washed with TBS for 5 min and incubated for 2 hr with the rimary antibody diluted in TBS containing 0.5% non at dry milk. After two washes, each for a period of 10 min in TBS supplemented with 0.05% Tween 20, the blots were incubated for 1hr in TBS supplemented with 0.5% nonfat dry milk and the secondary antibody conjugated to alkaline phosphatase as the detection enzyme (Goat antirabbit IgG (H + L) (Bio-Rad Lab., Richmond, CAj). The blots were washed twice in TBS supplemented with 0.05% Tween 20 (10 rnin for each wash) and once in 0.1 M Tris; pH 9.5 ( 5 rnin). The blots were developed by using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitro blue tetrazolium salt (NBT) (Bio-Rad Lab.) as the substrate for alkaline phosphatase. Blots were developed until clear purple bands appeared. For immunoblot analysis, the polyclonal antibody against the glutathione transferase subunit Y (kindly provided by Dr. K. Satoh) was used at a difution of 1:1,000. The polyclonal antibodies against the glutathione transferase subunits Y,, Yb2,and Y, (Medlabs, Dublin, Ireland) were used at dilutions of 1:250.
P
Lactate dehydrogenase LDH isozymes were separated electrophoretically on cellulose acetate membranes by using a standard isoenzyme kit (Sigma Chemical Co., St. Louis, MO); 10 pg of cytosolic protein was loaded on a cellulose acetate membrane (Super Sepraphore cellulose acetate mem-
336
BISGAARD AND THORGEIRSSON
brane (Gelman Sciences, MI). Electrophoresis was carried out at constant voltage (250 V) for 25 min in a resolution buffer containing 50 mM Tris, 12 mM citrate, 25 mM barbital. The isoenzyme pattern was visualized by staining with LDH Agaro-Stain@Reagent (Sigma).
Aldolase Cytosolic protein (25 pg) was subjected to electrophoresis on cellulose acetate membranes (Gelman Super Sepraphore) for 60 min at 250 V, in 60 mM high-resolution buffer (Gelman Sciences) containing 10 mM P-mercapto-ethanol. Isozymes were visualized by reactions coupled to the reduction of p-nitrotetrazolium blue as described by Penhoet et al. (1966). Immunofluorescence Indirect immunofluorescence was performed on ethanol-fixed monolayer cultures grown on glass slides (Miles Lab., Naperville, IL). Following rehydration in PBS for 30 min, the slides were incubated with the primary antibody diluted in PBS supplemented with 12% bovine serum albumin for 12 hr at 4°C. The monoclonal antibody against cytokeratin 8 (Amersham) was used at a dilution of 1:lOO. The monoclonal antibody against cytokeratin 14 (Sigma) was used at a dilution of 1:250. The slides were washed twice for 10 min in PBS followed by incubation with a 1:lOO dilution of biotinylated horse anti-mouse IgG (Vector Lab,, Burlingham, CA) in PBS supplemented with 12% albumin for 30 min at room temperature. The slides were washed as before and incubated with streptavidinconjugated Texas Red (Life Technologies Inc., Gaithersburg, MD) diluted 1:400 in PBS supplemented with 12%albumin for 30 min. After washing, the slides were mounted and examined on a Nikon fluorescent microscope. RESULTS Cell morphology Phase-contrast light microscopy of the newly established clonal lines RLEAM7, RLEAM12, RPEAM3, and RPEAM4 showed small cuboidal-shaped cells of relative uniform size (Fig. la,b,d,e). Typical electron micrographs representing the clonal lines RLEAMl2 and RPEAM4 are shown in Figure l c and If. The micrographs revealed cells with a large, rounded nucleus, a small amount of cytoplasm, little rough endoplasmic reticulum, and relatively few mitochondria, small in size. It has previously been reported that a typical feature of small epithelial cells isolated from liver and pancreas is their irregular, indented nucleus (Hayner et al., 1984; Tsao and Duguid, 1987). However, electron micrographs of small epithelial cells as bile duct cells, hepatic oval cells, and pancreatic duct cells in vivo show large, rounded nuclei (Hayner et al., 1984; Tsao and Duguid, 1987). The previous studies of isolated epithelial cells were done on cells fixed in suspension. In contrast, our studies were done on fixed monolayer cultures. Therefore, the irregular, indented nucleus can be attributed to an artifact of the fixation method used. The electron micrographs of the established hepatic and pancreatic cell lines all showed the presence of
tight junctions and desmosomes, supporting the epithelial origin of the cell lines.
Karyotyping The karyotypes of the clonal epithelial cell lines showed that all cell lines were rat-derived diploids with 42 chromosomes. The lines RLEAM7, RLEAM12, RPEAM3, and RPEAM4 were characterized as diploid male (XY) rat while RLESFl3 was characterized as a diploid female (XX) rat. No marker or unassignable chromosomes were present in the lines RLEAM7, RLEAM12, RPEAM3, and RPEAM4, typing these cell lines as apparent normal. A single marker chromosome (chromosome 1)was found in RLESF13. Growth characteristics The doubling times at early passage numbers varied among the epithelial cell lines. While RLESF13 had a doubling time of 18.3 +- 1.3 hr (n = 31, the two lines isolated from adult liver, RLEAM7 and RLEAM12, had doubling times of 31.2 2 2.9 hr (n = 3) and 23.5 2 1.0 hr (n = 3 ) . respectively. The cell lines isolated from adult pancreas, RPEAM3 and RPEAM4, proliferated with doubling times of 30 k 2.7 hr (n = 3) and 34.2 t 1.4 hr (n = 3), respectively. Proliferation of all the epithelial cell lines were inhibited to the same extent by the potent growth inhibitor TGF-P1. The concentration of TGF-P1 causing 50% inhibition of the DNA synthesis (IDs0) was 50-75 pgiml. The proliferation of the FR line established from rat epidermis was not inhibited by TGF-P1 at concentrations up to 1,000 pgiml. Expression of cytoskeletal proteins The two-dimensional " eel natterns of 35S-methioninelabelled intermediate fdaments extracted from RLEAM7, RLEAM12, RPEAM3. RPEAM4. RLESF13. and FR are shown in Figure 2. Relatively few interme: diate filament proteins were present in the cell lines. For identification, the polypeptides were transferred to nitrocellulose and their location was visualized by immunostaining with specific antibodies (data not shown). All the cell lines expressed vimentin, actin, and p-tubulin although the expression varied between the lines. In addition, the cell lines RLEAM12, RPEAM3, and RPEAM4 synthesized several other intermediate filament subunits. Using immunoblotting analysis of cytoskeletal extracts from RPEAM3, three cytokeratins were identified. In addition to the rat equivalents of human cytokeratins 7 and 8 an equivalent of human cytokeratin 14 was expressed. The normal type I partners for cytokeratins 8 and 7, cytokeratins 18 and 19, respectively, as well as the type I1 partner for cytokeratin 14, cytokeratin 5, could not be detected in any of the cell lines by immunoblotting analysis. The two-dimensional gel analysis indicated that cells from the different epithelial cell lines synthesized different quantities of cytokeratins. Using the more sensitive immunofluorescence analysis of cell monolayers with antibodies against cytokeratins 8 and 14, cells showing typical patterns of cytokeratin filaments were found to be present in all the liver and pancreatic cell lines although in varying numbers (Fig. 3). The overall
337
EPITHELIAL CELL LINES FROM LIVER AND PANCREAS
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BISGAARD AND THORGEIRSSON
Fig. 3. Immunofluorescent staining of epithelial cell lines at early passage number by cytokeratins 8 (a, c, e, $, i) and 14 (b, d, f, h, j), RLEAM7 passage 18 (a,b), RLEAMl2 pasage 19 (c,d). RpEAM3 passage 18 (e,D,RPEAM4 passage 18 (g,hi, RLESF13 passage 18 (i,jl.
number of cytokeratin-expressing cells correlated well with the amount of labelled protein detected on the two-dimensional els. Relatively few cells (< 10%) from the liver ce 1 lines RLEAM7, RLEAM12, and RLESF13 were found to express cytokeratins in contrast to a larger number of cells (> 40%) in the pancreatic cell lines RPEAM3 and RPEAM4. However, prolonged passage in vitro (> 35 passages) resulted in a dramatic increase in the number of cells showing filament formation in the liver cell line RLEAMl2 (> 90%) and in the pancreatic line RPEAM4 (> 75%) (Fig. 4c,d,g,h).Cells staining for cytokeratin 5,18, or 19 could not be detected by immunofluorescence in any of the epithelial cell lines at early passage numbers or after at least 35 passages in vitro. No cells stained in monolayers of the epidermal cell line FR.
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Fig. 4. Immunofluorescent staining of epithelial cell lines after prolonged Passage in vitro by cytokeratin 8 (a, c, e, g, i) and 1 4 (b, d, f~ h, j). RLEAM7 Passage 35 (ah), RLEAM12 Passage 39 (cjd)t RPEAM3 passage 33 (e,fl, RPEAM4 passage 37 (g,h), RLESF13 passage 35 (i,j).
Isozyme expression Lactate dehydrogenase. The relative distribution of LDH isoenzymes varies among both different organs and cell types and can therefore be used as a cell lineage marker (Hayner et al., 1984). Five different isoenzymes, LDH,, LDH4, LDH,, LDH2, and LDH1, were detected in the cytosolic fraction of whole homogenates from brain, kidney, and pancreas, while four isozymes-LDH,, LDH4, LDH,, and LDH,-were observed in whole liver homogenates (Fig. 5A). While the different isoenzymes were expressed in almost equal amounts in brain and kidney, the liver and pancreas expressed predominantly LDH,. All of the epithelial cell lines showed a similar expression pattern of LDH
339
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Fig. 6. Cellulose acetate electrophoresis of aldolase isozymes in supernatants from whole organ homogenates (A) and homogenates from epithelial cell lines isolated from liver and pancreas (B).
isoenzymes (Fig, 5B). The predominant isozymes expressed were LDH5, LDH,, and LDH,, with a lesser expression of LDH2. Only two isozymes, LDH5 and LDH,, were expressed in the cell line FR established from rat epidermis. Aldolase. Three types of aldolase subunits representing three different gene products designated A, B, and C are known to exist in mammals (Penhoet et al., 1966).Aldolase is a tetrameric enzyme and hybrids are readily formed in cells expressing more than one subunit gene (Penhoet et al., 1966; Hayner et al., 1984).In liver homogenates B, was the predominant isozyme, but A4 and AB, isozymes were also present (Fig. 6A). In homogenates from rat pancreas the hybrids AB,, A,B2, A,B, and A, were found. A and C subunits in various hybrid combinations were expressed in homogenates from whole rat brain. The only hybrid expressed in homogenates from all the cell lines including the FR line was A4 (Fig. 6B).
Glutathione S-transferase. Similar to LDH and aldolase, a complex mixture of different GST isoenzymes composed of different subunits is present in different organs and cell types (Abramovitz et al., 1989; Evarts et al., 1990).As shown in Figure 7, the subunits Y, and Y, were expressed in the adult rat liver and kidney but not in pancreas. The subunit Y,, was expressed in liver and pancreas but not in the kidney. The placental form Y, could not be detected in either organ by the method used in the present study. Newly isolated adult rat hepatocytes expressed Y,, Y,, and Ybz while Y, was absent. The cell line isolated from rat epidermis with a morphological appearance of fibroblasts showed a very low expression of the Y b 2 subunit and no expression of the other three subunits. All the epithelial cell lines showed an almost identical expression pattern of GST subunits with a strong expression of Y, and Y,, a lower expression of Yb2, and a very low expression of Y,.
340
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Expression of proteins related to cell-specific functions In the adult rat liver specific functions such as albumin production and secretion are features of the differentiated hepatocyte while differentiated bile duct cells express y-glutamyl transpeptidase. In the adult pancreas, elastase and amylase are produced specifically in the exocrine acinar cells while epithelial cells of ductal structures express y-glutamyl transpeptidase. None of the e ithelial cell lines showed expression of albumin, amy ase, elastase, or y-glutamyl transpeptidase, indicating that none of the cell lines expressed tissue- or cell-specific functions. The fetal liver protein a-fetoprotein was not detected in any of the cell lines.
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DISCUSSION In the present study, epithelial cell lines having extended capacity to proliferate in vitro were established from adult rat liver and pancreas. The cell lines exhibited almost identical phenotypes as judged by both morphological appearance and biochemical characterization, suggesting a common cell or origin for primitive epithelial cells isolated from rat liver and pancreas. Studies of the cytokeratin expression in the hepatic and pancreatic epithelial cell lines revealed a unique coexpression of the “simple” epithelial type I1
cytokeratins 7 and 8 with type I cytokeratin 14. The data suggest that cytokeratin 14 can act as an indiscriminate cytokeratin partner in filament formation with cytokeratin 8. In an attempt to investigate the possibility of a common cell of origin for the established hepatic and pancreatic epithelial cell lines a series of morphologic as well as biochemical parameters were used as cell lineage markers. The newly established clonal hepatic and pancreatic cell lines appeared similar with respect to both morphology and growth characteristics. All the cell lines had apparent normal diploid karyotypes and displayed a morphology similar t o what has been reported for other hepatic and pancreatic derived epithelial cells in culture (Hayner et al., 1984; Tsao et al., 1984; Tsao and Duguid, 1987). The epithelial nature of the cell lines was indicated by their cuboidal appearance and the presence of desmosomes and gap junctions. The cell lines showed similar proliferation rates and all were equally sensitive to the growth-inhibitory effects of TGF-P1. TGF-P1 has previously been shown to be a potent inhibitor of normal epithelial liver cell proliferation (McMahon et al., 1986; Nagy et al., 1989). The present data suggest that TGF-P1 is an equally potent inhibitor of normal epithelial pancreatic cell proliferation. The use of intermediate filament proteins as epithe-
EPITHELIAL CELL LINES FROM LIVER AND PANCREAS
lial cell lineage markers is well established (Moll et al., 1982; Osborn and Weber, 1982). Among this family of proteins, cytokeratins are of special interest since they are specifically and differentially expressed in different epithelial cell types (Moll et al., 1982).The cytokeratins are frequently expressed in distinct pairs consisting of one acidic (type I) and one basic (type III cytokeratin. In the rat liver two cytokeratins equivalent to the human cytokeratins 8 (type 11) and 18 (type I) are present in both mature hepatocytes and bile duct cells, while two cytokeratins equivalent to human cytokeratins 7 (type 11)and 19 (type I) are expressed specifically in bile duct and oval cells (Marceau, 1990).A similar distribution of simple cytokeratins seems to exist within pancreas where cytokeratins 8 and 18 are expressed in exocrine cells and cytokeratins 7 and 19 in addition to 8 and 18 in epithelial cells of the pancreatic ducts (Marceau, 1990). The two-dimensional gel analysis performed on our established cell lines showed a unique expression of cytokeratins 7, 8, and 14 in several of the cell lines. Immunofluorescent localization of cytokeratins 8 and 14 revealed formation of filament bundles expanding from the nucleus to the membrane. Filament formation of cytokeratins normally requires expression of distinct pairs of type I and type I1 cytokeratins, the normal counterpart for cytokeratin 8 being cytokeratin 18 (Hatzfeld and Franke, 1985). However, we were unable to detect an equivalent of cytokeratin 18 as well as 19 in the cell lines at early as well as late passage number in vitro. Cytokeratin 8 has been shown to be indiscriminate when forming filaments in reconstituted systems in vitro (Hatzfeld and Franke, 1985). In addition, it has been suggested to act as an indiscriminate type I1 cytokeratin in filament formation with other type I cytokeratins such as 19 in intestinal epithelia and MCF-7 cells (Moll et al., 1982). In view of the coexpression of cytokeratins 8 and 14 observed in the RLEAMl2 after prolonged passage in vitro and our inability t o detect cytokeratins 18,19, and 5, it seems reasonable to suggest that cytokeratin 8 acts as the type I1 partner in filament formation with cytokeratin 14 in all our epithelial cell lines. Whether cytokeratin 7 can act as an indiscriminate cytokeratin in filament formation in the epithelial cell lines is presently under investigation. All the established cell lines expressed a similar set of epithelia-specific intermediate filaments although to varying degrees (Fig. 2). In two of the cell lines, RLEAMl2 and RPEAM4, the extent of the cytokeratin expression seems to be related to the passage number of the cells (Fig. 3, 4). In both these cell lines prolonged passage in vitro resulted in an increased expression of cytokeratins 8 and 14. The unique phenotype of the established hepatic and pancreatic cell lines regarding the cytokeratin expression is supported by data provided on rat epithelial cell lines established from other tissues. Lobach and co-workers (1987) found that a newly established thymic epithelial cell line (IT26R21) expressed cytokeratins 5, 7, 8, and 14 and upon prolonged passage also 6 and 16. A rat kidney epithelial cell line was shown to express exclusively cytokeratins 8 and 18 (Storey et al., 1988). Moreover, in vitro transition of pulmonary type I1 epithelial cells to type I cells is accompanied by an induction of cytokeratins 8
341
and 18, and cytokeratins 5 , 7, 14, and 19 were not detected during this transition (Woodcock-Mitchell et al., 1989). In the present study the FR cell line was chosen as a control cell line because of its derivation from rat epidermis and possible relationship to keratinocytes. However, the morphological appearance and the expression of cytoskeletal proteins similar to a rat fibroblast cell line characterized by Garrels and Franza (1989)indicate that this cell line is of mesenchymal and not epithelial origin. Although a cytokeratin with a molecular weight identical to the rat cytokeratin 14 identified in the present study has been found in cells of early embryonic rat liver a definitive identification still is not complete (Germain et al., 1988a,b). To date the expression of cytokeratin 14 has not been reported in adult rat liver or pancreas. However, preliminary observations from our laboratory indicate that cytokeratin 14 is expressed during early stages of oval cell proliferation (Bisgaard, H.C., and Evarts, R.P., unpublished data). Whether expression of this unique set of keratins in the cell lines is merely related to the in vitro culture conditions or reflects origin from as-yet-undefined “stem” cell populations in vivo is presently under investigation. Similar to that observed with the cytoskeletal proteins, a complex mixture of different GST isoenzymes is also present in mammalian organs due to variable expression of some forms and the complete absence of other forms (for review see Boyer, 1989).Recent data on the expression of mRNA coding for the placental subunit of glutathione transferase (Y,) during early stages of hepatocarcinogenesis (Evarts et al., 1990) and data on the differential expression of mRNA coding for other subunits in primary hepatocytes (Abramovitz et al., 1989) indicate that GST can be used as a cell lineage marker in rat liver. Primary adult rat hepatocytes show transcripts for the Y,, Y,, Y b l , Yb2, and Yb3 subunits but not for Y,. However, during early stages of hepatocarcinogenesis Y, transcripts are found in bile ducts, ductal structures, oval cells, and basophilic foci of small hepatocytes but not in normal mature hepatocytes, endothelial cells, or the mesothelial cells of the Glisson capsula (Evarts et al., 1990).In addition, the Y, protein has been detected in human fetal liver (Guthenberg et al., 1986). Our data using immunoblotting analysis with antibodies specific for the Y,, Y,, Yb2, and Y, subunits confirmed that freshly isolated rat hepatocytes express the Y,, Y,, and Yb2subunits while Y, is absent. However, the different pattern of GST subunit expression in the liver-derived epithelial cell lines characterized by high expression of the Y, and Y, subunits and low expression of Y, and Yb2 indicates that these cell lines might be derived from cell populations located in the biliary structures of the normal rat liver. The lack of expression of Y, in endothelial and mesothelial cells of rat liver (Evarts et al., 1990) indicates that the established cell lines are not derived from these cell compartments. Rat liver epithelial cells in long-term culture have phenotypic traits almost identical to those of oval cells (Tsao et al., 1984; Grisham et al., 1974). However, the isoenzyme pattern of both LDH and aldolase present in the liver-derived cell lines differs significantly from the pattern previously found in isolated oval cells and bile
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BISGAARD AND THORGEIRSSON
duct cells (Hayner et al., 1984). While LDH,, LDH,, and LDH, are the main isoforms detected in our established cell lines, LDH4,LDH3, and LDHz were the main isoforms found in both oval and bile duct cells. Our data therefore support the suggestion that the propagable liver epithelial cell lines may originate from a compartment of cells located in the ductular structures (Grisham, 1980) but with phenotypic features that differ somewhat from oval cells and terminal bile duct cells. Our study was designed to investigate a possible relationship between epithelial cell lines isolated from liver and pancreas. During embryogenesis both the liver and pancreas develop from a common origin, the endoderm. Recent findings in rats have shown that a unique transdifferentiation of epithelial cells in pancreas into fully differentiated hepatocytes occurs after treatment with a copper-deficient diet (Reddy et al., 1984; Rao et al., 1989). The fact that populations of small epithelial cells with common morphological phenotypes arise after extensive hepatic and pancreatic injury led us to hypothesize that identical compartments of primitive epithelial cells capable of proliferating in vitro are present in these organs. The present findings that the phenotypic traits characteristic of epithelial cell lines isolated from pancreas closely resemble those observed in epithelial cells isolated from liver suggest that these cell lines may originate from a common rimitive cell population that resides in both organs. It as recently been shown that chemical transformation of rat liver epithelial cell lines results in the generation of a wide range of tumors including hepatocellular carcinomas, cholangiocarcinomas, and hepatoblastomas as well as sarcomas and mixed epithelial-mesenchymal tumors (Tsao and Grisham, 1987; Lee et al., 1989). Furthermore, we have recently shown that similar tumor types are generated when rat-liver-derived epithelial cells are transformed by selective oncogenes (Garfield et al., 1988). These data strongly indicate the blastic multipotent nature of the liver-derived epithelial cell lines. We are currently investigating whether transformation of the epithelial cell lines from pancreas results in formation of tumorigenic cells reflecting bhe same blastic multipotent nature.
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ACKNOWLEDGMENTS P.T. Ton is acknowledged for skillful technical assistance. We wish to thank Dr. P.J. Wirth and Dr. B.S. Warren for many helpful discussions and for critical reading of the manuscript. H.C. Bisgaard was supported by a grant from the Danish Research Academy.
LITERATURE CITED Abramovitz, M.M., Ishigaki, S., and Listowsky, I. (1989) Differential expression of glutathione S-transferase in cultured hepatocytes. Hepatology, 92235-239. Bowden, P.E., Quinlan, R., Breitkreutz, D., and Feusenig, N.E. (1984) Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis. Eur. J. Biochem., 142:29-36. Boyer, T.D. (1989) The glutathione S-transferases: An update. Hepatblogy, 9:486-497. Chapekar, M.S., Iluggett, A.C., and Thorgeirsson, S.S. (1989)Growth modulatory effects of a liver-derived growth inhibitor, transforming
growth factor p1, and recombinant tumor necrosis factor a, in normal and neoplastic cells. Exp. Cell Res., 185.247-257. Chiu, T. 11987) Focal eosinophilic hypertrophic cells in the rat pancreas. Toxicol. Pathol., 15:331-333. Dunsford? H.A., Karnasuta, C., Hunt, J.M., and Sell, S. (1989) Different lineages of chemically induced hepatocellular carcinoma in rats defined by monoclonal antibodies. Cancer Res., 49:48944900. Dunsford, H.A., and Sell, S. (19891Production of monoclonal antibodies to preneoplastic liver cell populations induced by chemical carcinogens in rats and t o transplantable Morris hepatomas. Cancer Res.; 49:48874893. Evarts, R.P., Nagy, P., Marsden, E., and Thorgeirsson, S.S. (1987) A precursor-product relationship exists b e h e e n oval cells and hepatocytes in rat liver. Carcinogenesis, 8:1737-1740. Evarts, R.P., Nagy, P., Nakatsukasa. H., Marsden, E., and Thorgeirsson? S.S. (1989) In vivo differentiation of rat liver oval cells into hepatocytes. Cancer Res., 49:1541-1547. Evarts, R.P., Nakatsukasa, H., Marsden, E., Hsia, C.-C., Dunsford, H.A., and Thorgeirsson, S.S. (1990) Cellular and molecular changes in the early stages of chemical hepatocarcinogenesis in the rat. Cancer Res., 50:3439-3444. Farber, E. 11956) Similarities in the sequence of early histological changes induced in the liver by ethionine, 2-acetylaminofluorene and 3'-methyl-4-dimethylaminoazobenzene. Cancer Res., 16:142148. Farber, E.(1984)The multistep nature of cancer development. Cancer Res., 44:42174223. Fausto, N., Thompson, N.L., and Braun, L. (1987) Purification and culture of oval cells from rat liver. In: Cell Separation: Methods and Selected Applications, vol. 4. T.G. Pretlow, and T.P.P. Pretlow, ed. Academic Press Inc., New York, pp. 45-77. Garfield, S., Huber, B.E., Nagy, P., Cordingley, M.G., and Thorgeirsson, S.S. (1988) Neoplastic transformation and lineage switching of rat liver epithelial cells by retrovirus-associated oncogenes. Mol. Carcinog., 1:189-195. Garrels, J.I., and Franza, B.R. (1989) Transformation-sensitive and growth-related changes of protein synthesis in REF52 cells. J. Biol. Chem., 26452994312. Germain, L., Blouin. M.-J.,and Marceau, N. (1988a) Biliary epithelial and hepatocytic cell lineage relationships in embryonic rat liver as determined by the differential expression of cytokeratins, a-fetoprotein, albumin, and cell surface-exposedcomponents. Cancer Res., 48:49094918. Germain, L.,Goyette, R., and Marceau, N. (1985) Differential cytokeratin and a-fetoprotein expression in morphologically distinct cells emerging at early stages of rat hepatocarcinogenesis. Cancer Res., 45:673-681. Germain, L.,Noel, M., Gourdeau, H., and Marceau, N. (198813) Promotion of growth and differentiation of ductular oval cells in primary culture. Cancer Res., 48:368-378. Grisham, J.W. (1980) Cell types in long term propagable cultures of rat liver. Ann. NY Acad. Sci., 349:128-137. Grisham, J.W., and Porta, E.A. (1964) Origin and fate of proliferated ductul cells in the rat: electron microscopic and autoradiographic studies. Exp. Mol. Patbol., 3:242-261. Grisham, J.W., Thal, S.B., and Nagel, A. (1974) Cellular derivation of continuously cultured epithelial cells from normal rat liver. In: Gene Expression and Carcinogenesis in Cultured Liver. L.E. Gerschenson and E. Thompson, eds. Academic Press, New York. Guthenberg, C., Warholm, M., Rane, A,, and Mannervik, B. (1986) Two distinct forms of glutathione transferase from human fetal liver. Biochem. J . , 235:741-745. Hatzfeld, M., and Franke, W.W. (1985) Pair formation and promiscuity of cytokeratins: Formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J. Cell Biol., 101r1826-1842. Hayner, N.T., Braun, L.? Yaswen, P., Brooks, M., and Fausto, N. (1984) Isoenzymes of oval cells, parenchymal cells and biliary cells isolated by centrifugal elution from normal and preneoplastic livers. Cancer Res., 44:332-338. Hochstrasser, D.F., Harrington, M.G., Hochstrasser, A.C., Miller, M.J., and Merril, C.R. (1988) Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal. Biochem., 173:424-135. Inaoka, Y. (1967) Significance of the so-called oval cell proliferation during azo-dye hepatocarcinogenesis. GANN, 68:355-366. Kimbrough, R.D., Linder, R.E., and Gaines, T.B. (1972)Morphological
EPITHELIAL CELL LINES FROM LIVER AND PANCREAS chances in liver of rats fed polychlorinated biphenyls. Arch. Environ. Health, 253354. Kraemer, B.L., Staecker, J.L., Sawada, N., Sattler, G.L., Hsia, M.T.S., and Pitot, H.C. (1986) Use of low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell. Dev. Biol.. 22:201-211. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 2273680-685. Lee, L.W., Tsao, M.S., Grisham, J.W., and Smith, G.J. 11989) Emergence of neoplastic transformants spontaneously or after exposure to N-methyl-N’-nitro-N-nitrosoguanidine in populations of rat liver epithelial cells cultured under selective or non-selective conditions. Am. J. Pathol., 135:63-71. Lobach, D.F., Itoh, T., Singer, K.H., and Haynes, B.F. (1987) The thymic microenvironment. Characterization of in vitro differentiation of the IT26R21 rat thymic epithelial cell line. Differentiation, 32~50-59. Marceau, N. (19901Biology of diseases: cell lineages and differentiation urotrrams in euidermal. urothelial and heuatic tissues and their neo&asms. Lab. &vest., 63.4-20. Marceau. N., Germain, L., Goyette, R., Noel. M., and Gourdeau, H. 11986) Cell of orinin of distinct cultured rat liver euithelial cells. as typed by cytokeitin and surface component selective expression. Biochem. Cell Biol., 64:788-802. McMahon. J.B.. Richards. D.W.. del Camuo, A.A.. Song. M.-K.H.. and Thorgeirsson; S.S.(1986) Differential effects of transforming growth factor$ on proliferation of normal and malignant rat liver epithelial cells in culture. Cancer Res., 46346674671. Moll, R., Pranke, W.W., Schiller, D.C., Geiger, B., and Krepler, R. (1982) The catalog of human cytokeratin: Patterns of expression in normal epithelia, tumors and cultured cell. Cell, 31:ll-24. N a g , P., Evarts, R.P., McMahon, J.B., and Thorgeirsson, S.S. (1989) Role of TGF-PI in normal differentiation and oncogenesis in rat liver. Mol. Carcinog., 2 3 4 5 3 5 4 . Osborn, M., and Weber, K. 11982) Intermediate filaments: celltype-specific markers in differentiation and pathology. Cell, 31:303-306. Penhoet. E.. Raikumar. T.. and Rutter. W.J. (19661 Multiple forms of fructose diphisphate aldolase in mammalian tissues. Proc. Natl. Acad. Sci. U S A . , 56:1275-1282. Rao, M.S., Bendayan, R.D., Kimbrough, R.D., and Reddy, J.K. (1986) Characterization of uancreatic-tvue tissue in the liver of rat induced by polychlorinated *biphenyls. j: Histochem. Cytochem., 34~197on1
343
Rao; M.S.: Dwiwedi, R.S., Yeldandi, A.V., Subbarao, V., Tan, X., Usman, M.I., Thangada, S., Nemali, M.R., Kumar, S., Scarpelli, D.G., and Reddy, J.K. (1989) Role of periductal and ductular epithelial cells of the adult rat pancreas in pancreatic hepatocyte lineage: A change in the differentiation commitment. Am. J. Pathol., 134:1069-1086. Reddy, J.K., Rao, M.S., Qureshai, S.A., Reddy, M.K., Scarpelli, D.G., and Lalwani, N.D. (1984) Induction and origin of hepatocytes in pancreas. J . Cell Biol., 982082-2090. Richards, W.L., Song, M.K., Krutzsch, H., Evarts, R.P., Marsden, E., and Thorgeirsson, S.S. (1985) Measurement of cell proliferation in microculture using Hoechst 33342 for rapid semiautomated microfluorimetric determination of chromatin. Exp. Cell Res., 159235246. Rubin, E. 11964)The origin and fate of proliferated bile ductular cells. Exp. Mol. Pathol., 3279-286. Seglen, P.O. (1979) Separation approaches for liver and other cell sources. Cell Popul. Methodological Surv., 8326-46. Sell, S. !19901 Is there a liver stem cell? Cancer Res.?50:38113815. Storey, A.?Pim, D., Murray, A.; Osborn, K., Banks, L., and Crawford, L. (1988) Comparison of the in vitro transforming activities of human papillomavirus types. EMBO J.: 7~1815-1820. Tatematsu, M., Thohru, K.. Medline, A., and Farber, E. (1985) Intestinal metaplasia as a common option of oval cells in relation to cholangiofibrosis in the livers of rats exposed to Z-acetylaminofluorene. Lab. Invest., 52:354. Tsao, M.A., and Duguid, W.P. (1987) Establishment of propagable epithelial cell lines from normal adult rat pancreas. Exp. Cell Res., 168:365-375. Tsao, M.-S., and Grisham, J.W. 11987) Hepatocarcinomas, cholangiocarcinomas and hepatoblastomas produced by chemically transformed cultured rat liver epithelial cells: A light and electron microscopic analysis. Am. J. Pathol., 127:16%181. Tsao, M.S., Smith, J.D., Nelson, K.G., and Grisham, J.W. (1984) A diploid epithelial cell line from normal adult liver with phenotypic properties of “oval” cells. Exp. Cell Res., 154:38-52. Wolf, H.K., Burchette, J.L., Garcia, J.A., and Michalopoulos,G. 11990) Exocrine pancreatic tissue in human liver: a metaplastic process? Am. J. Surg. Pathol., 14:590-595. Woodcock-Mitchell, J.?Rannels, S.R., Mitchell, J., Rannels, D.E., and Low, R.B. (1989) Modulation of keratin expression in type 11 pneumocytes by the extracellular matrix. Am. J . Respir. Dis., 1393343-351.
