Studies of hepatocyte polarity, an important property of liver epithelial cells, have been hampered by the lack of valid in vitro models. We report here that a new polarized hepatoma-derived hybrid cell line, called WIF-B, has improved characteristics to those of its parent, WIF12-1. This latter line originated from the fusion of non-polarized rat hepatoma Fao cells with human fibroblasts (WI-38) and selection for a polarized phenotype. We generated the WIF-B line by growing WIF12-1 cells as unattached aggregates for three weeks and selecting for survivors. Karyotype analysis showed a broad chromosome pattern in the initial WIF-B population, but this pattern stabilized after a few passages. The growth and phenotypic properties of these cells were quite different from those of their polarized WIF12-1 parent. WIF-B cells attained a 4-fold higher maximal density in monolayer culture, survived at this density for > 5 days rather than 1 day, and exhibited two to three times more apical structures during this period (80 to 95%). We compared several parameters of liver differentiation in the WIF-B cells with those of a related hybrid clone, WIF12-E, which is extinguished for most liver-specific functions, and with the common hepatoma parent, Fao. By immunoblot analysis, the levels of expression of eight plasma membrane proteins were higher in the WIF-B cells than in either of the other two cell lines and ranged from 10 to 200% of those in vivo. Two plasma membrane proteins were not detected in WIF12-E cells. By immunofluorescence, the apical membrane proteins in WIF-B displayed different cellular localizations than in either of the other two cell lines. In WIF-B cells, apical proteins were confined to a plasma membrane region that we have identified as the apical domain by several criteria (Ihrke, G., Neufeld, E.D., Meads, T., Shanks, M.R., Cassio, D., Laurent, M., Schroer, T.A., Pagano, R. E. and Hubbard, A. L. J. Cell Biol., 123, 1761–1765). The same molecules were distributed over the entire plasma membrane of Fao and WIF12-E cells and also (for Fao cells) in intracellular punctate structures that did not colocalize with the majority of structures containing a secretory protein, albumin. Our results indicate that the WIF-B cells are more highly differentiated than any of their ancestors (Fao or WIF12-1 cells) and thus, are promising candidates for in vitro studies of hepatocyte polarity.

Ahnen
D. J.
,
Brown
W. R.
,
Kloppel
T. M.
(
1985
).
Secretory component: the polymeric immunoglobulin receptor. What's in it for the gastroenterologist and hepatologist?.
Gastroenterology
89
,
667
682
Arends
M. J.
,
Wyllie
A. H.
(
1991
).
Apoptosis: mechanisms and roles in pathology.
Int.Rev. Exp. Pathol
32
,
223
254
Ash
J. F.
,
Louvard
D.
,
Singer
S.J.
) (
1977
).
Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts.
Proc. Nat. Acad. Sci. USA
74
,
5584
5588
Bailyes
E. M.
,
Newby
K.
,
Siddle
K.
,
Luzio
J. P.
(
1982
).
Solubilization and purification of rat liver 5-nucleotidase by use of a zwitterionic detergent and a monoclonal antibody.
Biochem. J
203
,
245
251
Barth
C. A.
,
Schwarz
L. R.
(
1982
).
Transcellular transport of fluorescein in hepatocyte monolayers: evidence for functional polarity of cells in culture.
Proc. Nat. Acad. Sci. USA
79
,
4985
4987
Bartles
J. R.
,
Braiterman
L. T.
,
Hubbard
A. L.
(
1985
).
Endogenous and exogenous domain markers of the rat hepatocyte plasma membrane.
J. Cell Biol
100
,
1126
1138
Bartles
J. R.
,
Braiterman
L. T.
,
Hubbard
A. L.
(
1985
).
Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane.
J. Biol. Chem
260
,
12792
12802
Bartles
J. R.
,
Hubbard
A. L.
(
1986
).
Preservation of hepatocyte plasma membrane domains during cell division in situ in regenerating rat liver.
Dev. Biol
118
,
286
295
Bartles
J. R.
,
Feracci
H. M.
,
Stieger
B.
,
Hubbard
A. L.
(
1987
).
Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation.
J. Cell Biol
105
,
1241
1251
Ben-Ze'ev
A.
,
Robinson
G. S.
,
Bucher
N. L. R.
,
Farmer
S. R.
(
1988
).
Cell-cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes.
Proc. Nat. Acad. Sci. USA
85
,
2161
2165
Boyer
J. L.
,
Gautam
A.
,
Graf
J.
(
1988
).
Mechanisms of bile secretion: insights from the isolated rat hepatocyte couplet.
Semin. Liver Disease
8
,
308
316
Brandtzaeg
P.
(
1981
).
Transport models for secretory IgA and secretory IgM.
Clin. Exp. Immunol
44
,
221
232
Brown
W. R.
,
Kloppel
T. M.
(
1989
).
The liver and IgA: immunological, cell biological and clinical implications.
Hepatology
9
,
763
784
Buys
C. H. C. M.
,
Aanstoot
G. H.
,
Nienhaus
A. J.
(
1984
).
The Giemsa-11 technique leading to dependable direct and sequential staining procedures.
Histochemistry
81
,
465
468
Cassio
D.
,
Hassoux
R.
,
Dupiers
M.
,
Uriel
J.
,
Weiss
M. C.
(
1980
).
Coordinate secretion of mouse alphafetoprotein, mouse albumin and rat albumin by mouse hepatoma-rat hepatoma hybrid cells.
J. Cell. Physiol
104
,
295
308
Cassio
D.
,
Hamon-Benais
C.
,
Guerin
M.
,
Lecoq
O.
(
1991
).
Hybrid cell lines constitute a potential reservoir of polarized cells: isolation and study of highly differentiated hepatoma-derived hybrid cells able to form functional bile canaliculi in vitro.
J. Cell Biol
115
,
1397
1408
Cassio
D.
,
Shanks
M. R.
,
Ihrke
G.
,
Laurent
M.
,
Hubbard
A. L.
(
1992
).
The WIF-B rat hepatoma hybrid cells exhibit plasma membrane polarity similar to that of hepatocytes in vivo. Mol. Biol.
Cell
3
,
304
–.
Chiu
J.-H.
,
Hu
C.-P.
,
Lui
W.-Y.
,
Lo
S. J.
,
Chang
C.
(
1990
).
The formation of bile canaliculi in human hepatoma cell lines.
Hepatology
11
,
834
842
Coon
H. G.
,
Weiss
M. C.
(
1969
).
A quantitative comparison of formation of spontaneous and virus-produced viable hybrids.
Proc. Nat. Acad. Sci. USA
62
,
852
859
Deschatrette
J.
,
Weiss
M. C.
(
1974
).
Characterization of differentiated and dedifferentiated clones from a rat hepatoma.
Biochimie
273
,
153
160
Deschatrette
J.
(
1980
).
Dedifferentiated variants of a rat hepatoma: partial reversion induced by cell aggregation.
Cell
22
,
501
511
Dunn
W. A.
,
Hubbard
A. L.
(
1984
).
Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics.
J. Cell Biol
98
,
2148
2159
Feller
A. C.
(
1982
).
Cytochemical reactivity of Tu lymphocytes in human lymphatic tissue for dipeptidylaminopeptidase IV.
Histochem. J
14
,
889
895
Feracci
H.
,
Connolly
T. P.
,
Margolis
R. N.
,
Hubbard
A. L.
(
1987
).
The establishment of hepatocyte cell surface polarity during fetal liver development.
Dev. Biol
123
,
73
84
Fesus
L.
,
Thomazy
V.
,
Autuori
F.
,
Ceru
M.
,
Tarcsa
E.
,
Piacentini
M.
(
1989
).
Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action.
FEBS Lett
245
,
150
154
Gorrell
M. D.
,
Wickson
J.
,
McCaughen
G. W.
(
1991
).
Expression of the rat CD26 antigen (dipeptidyl peptidase IV) on subpopulations of rat lymphocytes.
Cell. Immunol
134
,
205
215
Graf
J.
,
Gautam
A.
,
Boyer
J. L.
(
1984
).
Isolated rat hepatocyte couplets: a primary secretory unit for electrophysiologic studies of bile secretory function.
Proc. Nat. Acad. Sci. USA
81
,
6516
6520
Griffo
G.
,
Hamon-Benais
C.
,
Angrand
P. O.
,
Fox
M.
,
West
L.
,
Lecoq
O.
,
Povey
S.
,
Cassio
D.
,
Weiss
M.
(
1993
).
HNF4 and HNF1 as well as a panel of hepatic functions are extinguished and reexpressed in parallel in chromosomally reduced rat hepatoma-human fibroblast hybrids.
J. Cell Biol
121
,
887
898
Hanzon
V.
(
1952
).
Liver cell secretion under normal and pathological conditions studied by fluorescence microscopy on living rats.
Acta Physiol. Scand
28
,
101
–.
Hong
W.
,
Doyle
D.
(
1987
).
cDNA cloning for a bile canaliculus domain-specific membrane glycoprotein of rat hepatocytes.
Proc. Nat. Acad. Sci. USA
84
,
7962
7966
Hoppe
C. A.
,
Connolly
T. P.
,
Hubbard
A. L.
(
1985
).
Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway.
J. Cell Biol
101
,
2113
2123
Hubbard
A. L.
,
Wilson
G.
,
Ashwell
G.
,
Stukenbrok
H.
(
1979
).
An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. I. Distribution of 125I-ligands among the liver cell types.
J. Cell Biol
83
,
47
64
Hubbard
A. L.
,
Wall
D. A.
,
Ma
A.
(
1983
).
Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains.
J. Cell Biol
96
,
217
229
Hubbard
A. L.
,
Bartles
J. R.
,
Braiterman
L. T.
(
1985
).
Identification of rat hepatocyte plasma membrane proteins using monoclonal antibodies.
J. Cell Biol
100
,
1115
1125
Ihrke
G.
,
Neufeld
E. D.
,
Meads
T.
,
Shanks
M. R.
,
Cassio
D.
,
Laurent
M.
,
Schroer
T. A.
,
Pagano
R. E.
,
Hubbard
A. L.
(
1993
).
WIF-B Cells: An in vitro model for studies of hepatocyte polarity.
J. Cell Biol
123
,
1761
1775
Kuhn
L. C.
,
Kraehenbuhl
J.-P.
(
1983
).
Monoclonal antibodies recognizing the secreted and membrane domains of the IgA dimer receptor.
Ann. NY Acad. Sci
409
,
751
759
Landry
J.
,
Bernier
D.
,
Ouellet
C.
,
Goyette
R.
,
Marceau
N.
(
1985
).
Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.
J. Cell Biol
101
,
914
923
Laurell
C. B.
(
1966
).
Quantitative estimation of proteins by electrophoresis agarose gel containing antibodies.
Anal. Biochem
15
,
45
49
Lin
S. H.
,
Guidotti
G.
(
1989
).
Cloning and expression of a cDNA coding for a rat liver plasma membrane ecto-ATPase. The primary structure of the ecto-ATPase is similar to that of the human biliary glycoprotein I.
J. Biol. Chem
264
,
14408
14414
Maurice
M.
,
Rogier
E.
,
Cassio
D.
,
Feldmann
G.
(
1988
).
Formation of plasma membrane domains in rat hepatocytes and hepatoma cell lines in culture.
J. Cell Sci
90
,
79
92
Mevel-Ninio
M.
,
Weiss
M. C.
(
1981
).
Immunofluorescence analysis of time course of extinction, reexpression and activation of albumin production in rat hepatoma-mouse fibroblast heterokaryons and hybrids.
J. Cell Biol
90
,
339
350
Musat
A. I.
,
Sattler
C. A.
,
Sattler
G. L.
,
Pitot
H. C.
(
1993
).
Reestablishment of cell polarity of rat hepatocytes in primary culture.
Hepatology
18
,
198
205
Ogata
S.
,
Misumi
Y.
,
Ikehata
Y.
(
1989
).
Primary structure of rat liverdipeptidyl peptidase IV deduced from its cDNA and identification of the NH2-terminal signal sequence as the membrane-anchoring domain.
J. Biol. Chem
264
,
3596
3601
Olsen
J.
,
Lowell
G. M.
,
Konigshafer
E.
,
Danielsen
E. M.
,
Moller
J.
,
Laushen
L.
,
Hansen
O. C.
,
Welinder
K. G.
,
Engberg
J.
,
Hunziker
W.
,
Spiess
M.
,
Sjostrom
H.
,
Nosen
O.
(
1988
).
Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA.
FEBS Lett
238
,
307
314
Pignal
F.
,
Maurice
M.
,
Feldmann
G.
(
1982
).
Immunoperoxidase localization of albumin and fibrinogen in rat liver fixed by perfusion or immersion: effect of saponin on the intracellular penetration of labeled antibodies.
J. Histochem. Cytochem
30
,
1004
1014
Rodriguez-Boulan
E.
,
Salas
P. J. I.
(
1989
).
External and internal signals for epithelial cell surface polarization.
Annu. Rev. Physiol
51
,
741
754
Rogier
E.
,
Cassio
D.
,
Weiss
M. C.
,
Feldmann
G.
(
1986
).
An ultrastructural study of rat hepatoma cells in culture, their variants and revertants.
Differentiation
30
,
229
236
Roman
L. M.
,
Hubbard
A. L.
(
1983
).
A domain-specific marker for the hepatocyte plasma membrane: localization of leucine aminopeptidase to the bile canalicular domain.
J. Cell Biol
96
,
1548
1558
Roman
L. M.
,
Hubbard
A. L.
(
1984
).
A domain-specific marker for the hepatocyte plasma membrane. III. Isolation of bile canalicular membrane by immunoadsorption.
J. Cell Biol
98
,
1497
1504
Rotman
B.
,
Papermaster
B. W.
(
1966
).
Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters.
Proc. Nat. Acad. Sci. USA
55
,
134
141
Saison
M.
,
Verlinden
J.
,
Van Leuven
F.
,
Cassiman
J.-J.
,
Van den Berghe
H.
(
1983
).
Identification of cell surface dipeptidylpeptidase IV in human fibroblasts.
Biochem. J
216
,
177
183
Schell
M. J.
,
Maurice
M.
,
Stieger
B.
,
Hubbard
A. L.
(
1992
).
5nucleotidase is sorted to the apical domain of hepatocytes via an indirect route.
J. Cell Biol
119
,
1173
1182
Scott
L. J.
,
Hubbard
A. L.
(
1992
).
Dynamics of four rat liver plasma membrane proteins and polymeric IgA receptor. Rates of synthesis and selective loss into the bile.
J. Biol. Chem
267
,
6099
6106
Stanley
K. K.
,
Burke
B.
,
Pitt
T.
,
Siddle
K.
,
Luzio
J. P.
(
1983
).
Localization of 5-nucleotidase in a rat liver cell line using a monoclonalantibody and indirect immunofluorescent labelling.
Exp. Cell Res
144
,
39
46
Steer
C. J.
,
Ashwell
G.
(
1980
).
Studies on a mamalian hepatic binding protein specific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes.
J. Biol. Chem
255
,
3008
3013
Steer
C. J.
,
Weiss
P.
,
Huber
B. E.
,
Wirth
P. J.
,
Thorgeirsson
S. S.
,
Ashwell
G.
(
1987
).
Ligand-induced modulation of the hepatic receptor for asialoglycoproteins in the human hepatoblastoma cell line, Hep G2.
J. Biol. Chem
262
,
17524
17529
Sztul
E. S.
,
Howell
K. E.
,
Palade
G. E.
(
1983
).
Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes.
J. Cell Biol
97
,
1582
1591
Tong
J. Z.
,
De Lagausie
P.
,
Furlan
V.
,
Cresteil
T.
,
Bernard
O.
,
Alvarez
F.
(
1992
).
Long-term culture of adult rat hepatocyte spheroids.
Exp. Cell Res
200
,
326
332
Towbin
H.
,
Staehelin
T.
,
Gordon
J.
(
1979
).
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Nat. Acad. Sci. USA
76
,
4350
4354
Trowbridge
I.S.
(
1991
).
Endocytosis and signals for internalization.
Curr. Opin. Cell Biol
3
,
634
641
Wall
D. A.
,
Wilson
G.
,
Hubbard
A. L.
(
1980
).
The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes.
Cell
21
,
79
93
Wall
D. A.
,
Hubbard
A. L.
(
1985
).
Receptor-mediated endocytosis of asialoglycoproteins by rat liver hepatocytes: biochemical characterization of the endosomal compartments.
J. Cell Biol
101
,
2104
2112
Widnell
C. C.
,
Schneider
Y.-J.
,
Pierre
B.
,
Baudhuin
P.
,
Trouet
A.
(
1982
).
Evidence for a continual exchange of 5-nucleotidase between the cell surface and cytoplasmic membranes in cultured rat fibroblasts.
Cell
28
,
61
70
Worton
R. G.
,
Duff
C.
(
1979
).
Karyotyping.
Meth. Enzymol
58
,
322
344
Zijderhand-Bleekemolen
J. E.
,
Schwartz
A. L.
,
Slot
J. W.
,
Strous
G. J.
,
Geuze
H. J.
(
1987
).
Ligand-and weak base-induced redistribution of asialoglycoprotein receptors in hepatoma cells.
J. Cell Biol
104
,
1647
1654
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