[CANCER RESEARCH 49. 403-409, January IS. 1989]
Immortal Epithelial Cells of Normal C3H Mouse Liver in Culture: Possible
Precursor Populations for Spontaneous Hepatocellular Carcinoma1
Gang-Hong Lee,2 Norimasa Sawada, Yohichi Mochizuki, Kimie Nomura, and Tomoyuki Kitagawa
Departments of Pathology [G-H. L., K. N., T. K.] and Experimental Pathology ¡N.S.], Cancer Institute, Tokyo 170; and Department of Pathology, Cancer Research
Institute, Sapporo Medical College, Sapporo 060 [Y. M.J; Japan
ABSTRACT
Long-term culture of primary hepatocytes derived from normal young
male C3H/HeNJclMTV+ (C3H) and C57BL/6NJcl (C57) mice, respec
tively known for very high and low incidences of spontaneous hepatoma,
resulted in development of multiple slowly growing epithelial colonies in
the C3H case, the number of colonies being increased five-fold when 1.5
IHMphénobarbitalwas added to the culture medium. On the other hand,
the primary culture cells from C57 mouse liver gave rise to such epithelial
colonies only very rarely, even with phénobarbital.
Immunohistochemical investigation revealed a-fetoprotein and/or al
bumin production by the colony cells and ultrastructural analysis also
revealed some hepatocytic features in them. Subculturing of individual
colonies gave rise to cell lines which could be repeatedly passaged. Two
of five lines implanted into athymic nude mice manifested tumorigenicity,
the resultant neoplasms being diagnosed as a trabecular hepatocellular
carcinoma and an adenocarcinoma.
The experimental data suggest that the colony-forming immortal epi
thelial cells possibly represent early phase precursors of spontaneous
mouse hepatocellular carcinomas. This culture system is expected to be
useful for future elucidation of the mechanisms underlying spontaneous
mouse hepatocarcinogenesis.
INTRODUCTION
Investigation of so-called spontaneous tumorigenesis in ex
perimental animals is important since most human malignan
cies arise spontaneously. Hepatic tumorigenesis developing in
mice without carcinogen exposure would seem to be the most
useful model for this type of study because the genetics of
different mouse strains have been elaborately investigated and
both high and low incidence strains of spontaneous hepatomas
are available. Particularly C3H3 mouse strain is known for the
extremely high (up to 100%) incidence of spontaneous hepa
tomas, whereas C57BL strain for the low (<1%) incidence (1).
We have been interested in identifying the precursor cells of
spontaneous hepatomas as immortal cell population in culture
system. Previously it was shown in this laboratory that carcin
ogen-initiated hepatocytes in the rat are capable of growth in
culture, specifically in response to various promoters of hepa
tocarcinogenesis, to give rise to transplantable hepatocellular
carcinoma cell lines after several months (2, 3). If the two-step
concept of chemical carcinogenesis (4-6) is also applicable to
spontaneous carcinogenesis in mice, spontaneously initiated
hepatocytes should exist in the "normal" mouse liver, which
should be similarly detectable under culture system conditions.
This paper describes the successful culture of epithelial cells
with hepatocytic features from livers of young "normal" C3H
mice, and multicolony formation and progression to carcinoma
Received 3/18/88: revised 6/20/88, 9/15/88; accepted 10/18/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' Supported by Grant-in-Aids for Cancer Research from the Ministry of
Education. Science, and Culture and the Ministry1of Health and Welfare of Japan.
2 To whom requests for reprints should be addressed.
'The abbreviations used are: C3H, C3H/HeNJclMTV* C57, C57BL/6NJc,:
PB, phénobarbital;MLEC. mouse liver epithelial cell; AFP. a-fetoprotein; G6P,
glucose-6-phosphatase: GGT, -y-glutamyl transpeptidase; MTV, mammary tumor
virus.
cells in response to treatment with the hepatopromoter phé
nobarbital. Hepatocytes from young C57BL mice, in contrast,
gave rise to only very few colonies, even with phénobarbital
promotion. The described system should have a potential use
fulness for future analysis, both quantitative and qualitative, of
precursor cells of spontaneous hepatomas, from the very early
phase of their development.
MATERIALS AND METHODS
Animals. Five week old male C3H and C57 mice were purchased
from CLEA Japan Inc., Tokyo. They were maintained in plastic cages
and given diet (CE-II, CLEA Japan Inc., Tokyo) and water freely until
use.
Preparation and Culture of Primary Hepatocytes. When mice reached
8 to 12 weeks of age, hepatocytes were isolated by a collagenase
perfusion technique essentially according to the method of Berry' and
Friend (7). Livers were perfused in situ via the portal vein initially with
75 ml of Hanks' solution (Ça2*.Mg2*-free, GIBCO, OH), then with
100 ml of Hanks' solution containing collagenase (Sigma, MO) at a
dose of 150 units/ml. The flow rate was 10 ml/min. Digested livers
were then resected and minced on a No. 121 steel mesh and isolated
liver cells were collected by low speed centrifugation (500 rpm). Viable
hepatocytes were further purified by an isodensity Percoli centrifugation
method (8) and plated onto 60-mm plastic dishes at a density of 2.5 or
3 x 10s hepatocytes per dish. The basal culture medium was Waymouth's (Flow Laboratory, UK) supplemented with 10% fetal calf
serum (GIBCO, NY) and 60 ¿jg/mlkanamicin. 2 h after the plating,
attachment of more than 80% of viable hepatocytes was checked by the
trypan blue exclusion test and the culture medium was renewed. From
the second day on, PB (Tokyo Chemical Industry Co., Tokyo, Japan)
was added at a concentration of 1.5 HIM to half of the dishes, the
remainder continuing on phenobarbital-free basal media. Medium was
changed twice a week and cultures maintained for 3 months.
Counting of Epithelial Cell Colonies. After 3 months, the number of
MLEC colonies larger than 1 mm in their largest diameter was counted
under phase-contrast light microscopy. Occasionally, colonies were also
counted in formalin-fixed and Giemsa-stained dishes by routine light
microscopy.
Histochemical Examinations. For immunohistochemical demonstra
tion of albumin or AFP in the cultured cells, cells were fixed in chilled
absolute ethanol (—20°C),
and then air dryed and permeabilized with
0.25% Triton X in phosphate buffered saline. The routine avidin-biotin
complex method (Vectastatin ABC Kit: Vetor Laboratories Inc., CA)
(9) was performed using sheep anti-mouse albumin antibodies (1:2000
dilution) or rabbit anti-rat AFP antibodies (1:500 dilution) purchased
from Inter-Cell Technologies Inc., NJ, and Nihon Biotest Laboratory
Inc., Tokyo, Japan, respectively. BALB 3T3 cells were used as negative
control material.
For enzyme-histochemical demonstration of G6P and GGT activities
in the cultured cells, the methods of Wachstein-Meisel (G6P) (10) and
Rutenberg (GGT) (11) were used.
Establishment of MLEC Lines. Cells of each colony were isolated by
cylinder, treated with l IHMEDTA in phosphate buffered saline and
then 0.25% trypsin solution (GIBCO, NY), detached by pipetting, and
subcultured with basal medium. If contamination with other types of
cells was apprehended, these cells surrounding MLEC colonies were
scraped off with a rubber policeman before the isolation procedure.
Subsequent subculture was usually performed once a week. In some
cases when the MLECs showed low proliferative potential, 20 ng/ml
403
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IMMORTAL EPITHELIAL CELLS OF C3H MOUSE LIVER
^^^^^^^
ÂhW*î>«S
mm&7?^^
*
v
Fig. 1. Phase contrast photomicrograph of four representative MLEC colonies, a, an early colony showing cord like arrangement; b, a colony forming a large
sheet, reminiscent of cultured normal hepatocytes; r. a colony comprised of relatively large cells; </.a colony of flat type cells, x ISO.
of epidermal growth factor (Collaborative Research, Inc., MA) were
added.
Ultrastructural Analysis. Cells were fixed in 2.5% glutaraldehyde in
0.1 M cacodylate buffer (pH 7.4, 4"C) in situ, postfixed in 2% osmium
tetroxide. After fixation, cells were dehydrated in a graded series of
alcohol and hydroxy propyl methacrylate and embedded in Epon 812.
Ultrathin sections were cut horizontally, stained with uranyl acetate,
and observed by routine electron microscopy.
Tumorigenicity Test. A total of IO7 cells of each MLEC line at the
5th or 10th passage were innoculated into the back subcutis of individ
ual athymic nude mice (ICR nu/nu, male, CLEA Japan Inc., Tokyo).
The innoculation sites were grossly examined once a week for up to 6
months.
RESULTS
Cell Types in Long-Term Culture of Primary C3H Mouse
Hepatocytes. The morphology of the cultured liver cells in the
first week was similar to that described in the literature (7, 12)
with the mature hepatocytes forming cobblestone-type sheets.
By virtue of the Percoli centrifugation, only very few nonparenchymal cells were present. After 3 or 4 days the hepatocytes
gradually elongated and became flattened in shape with a re
duction in cytoplasmic granularity. In the second week, the
hepatocytes began to rapidly disappear, so that no sheets of
hepatocytes were visible by the end of the 3rd week. Instead,
stellate or amoeba-like cells were apparent forming multiple
colonies in every dish independent of PB treatment. The phagocytic nature of this type of cell was proven by uptake of India
ink added to the medium. These phagocytic cells became most
predominant for a while. Within 2 months, however, most of
these phagocytic cells stopped proliferating and gradually de
generated. Active growth of fibroblastic cells was only occasion
ally seen in focal areas. Rapidly growing small epithelial cells
with a similar morphology to established rat liver epithelial
cells as described by Klanning et al. (12) were never encountered
under our culture conditions, although such cells did appear
within 2 weeks when the isodensity Percoli purification proce
dure was omitted. No apparent differences were evident between
PB-positive and -negative dishes in the early phase.
From the end of the 1st month, on the earliest occasion, and
more commonly during the 2nd month, colonies with distinct
cobblestone-type epithelial morphology began to appear in PBpositive dishes. The cells comprising these colonies had dark
granular cytoplasms and round to oval nuclei with one or two
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IMMORTAL EPITHELIAL CELLS OF C3H MOUSE LIVER
prominent nucleoli (Fig. 1, a-c) and the colonies themselves
were clearly demarcated by smoothly curved or occasionally
tooth edge-like borders and similar in morphology to the col
ony-forming altered-hepatocytes observed in culture of carcin
ogen-treated rat liver cells (2, 13). In addition, colonies of flat
epithelial cells with rather clear cytoplasm were also observed
in PB-negative dishes (Fig. Id), making up about half of the
total. The epithelial colonies, including those comprising flat
cells, were designated as mouse liver epithelial cell colonies.
Biochemical Characteristics of MLECs and Effect of PB. The
results of analysis for AFP, albumin, G6P and GGT expression
are summarized in Table 1. All of the MLEC colonies in PBtreated dishes were positive for AFP immunohistochemically,
about two-thirds of them being strongly stained (Fig. 2). On
the other hand, only half of the colonies in PB-negative dishes
were positive for AFP and staining was generally weak. Most
of the AFP-negative MLEC colonies were composed of the flatTable 1 Biochemical markers expressed in MLEC colonies in the presence or
absence of PB
Composite data of three different culture experiments using male C3H mouse
liver cells.
No. (%) of colonies relative stain
degreeMarkersAFPAlbuminG6PGGTPB
ing
+++
no.
of colonies59
40 (68)
1(6)+
10(15)
0(0)+++19(32)
9(47)54(81)
9(47)3(4)
1967
Fig. 4. Comparative demonstration of MLEC colonies in PB-treated dishes
(left) and PB-negative dishes (right) at the end of the 3rd month. A",fibroblastic
0(0)3(5)
0(0)6(5)
12(100)52
(95)
12(100)108(95)
1255
colonies. Giemsa stained.
Table 2 Frequency of MLEC colony formation from strain C3H hepatocytes
1211430
(weeks)
Culture
number12345678Mean
of animals
PB8
3(10)±~-0(0)
27 (90)Total
colonies/
colonies/
no.
dishes488/10 1O4hepatocytes1.63index4.53.85.510.04.95.03.86.35.5
+8
108/10268/10
0.360.89
70/10381/10
0.231.27
69/10301/10
0.231.25
30/8227/7
0.131.08
+9
+10
+10
+12
53/8261/952/9208/9 0.221.16
+12
0.230.92
+12
Fig. 2. Immunohistochemical
treated MLEC colony, x 150.
demonstration
55/9184/9
of AFP production in a PB-
0.240.82
++No.
29/9No.
0.131.13
±0.25°
±1.9
0.22 ±0.07Promotion
" Statistically significant by Welch's test as compared to value without PB (P
< 0.001).
±SDAge
f >
Fig. 3. Immunohistochemical
treated MLEC colony, x 300.
demonstration
of albumin production in PB-
type cells. Albumin production was observed in most (96%) of
the MLEC colonies in PB-treated dishes (Fig. 3). In contrast,
it was conspicuously lacking in all of the MLEC colonies in
PB-negative dishes. Generally, MLEC colonies were negative
for G6P and GGT. Occasionally, however, some PB-treated
colonies demonstrated a distinct G6P activity and a weak GGT
reaction was demonstrable in a small proportion of MLEC. In
general, the demonstrated enzyme phenotype was comparable
with that of carcinogen-induced altered mouse hepatocytes
observed in vivo (14-16).
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IMMORTAL EPITHELIAL CELLS OF C3H MOUSE LIVER
Table 3 Frequency ofMLEC colony formation from strain C57 hepatocytes
(weeks)
Culturenumber123Mean
of animals
PB12
colonies/
no. dishes1/5
colonies/
IO4hepatocytes0.007
2/52/5
0.0130.013
+12
+12
0/50/5
00
+!No.
1/5No.
±SDAge
0.0070.007
±0.005
0.007 ±0.005
Effect of PB on MLEC Formation and Strain Differences.
With the C3H strain, eight separate culture experiments were
performed. Representative Giemsa-stained dishes are illus
trated in Fig. 4 and the results are summarized in Table 2.
Under the promotion pressure of 1.5 HIMPB, mean number of
MLEC colonies was 1.13 ±0.25/104 hepatocytes (mean ±SD),
while without PB it was only 0.22 ±0.07/104 hepatocytes. The
mean promotion index, defined as the number of PB-treated
MLEC colonies/number of PB-untreated MLEC colonies, was
5.5 ±1.9 (mean ±SD), this value being similar to the promo-
tion index for PB estimated in an in vivo-in vitro hepatocarcinogenesis system for rats (2).
With strain C57, three separate experiments were performed
(see Table 3). MLEC colonies were very rare, even in the
presence of PB promotion pressure, as was also reportedly the
case for carcinogen-untreated normal rat hepatocytes (2, 13).
Establishment of MLEC Cell Lines. Cells from more than 20
MLEC colonies of both PB-positive and -negative dishes were
isolated when they reached more than 1 cm in diameter, and
subcultured repeatedly to give rise to cell lines. Most of the cell
lines are now at more than the 30th passage and are, therefore,
considered to be immortal. Epidermal growth factor apparently
enhanced the growths of all MLEC lines.
Ultrastructural Characteristics of MLEC Lines. Cells of six
MLEC lines were examined by electron microscopy. They all
had abundant cytoplasmic organellae including numerous mitochondriae, well- to moderately organized rough endoplastic
reticulum and Golgi complexes, and a large number of free
ribosomes or polysomes. The plasma membranes possessed
many well-developed microvilli. Junction complexes were also
observed between adjacent cells. These characteristics are all
reminiscent of cultured carcinogen-initiated hepatocytes (2,17).
Fig. 5. Representative electron photomi
crographs of two different MLEC lines (a and
b) showing abundant cytoplasmic organellae
including mitochondriae, rough endoplasmic
reticulum and free ribosomes. Numerous mi
crovilli are seen on the cell membranes, x
6000. (Insets, phase contrast light photomicro
graphs of the lines, x 150.)
406
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IMMORTAL EPITHELIAL
CELLS OF C3H MOUSE LIVER
Fig. 6. Budding figures of retrovirus-like particles in a MLEC line, x 48000.
Representative electron micrographs of two different cell lines
are shown in Fig. 5.
In two cell lines, virus-like particles, each other similar in
morphology in each case (Fig. 6), were detected. Budding
figures of the particles were indistinguishable from those of
type C retroviruses.
Tumorigenicity of MLEC Lines. Cells of five cell lines, four
derived from PB-positive dishes and one derived from a PBnegative dish, were tested for their tumorigenicity in nude mice.
Three animals were used for each cell line. Within 6 months of
the innoculation, two PB-treated cell lines gave rise to tumors,
histologically diagnosed as a trabecular hepatocellular carci
noma and an adenocarcinoma, respectively (Fig. 7). Albumin
and AFP production was detected immunohistochemically in
the former, but not in the latter.
DISCUSSION
The present investigation has shown that: (a) cells obtained
from normal C3H mouse liver give rise to epithelial cell colonies
in culture; (b) PB enhances their appearance or growth; (c)
colony cells are immortal and can give rise to transplantable
cell line with time; and (d) such colonies are only very rarely
obtained from liver cells of C57 mice.
Although the cells comprising different colonies did show a
degree of heterogeneity, they were generally considered to have
hepatocytic character on the basis of the following evidence: (a)
light microscopically, they presented as hepatocyte-like cells
forming cobblestone-type monolayers in vitro and trabecular
hepatocellular carcinoma in nude mice; (b) electron microscop
ically, abundant cytoplasmic organellae including mitochondria
were presented, simulating the appearance of carcinogen-initi
ated hepatocytes in culture; (c) production of albumin and/or
o-fetoprotein was demonstrated, and (d) they responded to PB
with regard to both growth and phenotypic expression. Al
though one of the inoculated cell lines in nude mice gave rise
to a tumor of adenocarcinoma pattern, not a typical trabecular
hepatocellular carcinoma, such variation in histológica! appear
ance is frequently encountered in experimental hepatocellular
carcinomas in rodents (18-20).
The striking strain difference between C3H and C57 mouse
regarding incidence of spontaneous hepatic tumors (1) was
clearly reflected in the differential ability of isolated cells to
form colonies in vitro, demonstrated by the present investiga
tion. It is therefore tempting to consider that the colony forming
cells obtained almost exclusively from C3H mouse liver may
be the precursors of spontaneous hepatocellular carcinomas,
i.e., spontaneously initiated cells in the two-step concept of
carcinogenesis (4-6), and that the PB administration-associated
increase in number of colonies may be directly analogous to its
promotion of hepatocarcinogenesis in vivo (21-23). This view
is further supported by the evidence that initiated hepatocytes
induced in the rat liver by brief administration of a carcinogen,
but not normal hepatocytes, are capable of growing under
culture conditions and progressing to transplantable carcinoma
cells with time, especially in the presence of PB (2, 3, 13).
Similar observations have also been made regarding in vivo-in
vitro investigations of brain and trachéaltumorigenesis (24,25).
If this assumption is correct, it indicates that the C3H mouse
already has a considerable number of spontaneously initiated
cells in the liver at the age of 8-12 weeks with a rate of at least
one initiated cell per IO4hepatocytes, calculated on the basis of
the present results. This frequency may not be unreasonably
•¿
I
f
•¿;<
»»I': *
i*:^
¿i, ¿g*
#>
t>r
•¿*>•
Fig. 7. a, trabecular carcinoma developed in a nude mouse 6 months after inoculation of cells of a MLEC line (x 300); h. immunohistochemica) demonstration of
albumin production in the same tumor shown in a (x 400); c. adenocarcinoma developed in a nude mouse with cells of another MLEC line {H&E, x 300).
407
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IMMORTAL
EPITHELIAL
CELLS
high when one considers the possibility that initiation may
occur during embryogenesis or in early postnatal life and that
the initiated cells may proliferate considerably with the growth
of the organ. Although we couldn't identify altered-cell foci in
the liver of untreated young adult C3H mice either histologically or histochemically, in accord with other investigators (26),
this is not unexpected since in mice even many spontaneous
hepatomas do not differ very distinctly from normal tissue (20)
and small foci are often difficult to detect reliably (27).
The mechanism of induction of spontaneous initiated cells
and hepatomas is completely obscure, although an influence of
genetic factors has been shown (28). MTV, a common passenger
virus in C3H mice, is not a causal factor since spontaneous
hepatomas develop similarly in MTV-free C3H mice (29). In
the present study, no B-type virus particles were apparent in
the MLEC lines. Expression of retrovirus sequences has been
noted in both spontaneous and carcinogen-induced hepatic
tumors of B6C3F| mice and suggested to have a causal rela
tionship with tumorigenesis (30). Although C-type virus parti
cles were present in two of the MLEC lines investigated here,
in agreement with a previous report (31 ), this was not a constant
feature.
Another possible causal factor might be contamination with
carcinogens or procarcinogens in the food. Indeed, the suscep
tibility of C3H and C57BL strains to chemical carcinogens has
been shown to parallel their spontaneous rate of liver tumor
development (26, 28, 32). Since the incidence of spontaneous
liver tumor was observed to be much higher in gnotobiots and
conventionalized C3H mice than in germ-free counterparts
(33), endogenous production of carcinogens, especially nitrosamines, by bacteria (34) might also have importance.
It should be mentioned that our assumption of MLEC colo
nies being progenies of spontaneously initiated hepatocytes
currently lacks direct evidence for support. One might point
out the possibility that transformation giving rise to immortal
cells and transplantable cells are events that all occurred in
culture. On the other hand some investigators may suggest that
the colony-forming cells seen in our experiment might be de
rived from putative stem cells thought to exist in the terminal
bile ductules (35). In fact it has been shown that chemically
transformed rat liver epithelial cell lines, originally having
phenotypic resemblance to the so-called oval cells which are
thought to be derived from stem cells after specific stimuli (36,
37), gave rise to a variety of tumors including hepatocellular
carcinomas, adenocarcinomas, bile duct carcinomas and carcinosarcomas when inoculated into syngeneic rats (38). Appar
ently further investigations are necessary to settle all these
arguments. Transplantation study, for example, with the use of
PB as a promoter may add more information about the biolog
ical and morphological natures of MLECs in vivo. Since point
mutation of c-H-ras oncogene at codon 61 has been detected at
high frequency in spontaneous hepatocellular tumors of
B6C3F| mice (39), analysis of oncogene changes in MLEC cells
may also bring about an advance in our knowledge.
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Immortal Epithelial Cells of Normal C3H Mouse Liver in Culture:
Possible Precursor Populations for Spontaneous Hepatocellular
Carcinoma
Gang-Hong Lee, Norimasa Sawada, Yohichi Mochizuki, et al.
Cancer Res 1989;49:403-409.
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