Full text of DOI:10.1097/01.LAB.0000017363.11489.AD

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LABORATORY INVESTIGATION
Vol. 82, No. 6, p. 781, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
In Vivo Cell Lineage Analysis During Chemical
Hepatocarcinogenesis in Rats Using
Retroviral-Mediated Gene Transfer: Evidence for
Dedifferentiation of Mature Hepatocytes
Jérôme Gournay, Isabelle Auvigne, Virginie Pichard, Catherine Ligeza,
Marie-Pierre Bralet, and Nicolas Ferry
Laboratoire de Thérapie Génique (JG, IA, VP, CL, NF), Hôtel-Dieu, and Unité d’Hépatologie (JG), Centre Hospitalier
Universitaire de Nantes, Nantes, and Département de Pathologie (M-PB), Hôpital Henri Mondor, AP-HP, Créteil, France
SUMMARY: Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte
proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship
between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using
retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation
was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically
label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the
␤-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling
of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent
administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the
second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we
observed clusters of ␤-galactosidase–positive hepatocytes, some of them also expressing preneoplastic markers such as
gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that
preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature
hepatocytes may occur during the course of carcinogenic regimen. (Lab Invest 2002, 82:781–788).
epatocellular carcinoma is a tumor with an
increasing incidence worldwide. Various rodent
suggested that oval cells may derive from bone mar-
row cells (Petersen et al, 1999). Because the appear-
ance of oval cells is one of the earliest cellular re-
sponses to most carcinogenic regimens, it has been
suggested that the oval cell population is derived from
remnant liver stem cells still present in the adult liver
(Petropoulos et al, 1985). This hypothesis is reinforced
by the fact that oval cells are pluripotent, with the
ability to differentiate into both hepatocytes (Coleman
et al, 1993; Evarts et al, 1987) and bile duct cells (Lenzi
et al, 1992). Thus, oval cells may be directly involved in
the development of hepatocellular carcinoma by
blockade of their maturation (Sell and Dunsford, 1989).
The alternate hypothesis for liver tumor genesis is
based on dedifferentiation of mature hepatocytes,
which may account for tumor development without
the involvement of other cell types (Farber, 1984).
According to this latter model, cancer results from a
multistep process. Chemical carcinogens first act by
modifying DNA to form DNA adducts during the first
step of initiation. In the second step of carcinogenesis,
often referred to as promotion, other noncarcinogenic
compounds increase hepatocarcinogenic effects,
most of them being able to induce cellular prolifera-
tion. Arguments for this latter hypothesis are the
findings that exposure of rats to carcinogens results in
H
models of chemically induced liver tumors are cur-
rently used to study molecular and cellular events
during hepatocarcinogenesis. Many of these models
are characterized by the proliferation of unusual epi-
thelial cells called “oval cells” (Farber, 1956). For
example, administration of 2-acetylaminofluorene (2-
AAF) together with partial hepatectomy results in
suppression of hepatocyte proliferation and stimula-
tion of oval cell proliferation (Solt and Farber, 1976).
Oval cells arise in the periportal region of the liver
probably from cells of the Hering canal (Grisham and
Porta, 1964; Novikoff et al, 1991), from bile ducts
(Sarraf et al, 1994), from intraportal or periportal
ductules (Petersen et al, 1997), or from periductal cells
(Sell and Salman, 1984). Recently, it has also been
DOI: 10.1097/01.LAB.0000017363.11489.AD
Received March 12, 2002.
Supported by grants from the Association pour la Recherche contre le
Cancer, the Fondation pour la Recherche Médicale, and the Ligue dépar-
tementale contre le Cancer.
Address reprint requests to: Dr. Nicolas Ferry, Laboratoire de Thérapie
Génique, Hôtel Dieu, Centre Hospitalier Universitaire de Nantes, 44035
Nantes cedex 01, France. E-mail: nferry@sante.univ-nantes.fr
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Gournay et al
sequential liver alterations as follows: (a) focal prolif-
eration of altered hepatocytes, (b) appearance of
preneoplastic nodules, and (c) rise of cancer from
persistent nodules (Farber and Sarma, 1987).
differentiation of ␤-galactosidase–positive cells were
determined.
Results
Many studies have been performed in an attempt to
validate one of these two hypotheses by analyzing the
fate of different cell types in the liver during early and
late stages of carcinogenesis. Such cell lineage stud-
ies used various strategies: morphologic examination
(Dunsford et al, 1985; Grisham and Hartroft, 1961),
use of metabolic and phenotypic markers (Dempo et
al, 1975; Dunsford and Sell, 1989; Golding et al, 1995;
Hixson et al, 1990; Sell, 1978; Thompson-Hayner et al,
1984), analysis of transgenic mice models (Bennoun et
al, 1993; Schirmacher et al, 1991), and labeling of
dividing cells with [3H]thymidine (Evarts et al, 1987;
Sell and Salman, 1984). Also, cell lineage of oval cells
was studied after ex vivo labeling of oval cells that
were subsequently reimplanted into recipient animals
(Coleman et al, 1993; Dabeva et al, 1997). Most of the
studies of oval cells strengthened the hypothesis that
these cells have a potential role as a “facultative stem
cell” and serve as progenitors for hepatocytes, al-
though contradictory results were also obtained by
some authors (Gerlyng et al, 1994; Lenzi et al, 1992;
Tatematsu et al, 1984). However, it was suggested
that oval cell proliferation and development of hepa-
tocellular tumors are independent and result from
activation of a different cell lineage (Anilkumar et al,
1995; Steinberg et al, 1991; Tarsetti et al, 1993).
Genetic labeling represents an attractive method to
study in vivo cell lineage. Indeed, retroviral vectors
meet many requirements for cell lineage analysis (Bra-
let et al, 1994; Lemischka, 1993). First, infection with
recombinant retroviruses results in stable integration
of the marker gene in the infected cell. Such integra-
tion leads to replication of the transgene along with
the cellular genome and therefore to transmission of
the label to the progeny of one single cell. Second, the
expression of the marker gene is restricted to the cells
that have integrated the transgene. Third, the use of
A first series of animals (Group 1) received 2-AAF and
were subjected to a two-thirds hepatectomy at Day 7
according to the protocol described in Figure 1. As
expected, we observed oval cells as early as 4 days after
the two-thirds hepatectomy. The proliferation of oval
cells was initially localized around the portal tract and
subsequently invaded the hepatic lobule. According to
previous studies, we observed that the peak of oval cell
division occurred 7 days after partial hepatectomy
(Evarts et al, 1987). Because retroviral vectors are only
able to infect dividing cells (Hajihosseini et al, 1993; Roe
et al, 1993), they were administered to the animals at that
time, either via the common bile duct or via a peripheral
vein to label oval cells. We analyzed the number and the
type of ␤-galactosidase–positive cells in various sections
obtained from different lobes in animals killed 1 and 2
weeks after gene transfer (ie, 3 and 4 weeks after the
beginning of 2-AAF feeding). In animals injected via a
peripheral vein, we observed only ␤-galactosidase–pos-
itive hepatocytes by 5-bromo-4-chloro-3-indolyl-␤-D-
galactopyranoside (X-Gal) staining of cryostat sections
(Fig. 2A) or by immunohistochemistry (Fig. 2B). These
positive hepatocytes were sometimes arranged in small
clusters (up to 24 cells) in the livers of 4 of 5 animals. No
␤-galactosidase–positive oval cells were detected in the
livers of these animals. In contrast, after delivery of
a
marker gene, such as the Escherichia coli
␤-galactosidase coupled to a nuclear localization sig-
nal (Kalderon et al, 1984), allows precise immunohis-
tochemical or histochemical detection of the labeled
cells.
In the present report, our goal was to gain further
insights into the relationship between preneoplastic
foci and hepatocytes in the livers of rats treated with
sustained administration of 2-AAF using in vivo ge-
netic labeling. For this purpose, we performed
retroviral-mediated genetic labeling of cells in two
groups of rats: in the first group, genetic labeling was
performed after the introduction of the carcinogenic
regimen to target cells induced to proliferate by such
regimen. In the second group, genetic labeling was
performed after partial hepatectomy before introduc-
tion of the carcinogenic regimen to specifically label
mature hepatocytes. In both groups, animals were
killed at the end of 2-AAF administration and the
presence, the distribution, the type, and the level of
Figure 1.
Experimental procedures for the two groups of rats included in the study. In
Group 1, partial hepatectomy was performed 1 week after the onset of
2-acetylaminofluorene (2-AAF) feeding. Injection of retroviral vectors (Inj) was
performed either through the bile duct or intravenously 1 week after partial
hepatectomy. In Group 2, animals were hepatectomized and injected intrave-
nously with retroviral vectors 1 week before the beginning of 2-AAF adminis-
tration. Cyclophosphamide was injected at the time of virus administration (see
“Materials and Methods”). A liver biopsy (B) was performed 1 week after virus
injection at the start of 2-AAF regimen. Inj ϭ injection of retrovirus; 2/3 H ϭ
two-thirds partial hepatectomy; S ϭ sacrifice; B ϭ liver biopsy.
782 Laboratory Investigation • June 2002 • Volume 82 • Number 6

In Vivo Cell Lineage in Rat Liver Carcinogenesis
(100 mg/kg intravenously) 1 hour before the first
injection of virus to decrease the immune response
observed after systemic high-titer retroviral vector
delivery (Izembart et al, 1999). Indeed, it has been
demonstrated that this treatment allowed prolonged
expression of ␤-galactosidase in the liver after
adenoviral-mediated gene transfer (Jooss et al, 1996).
Seven days after retrovirus delivery, after completion
of liver regeneration, rats were fed with 2-AAF and
sequential biopsy specimens were obtained to detect
the presence of ␤-galactosidase–positive hepatocytes
(Fig. 1).
One week after the injection of ␤-galactosidase
retroviral vectors, immunohistochemical analysis of
liver biopsy specimens revealed the presence of an
average of 25% ␤-galactosidase–positive hepato-
cytes, indicating that mature hepatocytes had been
successfully infected (Fig. 3A). This figure is in keeping
with our previous studies of liver gene transfer using
high-titer retroviral vectors (Kitten et al, 1997). We
never detected other cell types that were positive for
␤-galactosidase, indicating that labeling was hepato-
Figure 2.
Histochemical and immunohistochemical analysis of livers in Group 1 animals.
Rats were fed 2-AAF and injected with retroviral vectors either via the portal
vein (A and B) or the bile duct (C and D). The presence of labeled cells was
analyzed by 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside staining on
cryostat sections (A and C) or by immunohistochemical detection of
␤-galactosidase on paraffin sections (B and D). Both techniques revealed the
presence of labeled hepatocytes (A and B) as well as labeled oval cells (C and
D). Magnification, ϫ1000.
retroviral vectors via the common bile duct, we observed
the presence of ␤-galactosidase–positive oval cells near
the portal tract either by histochemistry (Fig. 2C) or
immunohistochemistry (Fig. 2D). However, ␤-galactosi-
dase–labeled hepatocytes were always present along
with ␤-galactosidase–positive oval cells in these ani-
mals. At that time, we also observed hepatocyte mitosis,
indicating that 2-AAF had not completely blocked hepa-
tocyte regeneration and had allowed retroviral-mediated
gene transfer in hepatocytes.
To specifically label one cell population, we per-
formed prelabeling of mature hepatocytes. To this
end, a second series of rats (Group 2) were submitted
to a two-thirds partial hepatectomy and received
␤-galactosidase retroviral vectors by systemic injec-
tion 22 and 27 hours later. This protocol allows spe-
cific labeling of hepatocytes, which divide 24 hours
after partial hepatectomy, whereas nonparenchymal
cells divide later (Fabrikant, 1968; Grisham, 1962).
Because retroviral vectors only infect dividing cells,
hepatocytes are specifically labeled using this ap-
proach. Moreover, rats received cyclophosphamide
Figure 3.
Immunohistochemical analysis of liver in Group 2 rats. A, Liver biopsy
specimen obtained from a prelabeled rat, before administration of 2-AAF. Many
␤-galactosidase–positive hepatocytes are scattered throughout the lobule.
Hematoxylin counterstained. (Magnification, ϫ200). B and C, Serial sections of
a liver nodule within the hepatic lobule surrounded with oval cells (B). All
hepatocytes in the nodule are positive for ␤-galactosidase (C). (Magnification,
ϫ100). D, ␤-galactosidase–positive hepatocytes completely surrounded by
oval cells in liver specimen obtained at the time of death, 6 weeks after the
beginning of the carcinogenic regimen. (Magnification, ϫ200).
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Gournay et al
cyte specific. Rats were killed at 5, 6, and 10 weeks
after the beginning of 2-AAF administration. At the
time of death, we observed the presence of clusters of
hepatocytes, organized in foci within the hepatic lob-
ule and surrounded with oval cells (Fig. 3B). Some of
these clusters were also positive for ␤-galactosidase
by immunohistochemistry (Fig. 3C). Such large foci of
␤-galactosidase–positive hepatocytes indicated that
mature hepatocytes had divided during the course of
the 2-AAF regimen. The mean number of ␤-galactosi-
dase–positive foci was 3.6 Ϯ 3.2 (mean Ϯ SD) per cm2
of examined liver sections. As expected, we never
observed ␤-galactosidase–positive oval cells. We also
observed isolated ␤-galactosidase–positive hepato-
cytes that were sometimes completely surrounded
with oval cells (Fig. 3D), suggesting that not all hepa-
tocytes had divided.
To evaluate the state of differentiation of hepato-
cytes, we assessed the presence of gamma-glutamyl
transpeptidase (GGT) and of the placental form of
glutathione-S-transferase (GSTp) using immunohisto-
chemistry. Clusters of GGT- and GSTp-positive hepa-
tocytes were detected as early as 6 weeks after the
beginning of 2-AAF treatment. The cluster size varied
amongst experimental animals. The mean number of
GSTp-positive clusters recorded in more than 8 cm2 of
liver sections in the experimental animals was 12.6 Ϯ
10.1 per cm2 of examined liver sections.
than the proportion of initially labeled hepatocytes
(25%; Fig. 3A). Finally 80% of ␤-galactosidase–posi-
tive hepatocyte clusters were negative for GSTp and
GGT, suggesting that proliferation of mature normal
hepatocytes also occurred.
Discussion
In this study we analyzed cell lineage during a modi-
fied Solt-Farber regimen using in vivo retroviral-
mediated gene transfer. We demonstrated that prolif-
erating oval cells could be transduced with high-titer
recombinant retroviral vectors delivered via the biliary
tract but not via a peripheral vein. However, concom-
itant labeling of proliferating hepatocytes did not allow
us to study the fate of a specific cell population. In
contrast, genetic labeling of mature hepatocytes be-
fore administration of 2-AAF clearly demonstrated that
mature hepatocytes were able to dedifferentiate and
re-express GSTp and GGT. This demonstrates that
preneoplastic foci may arise from mature hepatocytes.
During the past years, many studies have ad-
dressed the lineage relationship between oval cells
and liver tumors during the course of carcinogenic
regimens. However, although evidence for a lineage
relationship between oval cells and mature hepato-
cytes as well as biliary cells has been provided, the
actual role of oval cells in the development of liver
tumors still remains obscure. Previous studies sug-
gested that oval cell proliferation and development of
hepatocellular tumors occur independently and result
from activation of different cell lineages (Anilkumar et
al, 1995; Steinberg et al, 1991; Tarsetti et al, 1993). To
document this hypothesis, we studied cell lineage
using genetic labeling of cells in a modified Solt-
Farber regimen. Previously, the use of tritiated thymi-
dine as a marker to follow the fate of labeled oval cells
yielded contradictory results (Evarts et al, 1987; Ger-
lyng et al, 1994). Moreover, reutilization of the marker
We analyzed the expression of ␤-galactosidase as
well as GGT and GSTp in serial sections of the same
foci. In most cases we found foci of GGT- and
GSTp-positive hepatocytes that were negative for
␤-galactosidase. However we also detected foci of
hepatocytes that were positive for ␤-galactosidase
(Fig. 4A) as well as GSTp (Fig. 4B) and GGT (Fig. 4C).
This clearly indicated that mature hepatocytes were
able to give rise to preneoplastic foci. We determined
that 3.9% of the GSTp-positive cluster was also
positive for ␤-galactosidase. This proportion is lower
Figure 4.
Immunohistochemical detection of ␤-galactosidase and preneoplastic markers in prelabeled rats. Serial sections of the liver obtained in a prelabeled rat at the end
of carcinogen administration showing a ␤-galactosidase–positive cluster (A), also positive for the placental form of glutathione-S-transferase (B) and gamma-glutamyl
transpeptidase (C). Hematoxylin counterstained. (Magnification, ϫ400).
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In Vivo Cell Lineage in Rat Liver Carcinogenesis
after cell death may occur when using tritiated thymi-
dine (Heiniger et al, 1971). Stable genetic labeling is a
powerful tool to study cell lineage, and we reasoned
that retroviral vectors, which are able to integrate the
marker gene in the infected cell’s genome, might
achieve such a goal. Indeed we previously studied the
fate of normal hepatocytes in long-term studies using
this strategy (Bralet et al, 1994). However, in a previ-
ous attempt to study cell lineage in two carcinogenic
models, we failed to infect oval cells with retroviral
vectors administered via the portal vein (Bralet et al,
1996; Kitten and Ferry, 1998).
Our present results demonstrated that preneoplastic
foci can arise from differentiated hepatocytes. Indeed we
observed hepatocyte foci that were positive for
␤-galactosidase as well as GSTp and GGT. In the normal
rat liver, GGT expression is restricted to bile duct cells,
and there is no expression of GSTp. These two enzymes
are commonly used markers of preneoplastic liver le-
sions and reveal dedifferentiation of hepatocytes (Tate-
matsu et al, 1985). Because only mature hepatocytes
expressed ␤-galactosidase before administration of
2-AAF, the presence of ␤-galactosidase–positive hepa-
tocytes that also expressed GGT and GSTp after 2-AAF
administration could only be explained by dedifferentia-
tion of mature cells. If all foci were derived from mature
hepatocytes, we should observe the same proportion of
foci expressing ␤-galactosidase as that of hepatocytes
expressing ␤-galactosidase before 2-AAF administra-
tion. Therefore, because the proportion of labeled hepa-
tocytes before administration of 2-AAF was 25%, we
expected that up to 25% of GSTp clones would express
␤-galactosidase. In contrast, we observed that the ma-
jority of GSTp foci were negative for ␤-galactosidase,
and only 4% of GSTp foci were ␤-galactosidase posi-
tive. Many factors may explain this discrepancy: (a) The
immune response could have eliminated ␤-galacto-
sidase clones; (b) ␤-galactosidase expression may be
switched off in some clones after inactivation of the
promoter after cell division, as has been demonstrated in
other tissues (Chen et al, 1997); (c) Finally, we cannot
exclude the possibility that oval cells may contribute to
the generation of some preneoplastic foci either directly
or by differentiation of oval cells in hepatocytes that will
subsequently dedifferentiate and give rise to foci. Obvi-
ously those preneoplastic foci deriving from oval cells
would not be prelabeled and express ␤-galactosidase.
Nevertheless, on a qualitative basis, our results are
consistent with the existence of a direct lineage between
mature hepatocytes and preneoplastic foci. Therefore,
our data agree with the hypothesis proposed by Farber
(1984) that dedifferentiation of mature hepatocytes oc-
curs during carcinogen administration.
Whether mature hepatocytes are directly involved in
tumor appearance could not be answered in the
present study. Indeed, remodeling of nodules resulting
in hepatocyte redifferentiation has long been de-
scribed (Tatematsu et al, 1983). According to this
hypothesis, we found here that most of the ␤-galac-
tosidase–positive foci were negative for GSTp. We
had observed similar ␤-galactosidase foci of appar-
ently normal hepatocytes in a previous study (Bralet et
al, 1996). This suggests that either normal mature
hepatocytes divide during exposure to 2-AAF or that
most preneoplastic foci arising from hepatocytes can
remodel. Long-term studies are therefore needed to
better clarify this issue and document the contribution
of redifferentiation or remodeling to carcinogenesis.
Finally, we observed that ␤-galactosidase–positive
hepatocytes may be completely surrounded with oval
cells. This also demonstrated that prelabeled hepato-
cytes may survive as single cells and that proliferation
of oval cells may surround preexisting hepatocytes
and exclude them from the lobule. Therefore no lin-
In the present study we report the successful trans-
fer and expression of a marker gene to oval cells
during the course of 2-AAF administration. ␤-galacto-
sidase–positive oval cells were present only when
high-titer retroviral solution was administered via the
common bile duct. However, despite the infusion of
retrovirus solution containing viral titers between 5 ϫ
107 and 5 ϫ 108 focus forming units/ml, we did not
observe a high number of ␤-galactosidase–positive
oval cells. Such low infectivity of oval cells and the
absence of labeled oval cells when retroviral vectors
were delivered intravenously may be related to the
presence of a basal membrane, which is known to
impair infection of cells with recombinant viral vectors
(Huard et al, 1996). In addition, we were unable to
specifically label oval cells, and we always concomi-
tantly labeled hepatocytes together with oval cells
after bile duct delivery of the vectors. Hepatocyte
mitosis was present at the time of injection, and this
may explain the presence of labeled hepatocytes in
our conditions. Indeed, biliary delivery of retroviral
vectors was found to be very effective to transduce
regenerating hepatocytes in a recent study (De Godoy
et al, 1999). This also demonstrated that, as previously
observed, 2-AAF is not able to completely block
hepatocyte division (Laws et al, 1952). Therefore la-
beled hepatocytes present 2 weeks after retroviral
injection might be derived either from labeled hepato-
cytes or from labeled oval cells that are known to be
able to differentiate into hepatocytes (Coleman et al,
1993; Evarts et al, 1987). It was therefore not possible
to specifically follow the fate of a particular cell type in
the liver using this labeling strategy.
To circumvent this drawback, we performed hepa-
tocyte prelabeling before administration of 2-AAF. To
this end retroviral vectors were injected at the peak of
hepatocyte division after a two-thirds hepatectomy.
Oval cells are not involved in this regenerative pro-
cess, which results in specific labeling of mature
hepatocytes. Injection of a single dose of cyclophos-
phamide was also performed to decrease immune
responses against ␤-galactosidase–positive cells as
previously demonstrated (Jooss et al, 1996). After
recovery from hepatectomy, animals harboring prela-
beled hepatocytes were treated with 2-AAF. We pre-
viously used this prelabeling strategy to analyze the
effect of galactosamine on mature hepatocytes and to
demonstrate that mature hepatocytes were able to
re-express GGT after galactosamine administration
(Kitten and Ferry, 1998).
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Gournay et al
eage relationship should be deduced from the contact
between hepatocytes and oval cells. In conclusion,
our present findings demonstrate the presence of a
direct lineage between hepatocytes and preneoplastic
foci in a modified Solt-Farber regimen and support the
view that the dedifferentiation hypothesis of liver can-
cer is still viable.
foci multiplied by the dilution factor. After concentra-
tion the titer of the viral stocks routinely reached 5 ϫ
107 to 5 ϫ 108 focus forming units/ml.
In Vivo Genetic Cell Labeling
Animals were divided in two groups (Fig. 1). In Group
1, genetic labeling was performed after the introduc-
tion of 2-AAF to target proliferating cells, mainly oval
cells, induced by such regimen. In Group 2, genetic
labeling was performed after a two-thirds hepatec-
tomy performed 1 week before introduction of 2-AAF
to target dividing hepatocytes.
In Group 1 (direct labeling), 2-AAF feeding was
started at Day 0, 7 days before a two-thirds hepatec-
tomy. Analysis of proliferating cells revealed that the
peak of oval cell division occurred at Day 7 after partial
hepatectomy. Therefore, because retroviral vectors
are only able to infect dividing cells (Hajihosseini et al,
1993; Roe et al, 1993), 7 days after two-thirds hepa-
tectomy (ie, at Day 14), the solution containing the
retrovirus (1 ml) was administered either intravenously
(in the dorsal vein of the penis) or into the common bile
duct to label preferentially oval cells. Rats were killed
1 or to 2 weeks later.
In Group 2 (prelabeled group), a two-thirds hepa-
tectomy was performed 1 week before 2-AAF admin-
istration. Recombinant retroviruses (1 ml) were in-
jected twice intravenously (in the dorsal vein of the
penis) at 22 and 27 hours after the hepatectomy. To
circumvent rapid immune elimination of ␤-galactosi-
dase–expressing cells, cyclophosphamide (100
mg/kg body weight) was injected intravenously imme-
diately before virus administration. At Day 7 after virus
injection (ie, after completion of liver regeneration), the
2-AAF regimen was started and rats were killed 5, 6, or
10 weeks later.
Materials and Methods
Animals and Surgical Procedures
Adult male Fischer rats, weighing 160 to 180 gm, were
used in this study. The rats were maintained under a
12-hour light/dark illumination cycle and received food
and drinking water ad libitum throughout the experi-
mental period. 2-AAF was incorporated into their
normal diet at 0.02% (w/w). All surgical procedures
were conducted on deeply anesthetized animals ac-
cording to the guidelines of the French Ministry of
Agriculture. Anesthesia was based on the use of
ketamine (50 mg/kg) combined with pentobarbital (15
mg/kg). Two-thirds hepatectomies were performed
according to the procedure of Higgins and Anderson
(1931).
Liver biopsy specimens were obtained from the
right lateral lobe after laparotomy. Each biopsy spec-
imen was cut into two fragments. One was snap-
frozen in dry ice-cold isopentane for cryostat section-
ing and ␤-galactosidase staining. The other was
immersed in formalin and paraffin embedded for rou-
tine histology and immunohistochemistry.
Rats were killed by injection of a lethal dose of
pentobarbital. The livers were washed in situ by per-
fusion of PBS through the portal vein. After washing,
fixation was obtained by perfusion with a 4% parafor-
maldehyde solution prepared in PBS. The livers were
removed and cut into 5-mm thick blocks. Some tissue
blocks were either snap-frozen in dry ice-cold isopen-
tane for cryostat sectioning or immediately processed
for ␤-galactosidase staining of whole fragment. The
other blocks were immersed in formalin and paraffin
embedded for immunohistochemistry.
Histology
Routine histology was performed on formalin-fixed/
paraffin-embedded sections.
Histochemical X-Gal Staining
Cell Culture and Virus Preparation
Beta-galactosidase activity was assessed on 10-␮m
cryostat sections. Sections were incubated for 6 hours
at 32° C in PBS containing 0.4 mg/ml X-Gal, 4 mM
potassium ferricyanide, 4 mM potassium ferrocyanide,
and 2 mM MgCl2. Sections were subsequently coun-
terstained with hematoxylin and eosin.
We used amphotropic recombinant retroviral vectors
containing the E. coli ␤-galactosidase gene coupled to
the nuclear localizing signal from an SV 40 large T
antigen (nls-lacZ gene) and produced by the TELCeB6
AF7-producing cell line (Cosset et al, 1995). Cells were
cultured at 37° C in DMEM containing 10% fetal calf
serum, 100 U/ml penicillin, and 100 mg/ml streptomy-
cin. Once the cells reached confluence, the medium
was harvested every day and filtered through a
0.45-␮m filter. The medium was used after concentra-
tion via tangential ultra filtration to achieve a higher
titer as previously described (Kitten et al, 1997).
The retroviral titer was determined by infection of Te
671 cells in 6-well dishes with 1 ml of serial dilutions of
the retrovirus-containing supernatant. The cells were
stained 48 hours later with X-Gal, and the titer was
defined as the number of ␤-galactosidase–positive
Immunohistochemistry
Formalin-fixed/paraffin-embedded sections (5 ␮m)
were deparaffinized, and endogenous peroxidase ac-
tivity was blocked by incubation for 30 minutes in a
3% H2O2 solution in PBS. Diluted primary antibodies
were applied either overnight at 4° C (diluted 1:1000
for the ␤-galactosidase antibody) or for 2 hours at
room temperature (diluted 1:150 for GGT; diluted 1:20
for GSTp). Dilutions were performed in PBS containing
bovine serum albumin (2% w/v) and Tween 20 (0.1%
786 Laboratory Investigation • June 2002 • Volume 82 • Number 6

In Vivo Cell Lineage in Rat Liver Carcinogenesis
injection of bile ducts with a pigmented barium gelatin
medium. Am J Pathol 118:218–224.
v/v). Positive cells were revealed with biotinylated
anti-mouse and anti-rabbit immunoglobulin and
streptavidin-peroxidase complex using amino-ethyl
carbazole as a chromogenic substrate. Slides were
counterstained with hematoxylin, and the number and
size of the positive clusters were recorded optically at
ϫ10 magnification.
Dunsford H and Sell S (1989). Production of monoclonal
antibodies to preneoplastic liver cell populations induced by
chemical carcinogens in rats and to transplantable morris
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Evarts RP, Nagy P, Marsden E, and Thorgeirsson SS (1987).
A precursor-product relationship exists between oval cells
and hepatocytes in rat liver. Carcinogenesis 8:1737–1740.
Acknowledgements
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The authors thank Dr. Yannick Laperche for the gift
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