[CANCER RESEARCH 48, 6882-6890, December 1, 1988]
Analysis of Hepatocyte Plasma Membrane Polarity during Rat Azo Dye
Hepatocarcinogenesis Using Monoclonal Antibodies Directed against
Domain-associated Antigens1
Jean-Yves Scoazec,2 MichèleMaurice, Alain Moreau, and GérardFeldmann
Laboratoire de Biologie Cellulaire and Inserm U24, Facultéde MédecineXavier Bichat, 16, rue Henri Huchard, 75018 Paris, France
ABSTRACT
While carcinogenesis is known to induce various alterations in epithe
lial cell polarity, little information is available about the fate of plasma
membrane polarity during the neoplastic process. Using three monoclonal
antibody-defined antigens as markers of the three plasma membrane
domains of rat hepatocytes, the sinusoidal, the lateral, and the canalicular,
we demonstrated by immunohistochemical techniques that changes in
hepatocyte plasma membrane polarity occur at every stage of .V-methyldimethylaminoazobenzene-induced hepatocarcinogenesis. At the preneoplastic stage, the division of hepatocyte plasma membrane into three
distinct domains was retained despite rearrangements in hepatocytic
architecture, characterized by the disorganization of hepatocytic plates
and the formation of pseudoacinar structures. The most striking change
was the distribution of the canalicular-associated antigen over the entire
plasma membrane in disorganized plates. At the neoplastic stage, changes
in plasma membrane polarity depended on the degree of morphological
differentiation of neoplastic cells. Poorly differentiated cells inconstantly
expressed the monoclonal antibody-defined antigens and showed no evi
dence of plasma membrane polarization. Differentiated cells constantly
expressed the three antigens, and their plasma membrane was divided
into antigenically distinct domains. The changes in hepatocyte plasma
membrane polarity demonstrated in situ during 3'-methyldimethylaminoazobenzene hepatocarcinogenesis may therefore be compared with
situations known to occur in vitro, in cultured epithelial cells presenting
varying degrees of polarization. Our observations suggest that these
alterations are of relevance to the in vivo biology of cancer.
number of domain-specific antigens can now be defined using
monoclonal antibodies. A previous report from this laboratory
(6) has described the production of a set of monoclonal anti
bodies characteristic of the three morphological and functional
domains of hepatocyte plasma membrane (7), the sinusoidal,
the lateral, and the canalicular. The sinusoidal domain, which
corresponds to the basal domain of simple epithelial cells, is
involved in exchanges with blood. The lateral domain is com
mitted in cell-cell adhesion and communication. The canalicu
lar domain, which corresponds to the apical domain of simple
epithelial cells, is specialized for bile secretion. The availability
of monoclonal antibodies against domain-associated antigens
of hepatocyte plasma membrane makes it possible to search for
alterations of plasma membrane polarity in a widely used model
of epithelial carcinogenesis, chemical hepatocarcinogenesis in
the rat. The present study was designed to investigate, by light
and electron microscopic immunohistochemistry,
the fates of
hepatocyte plasma membrane domains at the various stages of
hepatocarcinogenesis induced by a chemical carcinogen, 3'methyldimethylaminoazobenzene,
in order (a) to search for
alterations of hepatocyte plasma membrane polarity during the
neoplastic process and (b) to correlate those changes with the
alterations in cell polarity occurring in this in vivo model.
MATERIALS
INTRODUCTION
AND METHODS
Study Design. Male Fischer 344 rats (Animalerie de l'IRSC, Villejuif,
France) were used throughout the study. Animals were housed in an
air-conditioned room at controlled temperature and humidity, with a
12-h light-dark cycle. They received tap water ad libitum. The study
group, 38 rats, was fed a diet containing 0.06% 3MeDAB (Koch Light
Laboratories, London, England) from the age of 4 weeks to their
sacrifice, as described previously (8). Twelve rats, used as controls, were
fed a standard commercial diet (UAR, Villacoublay, France). The
animals were sacrificed at the following time points after initiation of
the intoxication: 4, 6, 10, 12, 14, 16, 18, and 22 weeks. At each time
point, 4 animals of the study group were sacrificed except at weeks 16
and 22 at which 7 animals were sacrificed. At least one rat from the
control group was sacrificed at each time point.
Histológica! typing of 3MeDAB-induced lesions was performed ac
cording to previously published classifications and descriptions of prelial cells (4) and HT29 cells (5). While alterations in cell polarity
neoplastic (9-12) and neoplastic (9, 12, 13) lesions.
induced in these /// vitro models share some similarities with
Monoclonal Antibodies. Three monoclonal antibodies, termed A39,
those observed in the neoplastic process, their relevance to in Bl, and BIO, were used. Their mode of production has been detailed
vivo biology of cancer is not assessed. Difficulties in the study
in a previous report (6). Briefly, BALB/c mice were immunized i.p.
of plasma membrane polarity in vivo mainly arose from the fact with plasma membranes of liver cells prepared according to the tech
that only a limited number of domain-specific markers was nique of Neville (14). Their splenic cells were fused with Sp2-0/Ag 14
available before the development of hybridoma technology. A mouse myeloma cells. Hybridomas were screened by indirect immunofluorescence on liver cryostat sections. The antigens defined by A39,
Received 12/30/87; revised 8/2/88; accepted 8/18/88.
Bl, and BIO have been characterized by immunoblotting (6) and
The costs of publication of this article were defrayed in part by the payment
immunoprecipitation (15) in Sprague-Dawley rats. A39 reacted with
of page charges. This article must therefore be hereby marked advertisement in
two main bands, corresponding to antigens with apparent molecular
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by grants from the Fondation pour la
weights between 30,000 and 36,000. Bl recognized a single band
Recherche Médicaleand from the Conseil Scientifique, FacultéXavier Bichat,
corresponding to an antigen with an apparent molecular weight of
UniversitéParis VII.
100,000. BIO reacted with two bands with molecular weights of 150,000
2To whom requests for reprints should be addressed.
and ^300,000. The three antigens have been shown to behave as integral
3 The abbreviations used are: MDCK, Madin-Darby canine kidney; 3MeDAB,
3'-methyldimethylaminoazobenzene;
endo F, endo-d-/V-acetylglucosaminidase F;
membrane proteins by alkaline extraction of a purified hepatocyte
SDS. sodium dodecyl sulfate; PBS, phosphate-buffered saline.
plasma membrane preparation (15).
6882
Most normal epithelial cells display a polarized organization.
One of the aspects of this asymmetry is the division of the
plasma membrane into morphologically, functionally, and bio
chemically distinct domains (1, 2). Carcinogenesis is known to
induce various alterations in epithelial polarity, which may
affect, according to the case, the overall cellular shape, the
intracellular distribution of organdÃ-es, or the secretory func
tions (3). However, little information is available about the fate
of plasma membrane polarity during the neoplastic process.
Most current knowledge about maintenance of plasma mem
brane domains in cells with altered polarity derives from in
vitro models using epithelial cell lines, such as MDCK3 epithe
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PLASMA MEMBRANE POLARITY IN HEPATOCARCINOGENESIS
The specificity of the monoclonal antibodies A39, HI, and BIO, first
documented in Sprague-Dawley rats, was reinvestigated in the Fischer
344 strain and is presented below.
Enzymatic Deglycosylation. Plasma membrane fractions were pre
pared according to the technique of Neville (14) from Fischer 344 rat
livers. Enzymatic deglycosylation was carried out with neuraminidase
from Vibrio cholerae (Calbiochem-Behring, La Jolla, CA) and with
endo-/3-A'-acetylglucosaminidase F (endoglycosidase F; Boehringer
Mannheim Biochemien, Mannheim, Federal Republic of Germany).
For neuraminidase treatment, plasma membrane samples containing
100 //g protein were incubated for 18 h at 37°Cwith 0.1 unit neuramin
idase in sodium acetate buffer, pH 5.5, containing 154 HIMNaCl and
2 mM CaCb. as described previously (16) with slight modifications.
Endo F digestion was performed according to the manufacturer's
recommendations, essentially as described previously (17). Plasma
membrane proteins were solubili/od in 0.5% SDS and boiled for 3 min.
The following additions were then made for 100 ^g of protein: (a) 75
n\ of a solution containing 100 HIM sodium acetate (pH 6), 45 HIM
EDTA, 1% Triton X-100, and 0.1% SDS; (b) 0.2 unit endo F. The
final solution was incubated for 18 h at 37°C.Plasma membrane
proteins incubated in corresponding buffers in the absence of enzyme
were used as controls. After incubation, samples were subjected to SDS
polyacrylamide gel electrophoresis on 5-15% gradient gels and immunoblotting as already described ( 15). For calibration, standard molec
ular weights (Bio-Rad, Richmond, CA; Pharmacia, Uppsala, Sweden)
were run in parallel.
Immiinoperoxidase Procedure. Immunoperoxidase was performed on
fixed liver samples, obtained from 3 rats at each time point, except at
the 16th and 22nd weeks at which 5 rats were studied. Rats were
anesthetized with sodium pentobarbital (Clin-Midy, Saint-Jean,
France). Through the portal vein, livers were washed for 4 min with
0.1 M PBS, bubbled with 95% O2 and 5% CO2, and then fixed for 10
min with 4% paraformaldehyde (Merck, Darmstadt, Federal Republic
of Germany) diluted in phosphate buffer, pH 7.4. Liver slices, 1 to 2
mm thick, were immersed in the same fixative for 2 to 4 h.
For each liver, 3 or 4 blocks from different slices were processed.
Blocks were snap frozen in isopentane, prechilled with liquid nitrogen.
Sections 8 ¿¿m
thick were cut with a cryostat. Liver sections were
incubated overnight at 4°Cin undiluted hybridoma culture supernatants. After being washed in PBS, sections were incubated for 3 h at
room temperature in peroxidase-labeled species-specific anti-mouse
immunoglobulin antibodies (Amersham, Little Chalfont, England),
diluted 1:50 in PBS. Peroxidase activity was revealed according to the
method of Graham and Karnovsky (18). For each block, at least two
sections, one of which lightly counterstained with hematoxylin, were
examined with a light microscope. The remaining sections were proc
essed for electron microscopy. They were post fixed for 30 min in 1%
phosphate-buffered osmium tetroxide solution, dehydrated through
graded alcohols and propylene oxide, and then embedded in epoxy
resin. Ultrathin sections were examined without additional staining
with a Siemens Elmiskop IA electron microscope. For each time of
sacrifice, one to four animals were investigated at the ultrastructural
level. For each of these animals, ultrathin sections from at least two
different blocks were examined.
For control reactions, primary monoclonal antibody was omitted
and replaced by either PBS or normal mouse serum diluted 1:500 in
PBS. Normal liver surrounding 3MeDAB-induced lesions was used as
a positive control.
Immunoblotting Procedure for Liver Tissue Homogenates. Imimmo
blotting was performed on frozen liver tissue obtained from 6 animals
presenting with preneoplastic lesions and 5 animals with hepatocarcinomas. After anesthesia, the liver was immediately removed. Slices 1
mm thick were snap frozen and stored in liquid nitrogen until use.
Tissue samples used for homogenization corresponded either to whole
liver slices for rats with preneoplastic lesions or to carcinomas excised
from the surrounding liver parenchyma for rats with neoplastic lesions.
Tissue samples were homogenized with a Potter homogenizer in ice
cold 1 HIMsodium bicarbonate, pH 8.2, containing 0.5 m\t phenylmethylsulfonyl fluoride. The homogenate was centrifugea for 30 min
at 10,000 x g at 4°C.The pellet was dissolved in 2% SDS-0.03 M Nethylmaleimide (Sigma, Saint Louis, MO)-10% glycerol-50 mM TrisHC1 buffer, pH 6.8. Samples containing 100 to 150 ^g of protein were
run through a 7.5 or a 9% polyacrylamide slab gel according to the
method of Laemmli (19). Electrophoreses were performed at room
temperature with water cooling, at 30 mA and for 5-7 h. Proteins were
then electrophoretically blotted onto a nitrocellulose sheet (Schleicher
and Schiill, Dassel, Federal Republic of Germany) as described by
Towbin et al. (20). Blotting was performed overnight at 0.15 mA. The
nitrocellulose filters were quenched for 1 h at 37"C in 20% newborn
calf serum (IBF, Paris, France) in PBS-0.1% Tween 20 (Merck, Darm
stadt, Federal Republic of Germany) and then incubated for 3 h at
room temperature in hybridoma culture supernatants diluted 1:2 in
PBS-0.1% Tween 20. After a washing, filters were incubated for 3 h at
room temperature with gold-labeled anti-mouse immunoglobulin anti
bodies (Janssen Biotech NV, Olen, Belgium), diluted 1:100 in PBS0.1% Tween 20 containing 20% newborn calf serum. Silver enhance
ment of gold staining was performed according to the manufacturer's
recommendations (Intense kit; Janssen Biotech NV). For calibration,
human erythrocyte membranes (kindly provided by Dr. Dhermy, Inserm
U160, Clichy, France) were run in parallel (21). Liver slices from
control rats were processed in the same way and run on the same gels.
RESULTS
Specificity of the Monoclonal Antibodies
Immunolocalization in Normal Fischer 344 Rat Liver (Table
1). As described previously in Sprague-Dawley rats (6), A39
labeled mainly the sinusoidal domain and occasionally the
canalicuhir membrane of hepatocyte plasma membrane (Fig. 1,
a and b). BIO labeling was restricted to the can alieu lar domain
(Fig. 1, e and /). The only significant differences between
Sprague-Dawley and Fischer 344 strains were observed for Bl.
While Bl mainly labeled the lateral domain in Sprague-Dawley
rats, this monoclonal antibody constantly labeled both the
lateral and the sinusoidal domains in Fischer 344 rats (Fig. 1,
c and d ). The basolateral domain of the biliary cells reacted
with A39 and Bl, and their apical membrane stained with BIO
and occasionally with A39.
Table 1 Results of immunolocalization
of
antigensConstant
ofdistinct
Presence
A39+
domains
Normal liver cells
Hepatocytes
cellsPreneoplastic
Biliary
ConstantConstant
b+asolat+
lesions
Disorganized plates
strNeoplastic
Pseudoacinar
ConstantInconstant
(can)—
+
of antigens defined by
sin," (can)
lat
basolatsin,
sin, (can)
sin,
lat
sin,
(lat)pericell
lat, sin
canpericell
lesions
pericell
Poorly diff carcinomas
Constant
+
basolat
basolat
apic
Adenocarcinomas
Well-diff carcinomasExpression
ConstantDistribution
+
basolatBlsin,
basolatBIOcanapiccan,
can
1sin, sinusoidal; lat, lateral; can, canalicular; basolat, basolateral; apic, apical; pericell, pericellular; str. structure; did. differentiated; +, present; —¿,
absent.
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PLASMA MEMBRANE POLARITY IN HEPATOCARCINOGENESIS
A39
(
•¿
<~
b
:•>Fig. 1. Distribution of immuno labeling for
A39, Bl, and BIO on normal hepatocytes by
light microscopy (a, x 550; c, x 450; e, x 550;
immunoperoxidase staining) and electron mi
croscopy (b, x 10,000; d, x 11,000;/, x 9,000;
immunoperoxidase staining without countercoloration). A39 labels mainly the sinusoidal
domain (a and ¿>)
and inconstantly the canalicular domain (/<). No labeling of the lateral
domain is observed. Bl labels the sinusoidal
and the lateral domains (c and d). BIO labeling
is restricted to the canalicular domain (c and
/). Arrow, sinusoidal domain; arrowhead, lat
eral domain; C, bile canaliculus; 5, sinusoidal
lumen. Bars: a, c, e, 25 *im; h.il.f. 1 *im.
B1
•¿
\
*
:
«.•
*
'
•¿<Vv
Characterization of the Corresponding Plasma Membrane An
tigens (Fig. 2). By immunoblotting, A39 was shown to react
with two main bands, corresponding to antigens with an appar
ent molecular weight of, respectively, 30,000 and 34,000. Bl
recognized a single band corresponding to an antigen with an
apparent molecular weight of 100,000. BIO reacted with a main
band with a molecular weight of 150,000.
Enzymatic deglycosylation with either neuraminidase or endo
F did not affect the immune detection of the three antigens
studied (Fig. 2). The mild alterations observed in reactivity
pattern and/or staining intensity for A39- and BlO-defined
antigens might be attributed to the differences existing between
buffer and incubation conditions used for enzymatic deglyco
sylation and those used in standard electrophoretic procedures
(22). In any case, our results strongly suggest that the epitopes
to which the three monoclonal antibodies bind are of peptidic
rather than of carbohydrate nature.
All three antigens were glycoproteins. As indicated by the
decrease in apparent molecular weight induced by neuramini
dase treatment, all three contained sialic acid residues (Fig. 2).
As shown by the results of endo F digestion, all three antigens
contained TV-linked oiigosaccharide chains. The decrease in
apparent molecular weight observed for A39-, Bl- , and BlOdefined antigens respectively accounted for approximately 20,
25, and 38% of the initial molecular weights calculated under
the same electrophoretic conditions (Fig. 2).
Immunolocalization in 3MeDAB-induced Lesions (Table 1)
Early Lesions (4-16 Weeks). The early lesions observed in
3MeDAB-induced hepatocarcinogenesis were characterized (a)
by a marked proliferation of oval cells and (/>)by the subsequent
development of preneoplastic foci and nodules constituted by
morphologically altered hepatocytes.
Oval Cell Proliferation. This was marked at 4 weeks and
maximal at 6 weeks. Subsequently, oval cells decreased in
number but remained present in significant amounts until 16
weeks. Typical oval cells presented a distinctive phenotype.
They constantly reacted with Bl, which labeled their whole
6884
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PLASMA MEMBRANE POLARITY IN HEPATOCARCINOGENESIS
Fig. 2. Characterization of A39-, Bl-, and
BlO-defined antigens. SDS-polyacrylamide gel
electrophoresis analyses and immunoblotting
of plasma membrane preparations. Lanes N,
nontreated plasma membrane fractions; Lanes
1, neuraminidase-treated plasma membrane
fractions; Lanes 2, plasma membrane fractions
incubated in neuraminidase buffer in the ab
sence of enzyme; Lanes 3, endo F-treated
plasma membrane fractions; Lanes 4, plasma
membrane fractions incubated in endo F buffer
in the absence of enzyme. Preparation of sam
ples, SDS-polyacrylamide gel electrophoresis,
and immunoblotting were performed as de
scribed under "Materials and Methods." Sam
ples were run on 5-15% gradient polyacrylamide slab gels. Standard molecular weights
are indicated by arrows and correspond to my
osin (M, 250,000), d-galactosidase
(M,
130,000), phosphorylase * (M, 94,000), bovine
serum albumin (M, 67,000), ovalbumin (M,
43.000) and carbonic anhydrase (M, 30,000).
In the normal state, A39 reacts with two main
bands with molecular weights of 30,000 and
34,000, Bl reacts with a M, 100,000 band, and
BIO reacts with a M, 150,000 band. In the
three cases, while reactivity pattern may be
modified by the incubation conditions, im
mune detection of the antigens is not affected
either by neuraminidase or by endo F diges
tions. The efficiency of enzymatic deglycosylation is assessed by the decrease in apparent
molecular weight of the antigens.
Fig. 3. Immunolabeling of oval cells by Bl
and B10. Light microscopy (a, x 180;*, x 160;
immunoperoxidase staining). Tracts of oval
cells (arrowheads) divide the liver lobules. In
tense Bl labeling is detected into areas corre
sponding to oval cell proliferation (a). The
same areas are unreactive for BIO (ft). Sur
rounding hepatocytes display a sinusoidal and
lateral Bl labeling reminiscent of that of nor
mal hepatocytes (a). BIO labeling on hepato
cytes is either canalicular or canalicular and
lateral (ft). Electron microscopy (c, x 6000; d,
x 7000; immunoperoxidase staining without
countercoloration). The entire plasma mem
brane of oval cells (II) is labeled by BI (double
arrows) (c). On d, several oval cells (O) are
visible between two hepatocytes (//). The iso
lated oval cell is not decorated by BIO, while
the oval cells organized in a duct-like structure
display an apical labeling. //. hepatocyte; O,
oval cell. Bars: a, ft, 50 urn; c, (/. 2 jim.
(150
130 f^
-«
94
«100
^
94 ^_
67 _
iâ
E:
^
4
30
N
».
1
2
3
4
N
B1
A39
B1
2
3
4
N
BIO
B10
b
B1
BIO
plasma membrane (Fig. 3, a and c). A39 labeling, distributed
all along the plasma membrane, was faint and inconstant. No
labeling was observed for BIO (Fig. 3, b and d). Numerous
duct-like structures resulting from oval cell maturation were
also observed. The cells lining these ductular structures were
polarized and their antigenic phenotype was reminiscent ofthat
of normal biliary cells. These cells possessed a basolateral
domain reactive with Bl and A39 and an apical domain faintly
reactive with A39. BIO labeling was rarely observed; when
present, it was restricted to the apical membrane (Fig. 3d).
Preneoplastic Foci and Nodules. These, consisting of a mix
ture of cytologically abnormal hepatocytes, were observed from
10 to 16 weeks. An average of 10 foci and/or nodules were
analyzed for each animal at each time point. As defined previ
ously (9), foci were smaller than a hepatic lobule, whereas
nodules had a diameter equivalent to or greater than the di
ameter of a lobule. No correlation was attempted between the
staining patterns and the cytological types usually described in
these lesions (9). Indeed, certain of the morphological charac
teristics used to define the various cell types observed within
preneoplastic lesions cannot be demonstrated in the frozen
sections used in this study. However, the technique allowed a
6885
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PLASMA MEMBRANE POLARITY IN HEPATOCARCINOGENESIS
B1
B10
Fig. 4. Preneoplastic lesions, disorganized
plates. Light microscopy (a, x 720; ft, x 450;
immunoperoxidase staining): the trabecular
organization of hepatocytes is maintained, but
plates vary in thickness and are irregularly
branched. BIO labeling (a) is observed on the
canalicular domain (short arrow) and is also
present on the lateral (arrowhead) and sinus
oidal (double arrow) domains. Bl labeling (/>)
remains sinusoidal (arrow) and lateral (arrow
head). Electron microscopy (c, x 15,000; d,
x 3,200; immunoperoxidase staining without
countercoloration): ultrastructural study con
firms that BIO labeling (c) is present not only
on the canalicular domain but also on the
sinusoidal (arrow) and on the lateral (arrow
head) domains. A39 labeling (</) remains si
nusoidal (arrow) and canalicular. No lateral
labeling is observed despite the dilation of in
tercellular spaces (arrowhead). S, sinusoidal
lumen; C, bile canaliculus; //. hepatocyte. Bars:
ti. h. 25 /'in; c, 1 Ã-iiiKd. 2 ¿jm.
s
B10
A39
'
H
.^Y\
-
f_
•¿/*
**
good correlation between staining patterns and rearrangements
in hepatocytic architecture. Two main architectural patterns
could be recognized, the disorganized plates and the pseudoacinar structures (11). In disorganized plates, the trabecular
disposition of hepatocytes was retained, but plates were irreg
ularly branched and of variable thickness. In pseudoacinar
structures, hepatocytes were organized in a tubular manner
around a stellate bile canaliculus. In both patterns, expression
of the three monoclonal antibody-defined antigens was con
stant. Hepatocyte plasma membrane retained a division into
three antigenically distinct plasma membrane domains. The
differences between the two architectural types lay in the pattern
of distribution of the domain-associated antigens. In disorgan
ized plates, the main abnormality was the labeling of sinusoidal
and lateral domains by BIO (Fig. 4, a and c). There were no
significant modifications of distribution patterns of Bl (Fig.
4b) and A39 (Fig. 4d). In pseudoacinar structures, Bl labeling
was modified. It was strong all along the sinusoidal domain but
was inconstant and faint along the lateral membrane (Fig. S, a
and c). Distributions of the two other monoclonal antibodies,
BIO (Fig. 5, b and d) and A39, were unchanged.
Neoplastic Lesions (16-22 Weeks). Hepatocarcinomas were
found in 13 animals from the 16th to the 22nd weeks of
intoxication. These malignant tumors displayed a wide range
of morphological features. Three types of hepatocarcinomas
were recognized, poorly differentiated carcinomas, adenocarcinomas, and well-differentiated hepatocarcinomas (12, 13). In
the same tumor, poorly differentiated areas usually coexisted
with areas presenting varying degrees of differentiation.
In poorly differentiated areas, neoplastic cells displayed no
evidence of morphological polarization. Expression of the
monoclonal antibody-defined antigens was inconstant. The loss
of reactivity for BIO was the most frequently observed and was
usually complete. For A39 and Bl, reactivity was always present
on a variable proportion of neoplastic cells (Fig. 6a). The
labeling, when present, revealed no antigenically distinct do
main and was distributed all along the plasma membrane (Fig.
6c). In certain neoplastic cells, intracellular lumina with nu-
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PLASMA MEMBRANE POLARITY IN HEPATOCARCINOGENESIS
'B,
*
•¿
l
»
>.
- *
«
*-
.
>
Fig. S. Preneoplastic lesions, pseudoacinar
structures. Light microscopy (a, X 550; ft,
x 400; immunoperoxidase staining): hepatocytes are organized in tubular structures cen
tered by dilated bile canaliculi. Bl labeling (a)
is mainly sinusoidal (arrow) and faintly lateral
(arrowhead). BIO labeling (ft) decorates only
bile canaliculi. Electron microscopy (c,
X 7000; ft, x 4500; immunoperoxidase stain
ing without countercoloration): BI labeling (c)
is mainly distributed on the sinusoidal or basal
domain (arrow). The lateral domain is incon
stantly labeled (arrowhead). BIO labeling (d)
remains restricted to the canalicular domain;
no labeling of the lateral (arrowhead) and si
nusoidal (arrow) domains is visible. 5, sinus
oidal lumen; C, bile canaliculus; H, hepatocyte.
liars: a, ft, 25 urn; c, d, 2 urn.
«¿r-.
*
S
B1
B10
H
1
A
H
:
mérousmicrovilli having features of the so-called vacuolar
apical compartment (23), have been observed. The membrane
of these organdÃ-es was labeled by BIO (Fig. 6e), but not by A39
and Bl.
In adenocarcinomas, expression of the three monoclonal
antibody-defined antigens was constant. Neoplastic cells were
morphologically polarized. Their organization was reminiscent
of that of simple epithelial cells, exemplified in the liver by
biliary cells. They presented an apical pole facing the glandular
lumen and a basolateral pole in contact with adjacent neoplastic
cells or with the extracellular matrix. These morphological
domains were antigenically distinct. Distribution of the three
monoclonal antibody defined antigens on neoplastic cells was
reminiscent of the normal distribution of these antigens on
simple epithelial cells. A39 and Bl labeled the basolateral
domain of neoplastic cells (Fig. db). B10 labeled only the apical
domain (Fig. 6d).
In well-differentiated hepatocarcinomas, the three mono
clonal antibody-defined antigens were constantly expressed.
Neoplastic cells were morphologically polarized and presented
an organization reminiscent of that of normal hepatocytes,
except that no distinction between a sinusoidal and a lateral
domain was usually possible because of the changes in microvascularization occurring within the neoplastic tissue. Two
antigenically distinct domains could be recognized. As in nor
mal hepatocytes, BIO labeling was restricted to the canalicular
membrane (Fig. 6/). A39 and Bl labeled the remaining plasma
membrane.
Control Reactions. All control reactions were negative.
Immunoblotting
In all cases, tissue samples from carcinogen-treated rats (6
with preneoplastic lesions and 5 with hepatocarcinomas) dis
played the same pattern of reactivity as did normal liver tissue
homogenates (Fig. 7). A39 reacted with two main bands, cor
responding to antigens with apparent molecular weights of,
respectively, 30,000 and 34,000. Bl recognized a single band
corresponding to an antigen with an apparent molecular weight
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PLASMA MEMBRANE POLARITY IN HEPATOCARCINOGENESIS
•¿
A39
B1
•¿â€¢â€¢i
Fig. 6. Neoplastic lesions. Poorly differen
tiated carcinomas: light microscopy (a, x 180;
immunoperoxidase staining). Neoplastic cells
are heterogeneous as regards the expression of
A39-defined antigen; numerous negative cells
coexist with positive cells. Electron microscopy (c, x 8,500; e, x 14,000; immunoperoxi
dase staining without countercoloration): Bl
labeling (c) is distributed all along the plasma
membrane of these unpolarized neoplastic
cells (arrows). BIO labels the membrane of an
intracellular vacuole lined by microvilli, resem
bling the so-called vacuolar apical compart
ment (<•).
Adenocarcinomas: light microscopy
(b, x 450 and d, x 450; immunoperoxidase
staining): polarized neoplastic cells line glan
dular structures. Their basolateral domain
reacts with Bl (ft) and their apical membrane
(arrow) is labeled by BIO (d). Well differen
tiated hepatocarcinomas: electron microscopy
(/ x 9,500; immunoperoxidase staining with
out countercoloration): BIO labeling of neo
plastic cells is restricted to a structure resembling a normal bile canaliculus, limited by well
formed tight junctions (short arrowheads). N,
nucleus. Bars: a, 50 urn; b, d, 25 um; c,e,f, l
b
3
-
B10
B1
L
».V
•¿
•¿
N
N
ç
B10
N
of 100,000. BIO reacted with one M, 150,000 band and an
additional Mr 210,000 band.
DISCUSSION
In this work, monoclonal antibodies against domain-associ
ated antigens were used to demonstrate, by an in vivo study
using immunohistochemical techniques, that changes in hepatocyte plasma membrane polarity occur during the course of
chemically induced hepatocarcinogenesis
and that these
changes correlate with rearrangements in hepatocyte architec
ture and with alterations in cell polarity.
Although the altered staining patterns observed for the three
monoclonal antibodies used in this study are likely to result
from an inappropriate distribution of the corresponding anti
gens, a cross-reactivity of the monoclonal antibodies with ab
normal membrane-associated proteins synthesized by trans
formed hepatocytes must be considered. Immunoblotting re
sults make this possibility unlikely. Indeed, the number of
reactive bands and the apparent molecular weights of the anti
gens detected in homogenates from preneoplastic and neoplas
tic lesions are similar to those detected in normal liver. Fur
thermore, the apparent molecular weights determined in this
study for both normal and carcinogen-treated Fischer 344 rats
compare well with previous results obtained by immunoblotting
(6) and immunoprecipitation (15) for normal Sprague-Dawley
rats. In addition, that the monoclonal antibodies used in this
study react with the protein core rather than with the carbohy
drate moieties of the corresponding plasma membrane antigens
contributes to reduce the probability of a cross-reactivity be
tween unrelated glycoproteins, which is most often due to
shared carbohydrate determinants (24).
Changes observed in 3MeDAB-induced preneoplastic and
neoplastic lesions are of a different nature. In preneoplastic
lesions, the division of hepatocyte plasma membrane into three
antigenically distinct domains (7, 25), is retained. However,
two of these domains, the lateral and the sinusoidal, are affected
by antigenic changes. The pattern of these changes is correlated
with the pattern of rearrangement in hepatocytic architecture
(11). The most striking antigenic changes involve both the
6888
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PLASMA MEMBRANE POLARITY IN HEPATOCARCINOGENESIS
1234
2
A39
3
B1
4 N
234
B10
Fig. 7. Immunoblotting results. SDS-polyacrylamide gel electrophoresis anal
yses and immunoblotting of tissue homogenates obtained from normal liver (Lane
N), from preneoplastic lesions (Lanes 1 and 2) and from neoplastic lesions (Lanes
3 and 4). Preparation of samples, SDS-polyacrylamide gel electrophoresis, and
immunoblotting were performed as described under "Materials and Methods."
Samples were run on 9% polyacrylamide slab gels (A39 and Bl) and 7.5%
polyacrylamide slab gel (BIO). Standard molecular weights are indicated by arrows
and correspond to spectrin (M, 220,000-240,000), component 4.la (M, 81,000),
component 4.2 (M, 72,000), actin (M, 43,000), and glyceraldehyde-3-phosphate
dehydrogenase (M, 35,000). Reactivity patterns are comparable for all samples.
A39 reacts with two main bands, corresponding to antigens with apparent
molecular weights of, respectively, 30,000 and 34,000. Bl recognizes a single
band corresponding to an antigen with an apparent molecular weight of 100,000.
BIO reacts with two bands with molecular weights of 150,000 and 210,000; the
apparent upper molecular weight band corresponds to nonspecific reactivity.
lateral and the sinusoidal domains in disorganized hepatocytic
plates. They are mainly due to the spreading over on the entire
plasma membrane of the canalicular-associated antigen defined
by BIO. These results are reminiscent of those obtained by
histoenzymological studies for two other canalicular-associated
proteins, alkaline phosphatase and an ATPase, which have been
shown to spread over on the entire hepatocyte plasma mem
brane in preneoplastic lesions induced by 3MeDAB or by
various other chemical carcinogens (26-28).
Our study, confirming previous reports (28), shows that
changes in the distribution of canalicular-associated proteins
occur early in the course of carcinogenesis. Therefore, these
changes cannot be considered markers of an imminent neoplas
tic transformation. Furthermore, these antigenic changes may
be not specific for the putative preneoplastic lesions. A similar
pattern of distribution for BIO labeling has been observed in
our laboratory in unrelated physiological and pathological con
ditions, i.e., a limited stage of rat liver development, from day
19 of gestation to day 5 postpartum (29), and experimentally
induced cholestasis in the rat (30). These concurrent observa
tions suggest that different cellular changes may result in similar
alterations of membrane protein processing by hepatocytes.
In neoplastic lesions, changes in plasma membrane polarity
depend on the degree of differentiation of neoplastic cells.
Poorly differentiated neoplastic cells, which are morphologi
cally unpolarized and exhibit reduced intercellular junctions,
inconstantly express the monoclonal antibody-defined antigens
and display no evidence of membrane polarization. In contrast,
differentiated neoplastic cells, which are morphologically po
larized and possess well-developed intercellular junctions, con
stantly express the monoclonal antibody-defined antigens and
display division of their plasma membrane into antigenically
distinct domains. These in vivo observations share similarities
with certain situations observed in cultured epithelial cells.
That the basolateral antigens defined in our study by the
monoclonal antibodies A39 and Bl remain unpolarized in the
absence of well-formed cell-cell interactions is in keeping with
observations performed in various in vitro models using primary
6889
cell cultures, such as primary hepatocyte monolayers (15), and
cultured cell lines, such as MDCK and HT29 cells (23, 31-35).
The inconstant detection of those antigens on the surface of
poorly differentiated neoplastic cells, as observed in our study,
may correspond to variations in the amount of protein actually
inserted in the plasma membrane or may be a further example
of the phenotypic heterogeneity which is known to occur in
many malignant clones (36). A lack of reactivity due to modi
fications in glycosylation, which are frequently observed for
glycoproteins synthesized by neoplastic cells (37), is unlikely
because the monoclonal antibodies used in this study react with
epitopes of peptidic rather than carbohydrate nature.
The apical marker defined in our study by the monoclonal
antibody BIO is also frequently undetected on the surface of
poorly differentiated neoplastic cells. This is in keeping with its
inconstant expression by certain hepatoma cell lines, as shown
by a previous work from our laboratory (15). These results
concur with numerous observations made on hepatoma cell
lines and human hepatocarcinomas for a variety of canalicularassociated antigens (38-43) to suggest that the expression of
canalicular proteins is highly sensitive to transformation. The
absence of detection of canalicular antigens may correspond
either to an actual lack of insertion or to a decreased amount
of the corresponding proteins in plasma membrane. The two
mechanisms have been demonstrated for apical membrane pro
teins in in vitro models using cultured epithelial cell lines (23,
44). It is of interest that in unpolarized MDCK cells, abnormal
targeting of apical markers is associated with appearance of a
cytoplasmic storage organdie, the so-called vacuolar apical
compartment, in which apically directed proteins may accu
mulate (23). Structures with comparable morphological fea
tures have been described in a variety of epithelial neoplasms
(45-47) and cancer cell lines (48). Our ultrastructural observa
tions show that such structures are actually present in chemi
cally induced poorly differentiated liver carcinomas. As for
vacuolar apical compartment in MDCK cells (23), the mem
brane of these organdÃ-es was found to be labeled by the mono
clonal antibody directed against an apical protein but was
unreactive with the antibodies binding to antigens associated
with the basolateral domain. Abnormal processing of apically
targeted proteins in a vacuolar apical compartment may there
fore be of relevance to explain impaired in vivo biogenesis of
apical membrane in neoplastic cells.
In conclusion, changes in hepatocyte plasma membrane po
larity can be demonstrated by in situ analysis at every stage of
the neoplastic process. They correlate well with the alterations
in cell polarity and in cell-cell interactions induced by the
neoplastic process. To a large extent, they may be compared
with the situations observed in vitro in cultured epithelial cells
with varying degrees of polarization. In situ demonstration of
changes in plasma membrane polarity during chemical hepatocarcinogenesis in the rat suggests that these alterations are of
relevance to the in vivo biology of cancer.
ACKNOWLEDGMENTS
The authors thank Paulette Echinard-Garin for her expert assistance.
They also thank JoséUriel for his helpful advice in the preparation of
the manuscript.
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Analysis of Hepatocyte Plasma Membrane Polarity during Rat
Azo Dye Hepatocarcinogenesis Using Monoclonal Antibodies
Directed against Domain-associated Antigens
Jean-Yves Scoazec, Michèle Maurice, Alain Moreau, et al.
Cancer Res 1988;48:6882-6890.
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