| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Tumor Biology |
1
Departments of Pathology [N. F.] and Molecular and Cellular Biology Program [M. E. V.], University of Washington, Seattle, Washington 98195-7610, and Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 [R. H. P.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
develop liver tumors between 12 and 15 months of age. Tumor development
is preceded by an overall increase in the rates of hepatocyte
proliferation and cell death. To examine the role of apoptosis in the
development of TGF--induced liver tumors, we generated TGF-/Bcl-2
double transgenic mice by crossing TGF- transgenic mice with Bcl-2
transgenic mice expressing a zinc-inducible Bcl-2 transgene.
Overexpression of the Bcl-2 transgene protected hepatocytes from
Fas-mediated apoptosis. We anticipated that hepatocytes in
TGF-/Bcl-2 double transgenic mice would be stimulated to proliferate
but would fail to undergo apoptosis, leading to increased liver weights
and accelerated tumorigenesis. At 4 weeks of age, both TGF- single
transgenic and TGF-/Bcl-2 double transgenic mice had elevated
hepatocyte proliferation and increased liver:body weight ratios.
However, by 8 months, the liver:body weight ratios had normalized in
both TGF- single transgenic and TGF-/Bcl-2 double transgenic
mice. Furthermore, Bcl-2 functioned as a tumor suppressor,
significantly decreasing the frequency and delaying the development of
TGF--induced liver tumors, despite having comparable levels
of TGF- transgene expression in both single and double transgenic
mice. Between 11 and 12 months of age, >80% of the TGF- single
transgenic mice had developed tumors, whereas only 54% of the double
transgenic mice had developed tumors after 13 months of age. The tumors
that eventually developed in the TGF-/Bcl-2 double transgenic mice
were histologically distinct and smaller in size and had lower
hepatocyte mitotic activity than tumors from TGF- single transgenic
mice. Furthermore, delaying Bcl-2 expression until 8.5 months of age
was sufficient to inhibit TGF--induced tumorigenesis. These results
indicate that Bcl-2 inhibits tumor progression in the liver, possibly
by interfering with hepatocyte proliferation. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Apoptosis occurs naturally in the liver, albeit at a very low rate (4 , 5) . Normal hepatocytes are sensitive to apoptosis induced by Fas agonist antibodies and by TNF given in conjunction with a transcriptional inhibitor (6 , 7) . Both Bcl-2 and Bcl-XL are expressed in the liver and may repress apoptosis through a common pathway (8) . Bcl-2 is normally expressed in bile duct cells but not in hepatocytes; however, its de novo expression, both in cultured cell lines and in transgenic mice, protects these cells against apoptosis (9 , 10) . The exact role of apoptosis in the development of HCC has not been fully elucidated. In studies of rat liver carcinogenesis, Grasl-Kraupp et al. (11) demonstrated that although the overall rate of hepatocyte proliferation increased during tumor development, so did the rate of apoptosis. This, and other works (3 , 12) , demonstrated that proliferation and apoptosis are linked and that the ratio between the two, rather than the absolute levels of proliferation and apoptosis, is important in liver tumorigenesis.
The development of HCC has been studied extensively in transgenic mice
overexpressing TGF- either as a single gene (13, 14, 15, 16, 17, 18)
or
in double transgenic mice that overexpress both TGF- and c-myc
(19
, 20)
. We have examined the pathogenesis of HCC in
transgenic mice that overexpress TGF- under the control of the MT1
promoter. In this model, 75–80% of male mice develop HCC after 12
months of age (13, 14, 15)
. In these animals, hepatocyte
proliferation is 3–4-fold higher than that in nontransgenic mice
during the first 2 months of life, and the liver:body weight ratio is
elevated. As the animals age, hepatocyte proliferation remains high,
but the liver:body weight ratio becomes similar to that of WT animals
in which hepatocytes are essentially quiescent. The increase in
hepatocyte proliferation in TGF- transgenic mice appears to be
compensated for by the increased rate of hepatocyte turnover that
occurs in these animals (16)
. Thus, liver mass homeostasis
is maintained for many months, presumably until a loss of the balance
between cell proliferation and apoptosis leads to tumor development.
Because cell proliferation and apoptosis appear to be linked during HCC
development, an analysis of the role of apoptosis in
hepatocarcinogenesis requires a system in which apoptosis can be
regulated independently of cell proliferation. To determine whether
prevention of apoptosis would alter tumor development in mouse livers,
we generated double transgenic mice that overexpress TGF- and Bcl-2.
Whereas both transgenes are driven by the MT1 promoter, the TGF-
gene construct is "leaky" (that is, it has a relatively high level
of expression in the absence of an inducer), whereas Bcl-2 expression
depends on induction by Zn or Cd. This feature provided the opportunity
to differentially regulate Bcl-2 in animals expressing TGF-. We
postulated that the antiapoptotic effect of Bcl-2 would cause liver
enlargement and accelerated tumorigenesis in TGF-/Bcl-2 double
transgenic mice. Instead, we found that tumor development in these
animals was delayed and decreased in comparison with TGF- single
transgenic mice or TGF-/Bcl-2 double transgenic mice in which Bcl-2
expression was not induced. Coexpression of TGF- and Bcl-2 also had
a striking effect on tumor cell proliferation, size, and morphology.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
transgenic mice of the MT42 line has been
reported previously (13)
. Heterozygous human Bcl-2
transgenic mice with the Bcl-2 transgene under the control of the mouse
MT1 promoter on a C57B6C3H background were provided by Dr. Stanley
Korsmeyer (Dana Farber Cancer Institute, Harvard Medical School,
Boston, MA 02115). Homozygous TGF- transgenic mice were crossed with
WT CD1 mice to generate TGF- heterozygotes. Female heterozygous
TGF- transgenic mice were mated to male heterozygous Bcl-2 mice to
generate the WT, TGF-, and Bcl-2 single transgenic and TGF-/Bcl-2
double transgenic mice, all in a hybrid (CD1 x C57/B6C3H) background. Genotypes were determined by PCR amplification
of genomic DNA obtained from mouse tails. To fully induce transgene
expression, mice were maintained on 25 mM
ZnSO4 drinking water (zinc water) from the time
of weaning (3 weeks of age). In addition to the mice that were
maintained continuously on zinc water, one group received water without
zinc addition for the duration of the study, and another group received
zinc water from 8.5 to 12.5 months. Between 25 and 30
F1 male mice from each genotype were used for the
long-term tumor studies. All animals were maintained and cared for in
accordance with NIH guidelines for animal care.
Histology, IHC, and ISH.
Livers were examined, and part of the tissue was fixed either in
neutral buffered formalin for 24 h for routine histology, IHC, and
ISH or in methacarne (70% methanol, 20% chloroform and 10% acetic
acid) for 1 h and transferred to 100% methanol for 24 h for
BrdUrd IHC. After fixation, tissue was embedded in paraffin. For
routine histological analysis, 5-µm sections were cut from
paraffin-embedded blocks and stained with H&E.
For Bcl-2 IHC, sections were deparaffinized, rehydrated, and treated for 30 min with 0.3% H2O2 to inactivate endogenous peroxidase activity. Sections were microwaved for 10 min in 10 mM sodium citrate buffer (pH 6.0) before incubation in primary antibody (mouse antihuman Bcl-2; clone N-100; Santa Cruz Biotechnology, Santa Cruz, CA) diluted1:500 in PBS containing 5% horse serum. Staining was detected using the Vectastain elite ABC kit (Vector Laboratories, Burlingame, CA). BrdUrd IHC was performed as described above with the following modifications: trypsin digestion was used in place of microwave retrieval, followed by incubation in 2.5 M HCl for 10 min at 37°C. Mouse anti-BrdUrd diluted 1:40 was used as the primary antibody (Dako, Carpinteria, CA).
ISH was performed as described by Alpers et al.
(21)
, except that slides were exposed in the dark for 5
days. A 600-bp 35S-UTP-labeled TGF- antisense
probe was transcribed from the same DNA template used for the Northern
blot analysis (see below). A TGF- sense probe was used as a negative
control.
Northern Blot Analysis.
Total liver RNA was isolated from snap-frozen tissue using a RNA
purification kit (RNAeasy mini kit; Qiagen, Chatsworth, CA). Total RNA
(10 µg) was separated on a 1.1% agarose/formaldehyde gel and
transferred to a nylon filter by capillary transfer. Membranes were
prehybridized for 1 h in NorthernMax Prehyb/hyb buffer (Ambion,
Austin, TX) and then hybridized overnight at 65°C with a
[32P]UTP-labeled TGF- antisense riboprobe
diluted in NorthernMax buffer. After hybridization with the TGF-
riboprobe, the membranes were rehybridized with either a
[32P]UTP-labeled ß-actin (Fig. 3)
or
cyclophilin (Fig. 7)
riboprobe to verify loading. The TGF-
riboprobe was synthesized off the T7 promoter from a PCR-generated DNA
fragment containing the T7 promoter ligated to a 600-bp TGF- cDNA
fragment (Lig n Scribe; Ambion). The ß-actin and cyclophilin
riboprobes were synthesized off the T7 promoters of the pTRI-ß-actin
and pTRI-cyclophilin templates (Ambion).
|
|
Induction of Fas-mediated Apoptosis and Determination of
Hepatocyte Proliferation.
One month before treatment, young adult Bcl-2 transgenic mice (C57B6C3H
background) and WT littermates were placed on zinc water to induce the
Bcl-2 transgene. Animals received an i.p. injection of 10 µg of mouse
anti-Fas antibody (clone Jo2; PharMingen, San Diego, CA) and were
killed 3 h after the injection. Livers were resected, fixed in
10% buffered formalin, and embedded in paraffin. Sections were stained
with H&E, and the number of apoptotic nuclei (determined by morphology)
per x400 high-power field was counted.
For proliferation studies, animals received an i.p. implant of Alzet osmotic 3-day pumps (model 1003D; Alza Corp. USA) that released a solution of 15 mg/ml BrdUrd (Boehringer Mannheim, Indianapolis, IN) at a rate of 1.0 µl/h. At the end of day 3, the mice were killed, the livers were removed, and sections were prepared for BrdUrd IHC. BrdUrd incorporation was measured by counting the number of positive nuclei labeled with a mouse monoclonal anti-BrdUrd antibody (1:40 dilution; Dako) and detected with Vector Laboratories ABC elite kit (as described above). A section of the duodenum was included on each slide to confirm the delivery of the BrdUrd. A minimum of three animals/group were counted, and a total of 3000 cells/animal were counted.
Statistical Analyses.
Statistical analyses were performed using the GraphPad Prism version
2.0 program (GraphPad Software, Inc. San Diego, CA). Student’s
t test analyses were used to calculate probability
(P) values. Results are presented as mean ± SE. For all tests, P < 0.05 was accepted as
significant.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
expression in the liver of TGF-
transgenic mice is high, although it can be increased by the addition
of zinc sulfate to drinking water (zinc water). By using Western blot
analysis, we determined the extent to which the expression of the Bcl-2
transgene could be modulated by placing Bcl-2 transgenic mice on zinc
water. Expression levels of the human Bcl-2 transgene in mice
maintained on either normal drinking water or zinc water for 30 days
were examined. There was no expression of Bcl-2 in the livers of WT
littermates of Bcl-2 transgenic mice, and little, if any, Bcl-2
transgene expression was detectable in Bcl-2 transgenic mice drinking
normal water. In contrast, expression was elevated approximately
10-fold in the Bcl-2 transgenic mice that received zinc water (Fig. 1A)
.
|
. Treatment with the Jo2 antibody
induced massive apoptosis in WT animals, such that more than 30
apoptotic nuclei/x400 field were found. In contrast, Bcl-2 transgenic
mice had, on average, only 2 apoptotic nuclei/x400 field. The
protection against Fas-induced hepatocyte cell death in vivo
in Bcl-2 transgenic mice was similar to the results reported by
Lacronique et al. (9)
.
Generation of TGF-/Bcl-2 Transgenic Mice, Transgene
Expression, and Hepatocyte Proliferation in 4-week-old Mice.
To examine the effects of Bcl-2 expression on TGF--mediated
hepatocarcinogenesis, we generated double transgenic mice capable of
expressing both TGF- and Bcl-2. This was accomplished by mating
heterozygous TGF- female mice of the MT42 line (CD1 background) to
heterozygous Bcl-2 males (C57B6/C3H background) to produce
TGF-/Bcl-2 double transgenic mice, TGF- and Bcl-2 single
transgenic mice, and WT littermates. A significant characteristic of
TGF- transgenic mice of the MT42 line on a CD1 background is the
highly elevated hepatocyte proliferation evident in young animals.
Although the proliferation diminishes with time, it remains elevated up
to the point of tumor development (16)
. Because the newly
generated lines of TGF- single transgenic mice and TGF-/Bcl-2
double transgenic mice have a different genetic background from the
MT42 mice that were previously studied, it was necessary to determine
the levels of hepatocyte proliferation in animals with the hybrid
background and to ascertain whether Bcl-2 expression would alter the
TGF- proliferative effect.
We measured hepatocyte proliferation in 4-week-old male TGF-/Bcl-2
double transgenic mice, TGF- and Bcl-2 single transgenic mice, and
WT controls (Fig. 2A)
that had been maintained on zinc water from the time of
weaning at 3 weeks of age. As measured by 3-day BrdUrd incorporation,
hepatocyte proliferation was approximately 4-fold higher in TGF- and
TGF-/Bcl-2 transgenic mice compared with Bcl-2 transgenic and WT
mice (Fig. 2A)
. The percentage of BrdUrd-positive cells in
TGF- single transgenic mice (18%), was similar to that in
TGF-/Bcl-2 double transgenic mice (17%), demonstrating that Bcl-2
expression did not affect the ability of TGF- to enhance hepatocyte
proliferation in 4-week-old mice. Although hepatocyte proliferation was
increased in the TGF- transgenic mice at 4 weeks of age, the
labeling index was approximately half of that observed previously with
the MT42 line (16)
. The amount of TGF- mRNA expressed
in the animals used for the present study was similar to that of MT42
mice (data not shown), indicating that the genetic background of the
mice can influence the extent of hepatocyte proliferation independent
of the amount of TGF- being expressed.
|
single transgenic and TGF-/Bcl-2
double transgenic mice, this ratio was significantly increased compared
with the ratio in WT and Bcl-2 single transgenic mice (Fig. 2B)
. The expression of Bcl-2 in the TGF-/Bcl-2 double
transgenic and Bcl-2 single transgenic mice was verified by Western
blot analysis (Fig. 2C)
. Bcl-2 was detected only in Bcl-2
single transgenic mice or in TGF-/Bcl-2 double transgenic mice.
Thus, at 4 weeks of age, mice expressing either TGF- alone or
TGF- and Bcl-2 had elevated levels of hepatocyte proliferation and
increased liver:body weight ratios.
Hepatocyte Proliferation and Liver:Body Weight Ratios in Older
Mice.
Previous work using TGF- transgenic mice of the MT42 line (CD1
background) showed that there was 10–12% hepatocyte proliferation
from 4 months of age to 8 months of age (16)
. In contrast,
in the hybrid background mice used in the present study, the rate of
hepatocyte proliferation was negligible in 4–8-month-old TGF- or
Bcl-2 single transgenic mice as well as in TGF-/Bcl-2 double
transgenic mice. In all of these lines, hepatocyte proliferation did
not differ from that of WT mice. Similarly, the liver:body weight
ratios in 8–10-month-old mice were similar in WT, single transgenic,
and double transgenic mice (Fig.
3A). To determine whether TGF- continued to be expressed in
TGF- single transgenic and TGF-/Bcl-2 double transgenic mice, RNA
was obtained from 8–10-month-old mice and analyzed by Northern
blotting. The data shown in Fig. 3B
demonstrate that TGF-
was expressed at very high levels in both TGF- single transgenic and
TGF-/Bcl-2 double transgenic mice, indicating that coexpression of
Bcl-2 (as determined by Western blot analysis, Fig. 3C
) with
TGF- did not affect the expression of the TGF- transgene.
Tumor Development in TGF- Single and TGF-/Bcl-2 Double
Transgenic Mice.
A total of 32 TGF-, 26 TGF-/Bcl-2, and 19 Bcl-2 transgenic mice
and 33 WT mice were examined for tumor development between 8 and 15
months of age. Livers of Bcl-2 transgenic mice showed no significant
histological alterations and were similar to those of WT mice
throughout the duration of the study. All TGF-/Bcl-2 double
transgenic mice killed at 8–10 months of age showed multiple foci of
basophilic hepatocytes containing dysplastic cells with abnormal
nuclear morphology (Fig. 4, B and D)
, and one of the animals had a small,
clearly demarcated adenoma. Unlike the TGF-/Bcl-2 double
transgenics, livers of the TGF- single transgenic mice showed no
basophilic foci (Fig. 4, A and C)
. However, one
of the nine mice killed at this time had a well-developed HCC with a
diameter of approximately 0.5 cm. Thus, at 8–10 months of age, the
livers of the TGF-/Bcl-2 double transgenic mice contained
potentially preneoplastic lesions detected by histological analysis.
Although a TGF- transgenic mouse had developed a HCC detectable at
gross examination, the morphology of the liver of TGF- transgenic
mice at this age was normal. The findings were entirely different in
animals killed at later times. In animals examined at either 11–12
months or 13–15 months of age, TGF- single transgenic mice had a
much higher incidence of HCC than double transgenic mice (Fig. 5, A and B)
.
|
|
transgenic mice had HCC, whereas
no tumors were detected grossly or histologically in
TGF-/Bcl-2 double transgenic mice. At 13–15 months, more than 80%
of TGF- transgenic mice had HCC, whereas only 54% of the double
transgenics had tumors. In addition, of the 20 TGF- single
transgenic mice that did develop liver tumors, 16 had tumors that were
larger than 1 cm in diameter. In contrast, of the seven TGF-/Bcl-2
double transgenic mice that developed liver tumors, only one had a
tumor diameter larger than 1 cm. Thus, coexpression of TGF- and
Bcl-2 in the double transgenic mice decreased the incidence of HCC,
delayed tumor development, and inhibited tumor growth.
We evaluated both mitotic and apoptotic activity in tumors from livers
of TGF- single transgenic mice and TGF-/Bcl-2 double transgenic
mice (Fig. 5C)
by determining the number of mitotic and
apoptotic bodies per 1000 cells in the tumors. The overall mitotic
activity in the tumors from TGF- single transgenic mice was nearly
three times higher than that of tumors from TGF-/Bcl-2 double
transgenic mice. Apoptotic bodies were also observed more frequently
(3.3 versus 0.7 cells/1000 cells counted) in tumors from the
TGF- transgenic mice than in tumors from TGF-/Bcl-2 double
transgenic mice. Overall, in mice maintained on zinc water from
the time of weaning, tumors from TGF-/Bcl-2 double transgenic mice
had less mitotic and apoptotic activity than tumors from TGF- single
transgenic mice.
Tumor Morphology and Transgene Expression.
In addition to the differences in tumor incidence and mitotic
activity observed between the TGF- single transgenic and
TGF-/Bcl-2 double transgenic mice, Bcl-2 had profound effects on the
morphology of TGF--induced liver tumors. Tumors that developed in
TGF- single transgenic mice were well-differentiated HCCs that
displayed either solid or trabecular patterns (Fig. 6, A and D)
. The morphology of these tumors was
similar to our previous observations (Ref. 13
; Fig. 6, A and D
). In contrast, tumors that developed in
TGF-/Bcl-2 double transgenic mice consisted of very large single or
binucleated hepatocytes that formed long, 1-cell-thick villiform
structures separated by blood-filled spaces and cavities. The
hepatocytes in these villi were often individually lined by endothelial
cells (Fig. 6, B and E)
. Although variation
occurred from animal to animal, the liver tumors that did arise in the
TGF-/Bcl-2 double transgenic mice were hemorrhagic and cystic, with
abundant endothelial cells in some areas of the tumors.
|
/Bcl-2 double transgenic mice that were not maintained on zinc
water. Because Bcl-2 expression is minimal or undetectable in these
animals (see below), they served as an excellent control for the
analysis of tumorigenesis in the TGF-/Bcl-2 double transgenic mice
described above, in which Bcl-2 was induced by maintaining the animals
on zinc water throughout their lifetime. Tumors that developed in
double transgenic mice in which Bcl-2 was not induced (Fig. 6, C and F)
were solid or trabecular HCC, similar to
those detected in TGF- single transgenic mice. No animal in this
group developed villous tumors with vascular spaces as observed in
TGF-/Bcl-2 double transgenic mice in which Bcl-2 was expressed.
These findings provide direct evidence that Bcl-2 expression modified
the morphology of liver tumors induced by TGF-.
Expression of the transgenes was examined in tumors and adjacent
tissue using Northern blot analysis and ISH for TGF- and Western
blot and IHC for Bcl-2. Compared with WT mice, expression of TGF-
mRNA was higher in the tumors and adjacent tissue of both TGF-/Bcl-2
double transgenic mice and TGF- single transgene mice (Fig. 7A)
. Although there was considerable variation among the
animals, TGF- mRNA expression was generally higher in TGF- single
transgenic mice than in double transgenic mice. Bcl-2 expression, as
determined by Western blot analysis, was relatively uniform in the
tumors and surrounding tissue of TGF-/Bcl-2 double transgenic mice
(Fig. 7B)
. Expression of Bcl-2 was weak or undetectable in
double transgenic mice that did not receive zinc water to induce this
transgene (Fig. 7B)
. IHC analysis of Bcl-2 expression was
consistent with the pattern of expression detected in Western blots,
that is, there was intense Bcl-2 staining in both tumor and adjacent
tissue (Fig. 8, A–C)
. Detection of TGF- by ISH revealed that TGF-
was more strongly expressed in HCC of TGF- single transgenic mice
than in the liver tumors that developed in TGF-/Bcl-2 double
transgenic mice.
|
/Bcl-2 double transgenic mice
maintained on zinc water were smaller in size and less mitotically
active than those of TGF- single transgenic mice. A possible
explanation for this finding is that Bcl-2 may inhibit tumor
progression. To investigate this hypothesis, exposure to zinc water in
a group of mice was delayed until they reached 8.5 months of age.
Because Bcl-2 expression in uninduced mice is very low or undetectable,
Bcl-2 expression in this group of mice is considered to have started at
8.5 months. Four months later, these animals were killed and examined
for tumor development. Grossly detectable HCCs developed in 50% (two
of four) of TGF- single transgenic mice that served as controls for
these experiments but were absent in all nine of the TGF-/Bcl-2
double transgenic mice examined (Fig. 9)
. Histological analysis of the livers of double transgenic mice
revealed no obvious abnormality, with the exception of a small
nonneoplastic proliferative lesion detected in an otherwise normal
liver section in one animal (data not shown). Therefore, late induction
of the Bcl-2 transgene reduced the tumorigenic effect of TGF-.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
transgenic mice with Bcl-2 transgenic mice that are protected against
Fas-mediated hepatocyte apoptosis. Overexpression of TGF-
in the livers of transgenic mice leads to the development of adenomas
and HCCs in more than 85% of these animals after 12 months of age
(13
, 15) . Contrary to the expectations, tumor development
was inhibited in the double transgenic mice that overexpressed both
TGF-, a potent hepatocyte mitogen, and Bcl-2. Expression of the
Bcl-2 transgene was inducible by adding zinc to the drinking water.
Using the inducible system for Bcl-2 expression, it was possible to
establish that inhibition of tumor growth in TGF-/Bcl-2 double
transgenic mice was a consequence of Bcl-2 expression. Tumors that
eventually developed in TGF-/Bcl-2 double transgenic mice were
smaller in size, had lower mitogenic and apoptotic activity than those
in TGF- single transgenic mice, and had an unusual morphology.
The inhibition of hepatocarcinogenesis in double transgenic mice was
particularly surprising because numerous foci of abnormal basophilic
cells were detected in the livers of these animals sampled between 6
and 10 months of age. These foci were not detected in either TGF- or
Bcl-2 single transgenic mice, suggesting that they resulted from the
combined expression of TGF- and Bcl-2 in the double transgenic mice.
Although basophilic foci observed during liver carcinogenesis are often
considered to be preneoplastic lesions (23
, 24)
, this was
not the case in TGF-/Bcl-2 double transgenic mice. The basophilic
foci that developed in these animals neither persisted nor progressed
into adenomas or carcinomas.
Inhibition of TGF- tumorigenesis in TGF-/Bcl-2 double
transgenic mice could be the consequence of at least two types of
effects: (a) inhibition of TGF- expression by Bcl-2; or
(b) a direct effect of Bcl-2 expression on cell
proliferation. Analysis of TGF- expression in TGF-/Bcl-2 double
transgenic mice did not reveal an obvious decrease of TGF-
expression relative to that in TGF- single transgenic mice. On the
other hand, a study of the timing of DNA replication during liver
regeneration in Bcl-2 single transgenic animals showed that the peak of
DNA synthesis was delayed in these
animals.4
These observations lead us to conclude that Bcl-2 can inhibit cell
cycle progression. If this is indeed the major mechanism by which Bcl-2
expression inhibits tumorigenesis in TGF-/Bcl-2 double transgenic
mice, it would be expected that Bcl-2 would have an effect on tumor
progression rather than initiation. This appears to be the case, as
demonstrated by experiments in which Bcl-2 expression initiated at 8.5
months of age proved to be sufficient to inhibit tumor development.
Similar to our results, recent studies using Bcl-2 transgenic
mice have revealed that Bcl-2 expression can inhibit tumorigenesis.
Murphy et al. (25)
found that that Bcl-2
expression delayed mammary tumor development in
dimethylbenz(a)anthracene-treated mice, and de La Coste
et al. (26)
demonstrated the ability of Bcl-2
to inhibit liver tumorigenesis in c-myc transgenic mice. These in
vivo studies correlate with tissue culture studies in which
overexpression of Bcl-2 was shown to delay cell cycle entry and
decrease cell proliferation (27, 28, 29)
. These results
provide an explanation for the tumor suppressor effects rather than the
oncogenic effects of Bcl-2 in TGF-/Bcl-2 double transgenic
mice.
The antiproliferative effect of Bcl-2 combined with its antiapoptotic
activity may explain some aspects of the atypical morphology of the
small numbers of tumors that eventually developed in the TGF-/Bcl-2
double transgenic mice. It is possible that the very large hepatocytes
present in these tumors resulted from the inhibition of both apoptosis
and replication of cells exposed to the constant TGF- mitogenic
stimulus. However, the antiapoptotic effects off Bcl-2 are unlikely to
have contributed to the proliferation of endothelial cells and the
formation of large vascular spaces observed in these tumors. It is of
interest that TGF- can have angiogenic effects (30)
.
However, the tumors formed in TGF- single transgenic mice were
typical solid or trabecular HCCs without vascular features.
Our previous studies of liver tumorigenesis in TGF- transgenic
mice were done with the MT42 line in a CD1 background. In these
animals, TGF- enhanced hepatocyte proliferation at 1 month of age,
and elevated hepatocyte replication persisted until tumors emerged.
Because the TGF-/Bcl-2 double transgenic mice had a CD1 x C57B6C3H background, we produced TGF- single transgenic mice
with this new genetic background to serve as controls for tumorigenesis
experiments. TGF- transgenic mice resulting from the CD1 x C57B6C3H cross had enhanced hepatocyte proliferation at 1–2
months of age, but no later. In these animals, tumors emerged in livers
that were morphologically normal and had no increased hepatocyte
proliferation. The mechanisms by which neoplasms developed in the
absence of elevated hepatocyte proliferation preceding tumor formation
in TGF- transgenic mice remain to be established. One possibility is
that the enhanced hepatocyte proliferation that occurs during the first
2 months of life in these animals is sufficient to cause genomic damage
that results in tumorigenesis many months later.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by National Cancer Insitute Grant
CA74131, USPHS National Research Grant T32 GMO7270 (to M. E. V.), and
the Irwin M. Arias Postdoctoral Research Fellowship from the American
Liver Foundation (to R. H. P.). 
2 To whom requests for reprints should be
addressed, at Department of Pathology, University of Washington, K-078
Health Sciences Building, Box 357705, Seattle, WA 98195-7470. Phone:
(206) 686-1221; Fax: (206) 616-1943; E-mail: nfausto{at}u.washington.edu
3 The abbreviations used are: HCC, hepatocellular
carcinoma; TGF, transforming growth factor; MT1, metallothionein; WT,
wild-type; IHC, immunohistochemistry; ISH, in situ
hybridization; BrdUrd, bromodeoxyuridine.
4 M. E. Vail and N. Fausto. Bcl-2 delays liver
tumor promotion and inhibits hepatocyte cell cycle progression,
manuscript in preparation.
Received 8/ 1/00. Accepted 11/13/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell, 61: 1137-1146, 1990.[Medline]
transgenic mice. Cancer Res., 52: 5162-5170, 1992. transgenic mice. Cancer Res., 52: 5171-5177, 1992. causes liver enlargement and increased hepatocyte proliferation in transgenic mice. Am. J. Pathol., 145: 398-408, 1994.[Abstract]
in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia and carcinoma of the breast. Cell, 61: 1121-1135, 1990.[Medline]
dramatically enhances oncogene-induced carcinogenesis in transgenic mouse pancreas and liver. Mol. Cell. Biol., 13: 320-330, 1993./c-myc double-transgenic mice. Arch. Toxicol. Suppl., 19: 359-366, 1997.[Medline]
: a more potent angiogenic mediator than epidermal growth factor. Science (Washington DC), 232: 1250-1253, 1986.This article has been cited by other articles:
|
|
 |
|
  Y. Janumyan, Q. Cui, L. Yan, C. G. Sansam, M. Valentin, and E. Yang G0 Function of BCL2 and BCL-xL Requires BAX, BAK, and p27 Phosphorylation by Mirk, Revealing a Novel Role of BAX and BAK in Quiescence Regulation J. Biol. Chem., December 5, 2008; 283(49): 34108 - 34120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Bailly-Maitre, E. Bard-Chapeau, F. Luciano, N. Droin, J.-M. Bruey, B. Faustin, C. Kress, J. M. Zapata, and J. C. Reed Mice Lacking bi-1 Gene Show Accelerated Liver Regeneration Cancer Res., February 15, 2007; 67(4): 1442 - 1450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Bai, H.-M. Ni, X. Chen, D. DiFrancesca, and X.-M. Yin Deletion of Bid Impedes Cell Proliferation and Hepatic Carcinogenesis Am. J. Pathol., May 1, 2005; 166(5): 1523 - 1532. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. H. Pierce, M. E. Vail, L. Ralph, J. S. Campbell, and N. Fausto Bcl-2 Expression Inhibits Liver Carcinogenesis and Delays the Development of Proliferating Foci Am. J. Pathol., May 1, 2002; 160(5): 1555 - 1560. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Mitchell, V. O. Mallet, J. E. Guidotti, C. Goulenok, A. Kahn, and H. Gilgenkrantz Liver Repopulation by Bcl-xL Transgenic Hepatocytes Am. J. Pathol., January 1, 2002; 160(1): 31 - 35. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
, adenomas; 
, tumor
volume <1 cm3; and 
, tumor volume >1
cm3. C, mitotic and apoptotic bodies were
counted in H&E-stained liver sections of tumors obtained from three
TGF-
