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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 6, 2001 as doi:10.1096/fj.00-0487fje. |
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,3
,4,¶,5
* Fraunhofer Institute of Toxicology and Aerosol Research, Department of Cell Biology, D-30625 Hannover, Germany;
Freie Universität Berlin, Institute of Veterinary Pathology, D-14163 Berlin, Germany;
HepaVec AG fuer Gentherapie, D-13125 Berlin, Germany; and
¶ Humboldt University, Molecular Cell Biology Group at the Max-Delbrück Center for Molecular Medicine, D-13122 Berlin-Buch, Germany
2Correspondence: Ingenium Pharmaceuticals AG, Fraunhoferstrasse 13, D-82152 Martinsried, Germany. E-mail: rainer.klocke{at}ingenium-ag.com
SPECIFIC AIMS
The aim of this study was to elucidate the contribution, if any, of the tumor suppressor function of p53 to the process of hepatocellular carcinoma (HCC) formation in mice. Its role within networks controlling either cell proliferation or cell death during deterministic liver tumorigenesis in mice overexpressing in the liver transgenes encoding murine c-myc, or c-myc plus the secretable human epidermal growth factor (EGF) analog IgEGF was analyzed.
PRINCIPAL FINDINGS
1. Lack of p53 caused an increase of relative liver weight in c-myc and c-myc/IgEGF transgenics as well as reduced life spans in c-myc/IgEGF transgenics
p53-deficient c-myc and c-myc/IgEGF
transgenics exhibited 
twofold increased relative liver weight
compared with age-matched c-myc/p53+/+
and c-myc/IgEGF/p53+/+ mice.
In contrast, the relative weight of IgEGF/p53KO mouse livers remained unchanged compared with their p53 positive counterparts, indicating that a lack of the p53 tumor suppressor function caused increased hepatocyte growth and oncogenic responses of the liver to overexpression of transgene-encoded c-myc, but not of IgEGF.
A significant effect of p53 deficiency on hepatocarcinogenesis could
also be deduced from a comparison of survival times of
c-myc/IgEGF/p53KO with those of
c-myc/IgEGF/p53+/+ mice. Whereas the latter displayed an
average survival time (±SD) of 134 ± 18 days, which
already reflects a drastic cooperative effect of c-myc and
IgEGF overexpression on hepatocarcinogenesis, the former died on
average after 58 ± 7 days (Fig. 1
) as a consequence of accelerated HCC growth. In p53-deficient
c-myc and IgEGF single transgenics, however, average life
spans were probably limited by other types of tumors caused by the p53
knockout background since they were not different from that of
nontransgenic p53KO mice (Fig. 1)
, but significantly shorter
than those of p53 positive c-myc and IgEGF transgenics.
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2. Lack of p53 caused enhanced malignancy in HCCs of c-myc or c-myc/IgEGF transgenics
Comparative histopathological analyses of HCCs of IgEGF/p53+/+ vs. IgEGF/p53KO mice revealed no significant difference in the appearance of the tissue architecture. In contrast, HCCs from 4- to 6-month-old c-myc/p53+/+ mice were clearly different from those of age-matched c-myc/p53KO mice. Whereas HCCs of c-myc/p53+/+ trangenics showed a well-differentiated trabecular architecture, those of c-myc/p53KO mice displayed a pronounced malignant structure and an invasive growth pattern that led to the displacement or destruction of most normal, nontumorous tissue in the liver. In summary, the histological features classify the tumors of c-myc/p53KO mice as moderately differentiated HCCs.
Enhanced tumor growth, as indicated by a significantly higher number of individual tumor nodules and drastically more extensive hyperplastic liver areas, was also correlated with p53 deficiency in livers of c-myc/IgEGF double transgenics.
3. Proliferative activities were increased in tumorous livers of p53-deficient c-myc/IgEGF transgenics compared with their p53 positive counterparts
To elucidate the cellular basis of the enhanced tumor load and malignancy in livers of c-myc/p53KO and c-myc/IgEGF/p53KO mice compared with their p53 positive counterparts, cell death (apoptosis) and cell proliferation rates were analyzed. Using the TUNEL assay, apoptotic activity was shown to be drastically increased in a p53-independent manner in HCCs of c-myc and of c-myc/IgEGF transgenics. These findings indicate that the acceleration of hepatocarcinogenesis observed in p53-deficient c-myc or c-myc/IgEGF mice was not due to reduced apoptosis rates in the developing HCCs.
On the basis of mitotic indices, increased HCC cell proliferation was
observed in c-myc/p53KO mice compared with
c-myc/p53+/+ mice. The proliferative activity of
HCCs as well as of non-HCC regions as determined by BrdU labeling
indices was increased by twofold in livers of
c-myc/IgEGF/p53KO vs.
c-myc/IgEGF/p53+/+ mice.
Taken together, the elevated proliferative activity observed in HCCs of p53-deficient c-myc and c-myc/IgEGF mice and the fact that it was not counterbalanced by comparable increases in apoptotic activity were responsible for the observed acceleration of the growth of these tumors compared with their p53 positive counterparts.
4. p53 expression is increased in tumors of c-myc/IgEGF double transgenics and correlates with enhanced p21 expression
Western blot analyses of whole tumorous liver lobes of age-matched
(7-wk-old) c-myc/IgEGF/p53+/+ and
c-myc/IgEGF/p53KO mice revealed that levels of
p53 were increased in c-myc/IgEGF/p53+/+ mice
threefold over nontransgenic controls. Similarly, levels of the cdk
inhibitor p21 were concomitantly increased. In contrast, in
c-myc/IgEGF/p53KO livers p21 levels were
drastically reduced (Fig. 2
), suggesting that the stringency of cell cycle control exerted in the
presence of high p21 levels in
c-myc/IgEGF/p53+/+ livers was reduced in
c-myc/IgEGF/p53KO livers, causing increased rates
of HCC cell proliferation and thus increased size of HCCs in these
mice.
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Apoptosis, the second type of cellular response to overexpression of
p53, is known to be mediated at least in some cases by
p53-dependent transcriptional activation of the bax gene. In
accordance with the p53-independent apoptotic activities in HCCs of
c-myc and c-myc/IgEGF transgenics (see above),
levels of Bax in livers of c-myc/IgEGF/p53+/+ and
of c-myc/IgEGF/p53KO mice were similar (Fig. 2)
.
CONCLUSIONS
The possible roles of functional p53 deficiency during the process of hepatocarcinogenesis remain unresolved in view of uncertainties with respect to the phenotype of most p53 mutations found in human HCCs. The p53ser249 mutation, which predominates in human HCCs associated with aflatoxin exposure, exemplifies that loss of the transcription factor activity can be superimposed by a gain-of-function, which converts the tumor suppressor p53 into a dominant oncoprotein. Furthermore, apparent inconsistencies between presumed roles of p53 in the development of HCCs in humans and mice have been substantiated. For example, p53 mutations were not found in carcinogen-induced HCCs in mice and DEN-induced HCC development is not affected by lack of p53.
We therefore asked the question of whether the p53KO
genotype in the mouse, which imparts a clear-cut loss-of-function,
exerts tumor promoting effects during multistage carcinogenesis in the
liver of transgenic mice overexpressing c-myc and/or a
secreted version of IgEGF. These were originally designed to mimic in
the mouse the frequently observed overexpression in human HCCs of c-MYC
and TGF-
(an analog of IgEGF used here).
The results of the present study show that lack of p53
accelerates hepatocarcinogenesis in mice provided that c-myc
is constitutively overexpressed in the liver. In contrast, lack of
p53 remained without apparent effect on HCC development in
transgenics constitutively expressing the IgEGF transgene. This
suggests that the biochemical pathway driven by constitutive IgEGF
overexpression is distinct from that activated by overexpression of
c-myc and implies that the sequence in which the genetic
alterations led to progressive selective growth advantages of
hepatocytes during HCC development is presumably not random. If the
first of several genetic changes in hepatocytes leads to overexpression
of EGF or TGF- and thus to increased mitogenic signaling in
hepatocytes, an additional somatic mutation causing overexpression of
c-myc would be required before loss of p53 would
result in selective growth advantage of the affected cells. Thus,
deterministic HCC development occurs not only as a result of the
accumulation of somatic mutations in individual hepatocytes, but the
order in which they occur also determines the propensity for liver
neoplasia.
Our data reveal that the accelerated HCC development observed in
c-myc/p53KO or in c-myc/IgEGF/p53KO
mice compared with c-myc or c-myc/IgEGF mice was
already evident in the early progression phase of HCCs. Specifically,
introduction of the p53 null alleles into
c-myc/IgEGF double transgenics caused the widespread
disappearance of normal hepatic tissue in the liver soon after birth
and led to considerably accelerated HCC growth, which in turn resulted
in drastically reduced life spans of only 58 days. Together with the
low spread of survival times of these mice (54 to 63 days, cf. Fig. 1
),
these results suggest, that three genetic alterations—namely,
overexpression of c-myc and of IgEGF and lack of the
molecular gatekeeper p53—might be sufficient to deterministically
create transformed hepatocytes in vivo that are capable of forming
multiple HCCs in the liver.
Furthermore, our findings indicate that the accelerated increases in
size and malignancy of HCCs in c-myc/p53KO and
c-myc/IgEGF/p53KO mice were not caused by reduced
levels of apoptotic activity. Rather, an increase of HCC cell
proliferation in c-myc/IgEGF/p53KO mice that was
not counterbalanced by corresponding increases in apoptotic activity
resulted in net increases of cell multiplication rates and of total
liver mass in these mice (Fig. 3
). As suggested by our Western blot data, at least in HCCs of
c-myc/IgEGF/p53KO mice, this might be due to
reduced levels of p21 compared with HCCs in
c-myc/IgEGF/p53+/+ mice. This notion is
consistent with previous findings that constitutive overexpression of
p21 in the transgenic mouse liver blocks hepatocyte cell
cycle progression, normal postnatal liver development, and liver
regeneration and that the occurrence of p53 mutations in
HCCs is frequently correlated with reduced expression levels of p21.
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In summary we conclude that p53 is a bona fide tumor suppressor gene in HCC formation.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0487fje ; to cite this
article, use FASEB J. (April 6, 2001) 10.1096/fj.00-0487fje 
3 Present address: Aventis Pharma, Inc., D-65795 Hattersheim, Germany.
4 Present address: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
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