Deficiency of the Promyelocytic Leukemia Protein
Fosters Hepatitis C-Associated Hepatocarcinogenesis in
Kerstin Herzer1,6*, Anna Carbow2, Svenja Sydor1, Jan-Peter Sowa1, Stefan Biesterfeld3, Thomas-
Georg Hofmann4, Peter-Robert Galle5, Guido Gerken1, Ali Canbay1
1Department of Gastroenterology and Hepatology, University Hospital, Essen, Germany, 2Spital STS Ag, Department of Surgery, Zweisimmern, Swizerland, 3Department
of Cytopathology, University Hospital of Du ¨sseldorf, Du ¨sseldorf, Germany, 4Research Group Cellular Senescence, Deutsches Krebsforschungszentrum, Deutsches
Krebsforschungszentrum- Zentrum fu ¨r Molekulare Biologie der Universita ¨t Heidelberg Alliance, Heidelberg, Germany, 51stDepartment of Medicine, University Medicine
of the Johannes Gutenberg University, Mainz, Germany, 6Gereral-, Viszeral and Transplantation Surgery, University Hospital, Essen, Germany
Overwhelming lines of epidemiological evidence have indicated that persistent infection with hepatitis C virus (HCV) is a
major risk for the development of hepatocellular carcinoma (HCC). We have recently shown that HCV core protein mediates
functional inactivation of the promyelocytic leukemia (PML) tumor suppressor pathway. However, the role of PML in HCC
development yet remains unclear. To clarify the function of PML in liver carcinogenesis and HCV-associated pathogenesis
we crossed PML-deficient mice with HCV transgene (HCV-Tg) expressing mice and treated the resulting animals with DEN/
Phenobarbital, an established protocol for liver carcinogenesis. Seven months after treatment, livers were examined
macroscopically and histologically. Genetic depletion of the tumor suppressor PML coincided with an increase in
hepatocyte proliferation, resulting in development of multiple dysplastic nodules in 100% of the PML-deficient livers and of
HCCs in 53%, establishing a tumor suppressive function of PML in the liver. In animals expressing the HCV-transgene in PML-
deficient background, HCC development occurred even in 73%, while only 7% of their wildtype littermates developed HCC.
The neoplastic nature of the tumors was confirmed by histology and expression of the HCC marker glutamine synthetase.
Several pro- and antiapoptotic factors were tested for differential expression and liver carcinogenesis was associated with
impaired expression of the proapoptotic molecule TRAIL in PML-deficient mice. In conclusion, this study provides first in vivo
evidence that the tumor suppressor PML acts as an important barrier in liver carcinogenesis and HCV-dependent liver
Citation: Herzer K, Carbow A, Sydor S, Sowa J-P, Biesterfeld S, et al. (2012) Deficiency of the Promyelocytic Leukemia Protein Fosters Hepatitis C-Associated
Hepatocarcinogenesis in Mice. PLoS ONE 7(9): e44474. doi:10.1371/journal.pone.0044474
Editor: Taro Yamashita, Kanazawa University, Japan
Received May 13, 2012; Accepted August 8, 2012; Published September 11, 2012
Copyright: ? 2012 Herzer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: FundingprovidedbyDeutscheKrebshilfegrant no.108990. Thisworkwas furthersupportedbytheDeutscheForschungsgemeinschaft(DFG,grant 267/8-1),
and the Wilhelm Laupitz Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Liver cancer is the fifth most common cancer worldwide and
the third most common cause of cancer mortality. Hepatocellular
carcinoma (HCC), which accounts for 80%–90% of primary liver
tumors, is characterized by a very poor prognosis and is associated
with high mortality . Chronic Hepatitis C and associated liver
cirrhosis represent major risk factors for HCC development, being
implicated in more than 70% of HCC cases worldwide with
increasing incidence in the western world . About 170 million
people are infected with the hepatotropic Hepatitis C virus (HCV).
HCV is a small RNA virus coding for a limited number of four
structural and six nonstructural polypeptides, which regulate HCV
replication and encapsulation of the viral genome . Several viral
proteins have been implicated in liver carcinogenesis with
emphasis on the HCV core protein. For example, HCV core
protein has been described to facilitate cellular transformation .
Among cellular host factors, which interact with HCV core is the
tumor suppressor protein p53, a key regulator of the cellular
response to genotoxic stress and antiviral response . Despite our
growing knowledge about HCV-host cell interaction, the molec-
ular mechanisms which contribute to HCV-mediated transforma-
tion and carcinogenesis are still incompletely understood. Several
studies using transgenic mouse models indicate that HCV is
directly involved in hepatocarcinogenesis, although other factors
such as continuous inflammation or environmental factors seem
also to play a role ]. The downstream events of the HCV
protein expression in the transgenic mouse HCC model are
segregated into two pathways. One is augmented oxidative stress
in the absence of inflammation along with the attenuation of some
scavenging systems in the putative preneoplastic stage with
steatosis in the liver. The other pathway is the alteration in
cellular gene expression and intracellular signalling, including the
mitogen-activated protein kinase cascade .
By focusing on the cellular function of HCV core protein we
recently uncovered a previously unidentified link between HCV
core and promyelocytic leukemia-nuclear bodies (PML-NBs). We
found that HCV core protein targets PML-NBs and inactivates the
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PML tumor suppressor pathway through interfering with the
apoptosis-inducing function of PML isoform IV . PML-NBs are
present in almost every human cell type analyzed so far and
appear as discrete nuclear domains in immunofluorescence. PML
exerts potent growth suppressive and apoptosis-inducing activities
, and PML-deficient mice and cells exhibit defects in multiple
apoptosis pathways . Furthermore, PML deficiency has been
linked to increased susceptibility to viral pathogens [11,12]. A
large number of proteins with diverse functions have been found to
localize to PML-NBs and their central role in multiple cellular
processes such as proliferation, apoptosis, and regulation of
transcription is well established . Moreover, comprehensive
studies have shown that the PML protein is frequently lost in
human cancers of various origins . So far, a functional role for
PML in HCC has not been defined.
In this study, we used transgenic mice with liver specific
expression of HCV RNA corresponding to the full-length open
reading frame (ORF) of the hepatitis C virus . These mice
were crossbread with PML2/2 mice  to achieve a HCV-
transgenic and PML2/2 genotype. For both parental strains, no
spontaneous liver tumor development has been described so far.
an indispensable factor in the complex interplay of liver tumor
development in HCV-dependent liver carcinogenesis.
Materials and Methods
Animals and Genotyping
PML2/2 mice (within a 129Sv genetic background) (kindly
provided by Hans Will, Heinrich Pette-Institut, Hamburg,
Germany) were generated by Pier Paolo Pandolfi (Beth Israel
Deaconess Medical Center, Boston, USA) and have been
described previously . HCV transgenic FL-N/35 mice (within
a C3H/C57BL6 genetic background) (kindly provided by Ula
Hibner, IGMM, Montpellier, France) were generated by Herve
Lerat (INSERM, Paris, France) and Stanley M. Lemon (UTMB,
Galvestone, USA) . The two mouse strains were crossed and
the following genotypes were used for this study: (1)WT; PML+/+,
(2)HCV; PML +/+, (3)WT; PML2/2 and (4)HCV; PML2/2.
Genotyping was performed with the following primers: PML: R:
59 TTG GAC TTG CGC GTA CTG TC-39, F1: 59-TTT CAG
TTT CTG CGC TGC C-39, F2: 59- CGA CCA CCA AGC GAA
ACA -3. HCV: NS4b forward MWG Biotech AG # 27-5072 1/4
59-TAT TGC CTG ACA AGA GGC AGT GTG GTT ATC-39,
NS4b reverse MWG Biotech AG # 27-5072 2/4 59-GAT GAA
ATT CCA CAT GTG CTT TGC CCA G-39.
PCR was performed in a standard thermocycler and analyzed
on 2% agarose gels.
About 100 mL of blood was collected from the tail vein. Alanine
aminotransferase (ALT) and aspartate aminotransferase (AST)
were measured in the Institute of Clinical and Laboratory
Medicine at the University Hospital Mainz by standard proce-
Transgenic mice as well as WT mice were treated according to
a hepatocarcinogenesis protocol consisting of tumor-initiation with
diethylnitrosamine (DEN) and tumor-promotion with phenobar-
bital (PB) as described . In brief, DEN (Sigma) was dissolved in
PBS and given twice i.p. at a dose of 5 mg/g bodyweight at the age
of 7 and 10 days post partum. Initiation was followed by PB
treatment (Sigma) in a concentration of 0.05% continuously added
to drinking water. Only male mice were included into the study.
Fifteen mice of each group were sacrificed at the indicated time
point after the beginning of the treatment with PB and were
further analysed. Age matched WT and transgenic littermates
which were left untreated were used as controls. Liver and body
weight were determined. At month 7, all remaining mice were
killed, since the incidence of hepatocellular carcinoma increased
and a high percentage of transgenic mice started to suffer from
cachexia or tumor burden.
Histopathology and Histology
All mice in the carcinogenesis protocol were subjected to a
complete necropsy and tissues were immediately fixed in 4%
neutral-buffered formaldehyde, embedded in paraffin, sectioned at
2 mm and stained with hematoxylin/eosin (HE) by standard
methods. For assessment of liver pathology at least eight
representative sections of each liver were prepared and evaluated
by a senior pathologist in a blinded fashion. Liver pathology was
scored into preneoplastic lesions (dysplastic nodules) and hepato-
Gomori, methenamine silver, and periodic acid-Schiff were
performed to allow histological evaluation of liver fibrosis (staging),
inflammatory activity and tumor (grading), respectively. The
histology of specimens was independently and blindely assessed by
two board certified pathologists.
In addition, we sacrificed the corresponding untreated trans-
genic mice at the age of 2 or 7 months. A complete autopsy with
inspection of the liver, lung, kidneys, intestine, spleen and heart for
macroscopically visible tumors was performed. Suspicious tissue
lesions were examined histopathologically.
Histology and Immunohistochemistry
In addition to the above mentioned staining techniques, slides
were immunostained for Ki67 (monoclonal rabbit, 1:100 dilution;
NeoMarkers) using an automated staining system with an iView
DAB kit (Ventana, Tucson, AZ). All sections were counterstained
with hematoxylin. The whole section was evaluated for the
number of positive hepatocytes, and pictures were taken from
representative high-power fields.
Immunohistochemical stainings of all liver specimens were
performed in order to investigate expression of glutamin synthe-
tase and proliferating cell nuclear antigen (PCNA) using the
Envision staining method (Dako, Denmark) as described earlier
. The antigen retrieval was carried out by steamer treatment in
pH 6 citrate buffer and mouse-anti-glutamine synthetase antibod-
ies (diluted 1:500) and rabbit-anti PCNA antibody (diluted 1:1000)
was applied, respectively. Specimens were then incubated with
anti-mouse or anti-rabbit envision secondary antibodies conjugat-
ed with horse raddish peroxidase (HRP) and immunostainings
were developed using diaminobenzidine as a substrate.
Cell death was detected by staining cell nuclei with DNA strand
breaks (TUNEL technology) using the in situ Cell Death Detection
Kit, Fluorescein (Roche), according to the manufacturer’s
instructions. Counterstaining and further procedure was per-
formed as described above.
Tissue Lysis and Western Blotting
About 20 mg of shock-frozen liver tissue were minced,
transferred into ice-cold lysis buffer containing 20 mM Tris-HCl
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(pH 8.0), 5 mM EDTA, 0.5% Triton-X 100, and 16 Protease
inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and
incubated on ice for 15 min. Western blotting was performed as
described . Immunodetection was performed using the
following primary antibodies: PML (Santa Cruz Biotechnology;
H-238 and PG-M3), HCV core (MA1-080 obtained from ABR,
Golden, CO, USA), TRAIL (Novus Biologicals, Littleton, USA)
and anti-tubulin (Sigma). Peroxidase-conjugated species-specific
secondary antibodies (Santa Cruz Biotechnology) were used at a
dilution of 1:10000. Western blots were performed for at least two
mice at each age indicated.
For isolation of total RNA, 20 mg of shock frozen liver tissue
were homogenized in 1 mL TRI-Reagent (Sigma), and further
isolated according to the manufacturers instructions. RNA
concentration was measured in a NanoDrop photometer (Peqlab,
Erlangen, Germany) and 1 mg of total RNA was subjected to
reverse transcription using oligo-dT primers. Isolated DNA and
complementary DNA (cDNA) were used for PCR (see above) and
real time (RT) PCR approaches, respectively.
RT Quantitative PCR
Relative target messenger RNA (mRNA) expression was
analyzed by RT-quantitative PCR using the QuantiTect SYBR
Green PCR Kit and QuantiTect primers (Quiagen, Hilden,
Germany) for murine TRAIL, TRAIL-R, CD95, CD95R,
NOXA, PUMA, Mcl-1, bcl-2, bcl-xL, survivin, XIAP, and
Succinate dehydrogenase complex, subunit A (SDHA) as house-
keeping gene. The relative increase in reporter fluorescent dye
emission was monitored. The level of target mRNA, relative to
SDHA, was calculated using the standard formula.
Validation of PML-deficiency in the Liver of HCV-Tg Mice
After breeding Pml 2/2 mice to HCV-Tg mice, homozygous
offspring were screened for deletion of PML and expression of the
HCV-Tg. We analysed PML and HCV expression in liver lysates
of 4-week-old mice (Fig. 1a). Immunoblotting demonstrated absent
PML expression in liver lysates of PML 2/2 as well as PML 2/
2/HCV-Tg mice. In addition, RT-PCR showed loss of PML
mRNA expression in total liver lysates of PML 2/2 mice in
absence and presence of the HCV transgene (Fig. 1b). We used the
following genotypes for further analysis: (1)WT; PML+/+,
(2)HCV; PML +/+, (3)WT; PML2/2 and (4)HCV; PML2/2
(Fig. 1). Of each genotype, 15 male mice were submitted to the
DEN/Phenobarbital hepatocarcinogenesis protocol  in order
to achieve initiation of liver carcinogenesis during a considerable
life span. The same number of mice of each group were left
untreated as control. Mice were sacrificed at the age of 7 months.
Basal Liver Damage and Increased Apoptosis in PML-
deficient HCV-transgenic Mice
The impact of PML deficiency in the presence or absence of
the HCV-Tg for liver homeostasis after 7 months of treatment
was assessed. In general, body weight of the mice was decreased
in the DEN/Phenobarbital treated group, presumably due to
tumor burden and cachexia (Fig. 2a). In contrast, liver weight
was considerably higher in treated mice, which was more
evident in PML-expressing animals and less in PML-deficient
mice (Fig. 2b). HCV;PML+/+ and WT;PML+/+ animals
showed under treatment an increased liver weight compared
to age-matched PML-deficient and untreated controls, presum-
ably due to deregulated apoptosis and regeneration, which may
be compromised when the tumor suppressor PML is lacking
(Fig. 2b). As a consequence, all animals showed under treatment
Figure 1. Characterisation of PML-deficient HCV-transgenic mice. (A) Whole liver extracts derived from 4 weeks old wild-type and HCV-PML+
mice were analysed by immunoblot analysis for the protein expression of PML and HCV-core as well as tubulin (loading control). (B) Whole liver
extracts from 4 weeks old PML-HCV+ mice as well as WT mice were analysed for the mRNA expression levels of PML and HCV core by quantitative RT-
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an increase of liver/body weight ratio compared to age-matched
untreated controls (Fig. 2c).
Aminotransferase levels were determined as a surrogate marker
for liver tissue damage. As expected, liver enzymes were
specifically increased in all animals treated with DEN/PB,
Figure 2. PML-expression influences liver weight. (A) Body weight, (B) liver weight as well as (C) liver/body weight ration of all mouse strains,
either treated or not, was determined.
Figure 3. PML-expression influences transaminases. Serum AST and ALT levels of treated (white bars) and untreated (grey bars) mice. Mean
levels of 15 mice per group 6 SD are shown.
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whereas untreated mice showed comparably low transaminase
concentrations (Fig. 3). Interestingly, PML-deficient mice exhib-
ited a more than 4-fold increase in serum ALT levels and a 6–8-
fold increase in Serum AST levels. In contrast, PML-deficient
mice bearing the HCV-Tg displayed a non-significant but
considerable higher concertration in both transaminases (Fig. 3).
An increase in transaminases indicates a damage of liver cells,
therefore these results suggest an additional liver damage in
We next asked whether this coincides with hepatocyte cell
death. Therefore, we analyzed liver sections of 7-months-old
untreated mice for DNA strand breaks using the TUNEL assay.
Intact expression of PML was associated with more TUNEL-
positive cells (Fig. 4a). Quantification demonstrated that the
TUNEL-index was decreased in PML2/2livers. Among PML-
positive hepatocytes, independend of the HCV transgene status,
TUNEL-index reached up to 3%. In contrast, liver sections of
PML-deficient mice revealed hardly any or no TUNEL-positive
hepatocytes (Fig. 4a).
Next we tested if reduced apoptosis activity in HCV;PML2/2
mice was accompanied by increased hepatocyte proliferation by
Ki67 staining. Ki67 expression
HCV;PML2/2 hepatocytes (median: 9,8%) as well as in
WT;PML2/2 hepatocytes (median: 6,2%) when compared to
WT-littermates. Furthermore, liver cells of mice expressing HCV-
transgene also displayed a slight increase in proliferation when
compared to PML-deficient wildtype mice (median: 5,1% versus
3,5%) (Fig. 4b). Taken together, our results indicate that both
PML deficiency and HCV-transgene expression contribute to the
strong proliferation rate of HCV;PML2/2 hepatocytes.
Next, we analysed expression of inflammatory mediators in liver
lysates, to determine if the increase in liver damage in PML2/2
hepatocytes leads to an inflammatory response. No difference in
IL-6 or TNFa mRNA expression in untreated mice was observed
(data not shown). However, expression of both inflammation
markers was increased after 7 months in DEN/PB treated mice,
most likely due to the carcinogenesis treatment.
Figure 4. Apoptosis and proliferation in PML-deficient livers. (A) Liver sections were stained for DNA strand breaks by TUNEL assay. Five
independent fields (406) of five mice per genotype were quantified. The ratio of TUNEL-positive nuclei per 100 cells (TUNEL index) was calculated. (B)
To assess hepatocyte proliferation Ki67 staining was performed. Five independent fields (406) of five mice per genotype were quantifiend for Ki67-
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PML Deficiency Potentiates Tumor Development in HCV
Deletion of the tumor suppressor PML did not induce
spontaneous development of liver tumors up to an age of 7
months, independent of HCV transgene status. However,
chemical induction of tumor development with DEN/PB treat-
ment lead to a clear increase of liver tumors in HCV+;PML2/2
animals when compared to identically treated WT mice (Fig. 5).
In all treatment groups, livers exhibited abnormal morphology.
All livers displayed numerous pleomorphic and atypical hepato-
cytes and an altered, remarkably nodular liver structure, which
was in contrast to livers of age-matched untreated WT mice
(Fig. 5a). In addition to macroscopic quantification of tumors in
mouse livers, we also measured the size of the individual lesions.
To this end, five representative liver slides of every mouse of the
four treatment groups as well as untreated control groups were
subjected to morphometric analysis of the size of the tumor area.
Interestingly, PML-negative livers displayed strikingly more lesions
(median: 2065) with smaller diameter (,3 mm) in average after
treatment (Fig. 5b), while PML-positive mice developed relatively
more lesions with a diameter .3 mm (Fig. 5c). Furthermore,
average tumor mass was calculated in relation to liver mass and
given as relative tumor mass in Fig. 5d. In WT mice the relative
tumor mass counts for 6,861,1, whereas in HCV;PML2/2 mice
the relative tumor mass is 5-fold as high (25,366,1). Therefore, in
PML-deficient mice not only the number of tumor lesions in total
was considerably increased but also the cumulative tumor area was
To determine whether the observed tumors are indeed HCCs,
livers were analysed by board certified pathologists. Histopatho-
logical evaluation of the livers after 7 months yielded at least one
HCC in 73% of HCV;PML2/2 mice. In contrast, only 7% of the
WT;PML+/+ mice exhibited HCC formation (Fig. 5e).
WT;PML2/2 mice developed at least one HCC in 53% of
animals even in absence of the HCV-Tg and HCV;PML+/+ mice
in 27%. Thus, PML-deficiency was associated with a significant
Figure 5. Liver tumors develop predominantly in HCV+ +PML- mice. Macroscopic inspection and HE staining of livers of all analysed mice
revealed a spectrum of findings ranging from a macroscopically unremarkable (untreated) to a strongly nodular structure (PML-deficient with HCV-
Tg) at the age of 7 months (A). Macroscopic analysis of all 4 DEN/PB-treated mouse strains as indicated, with 15 mice in every group. PML-deficient
mice are more susceptible towards carcinogenic stimuli and develop dysplastic nodules evolving to carcinomas after 7 months. Tumorous lesions
were macroscopically analysed and quantitatively evaluated as lesions ,3 mm in (B) and .3 mm in (C). Relative tumor mass was calculated as
number of lesions in relation to average size of lesions and liver mass (D). The data displayed in (B), (C) and (D) refer to the mice which displayed
lesions. The number of mice displaying lesions pro rata 15 mice in every group is given as percentage of the livers showing at least one dysplatic
focus or HCC in (E).
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increase in HCC incidence which was further aggravated by the
However, after 7 months of treatment at least one dysplastic
focus was found in livers of all PML-deficient mice. In contrast,
only 42% of WT mice displayed at least one dysplastic focus.
The larger nodules found in PML-positive livers where either
dysplasias or unspecific fibrotic nodules most likely due to
regenerative processes. In contrast, PML-deficient livers develop
a higher number of rather small dysplasias that develop to HCC at
an early stage of growth. Untreated age-matched mice in the
control groups revealed macroscopically normal livers.
In summary, we observed that PML-deficient mice were
markedly more sensitive towards carcinogenic stimuli, which was
further pronounced in presence of the HCV transgene.
Histologic analysis confirmed that all of the HCV;PML2/2
mice displayed liver tumors at the age of 7 months (Fig. 4). Those
tumors were characterized by cellular atypia, altered liver
architecture with broadening of liver cell cords and loss of
reticulin fibers (shown by Gomori staining; Fig. 6a). In addition,
the proliferation rate was increased compared to non-tumorous
areas, as a focal pattern of strong immunoreactivity for glutamine
synthetase was observed (Fig. 6c) paralleled by more PCNA
positive cells (Fig. 6d). Taken together the histological findings
qualify the tumors as HCC.
Liver Carcinogenesis is Accompanied by Downregulated
To obtain insight into the molecular mechanisms of tumor
development in PML2/2 livers with or without the HCV
transgene, mRNA expression of several apoptosis-related factors
Elevated transcript levels of survivin were detected in livers of 7
months old mice compared to untreated controls, with a non-
significant but considerable increase in PML2/2 livers only for
survivin and bcl-2 (Fig. 7a). However, neither transcript levels of
Mcl-1, Bcl-xL or XIAP were significantly different between
treatment groups, whether expressing PML or the HCV transgene
Differential expression of the proapoptotic genes NOXA,
PUMA, CD95L/CD95R and TRAIL-R between treated mice
versus untreated controls could not be found (data not shown).
However, the expression of the proapoptotic factor TRAIL, a
protein of the TNF-family associated with hepatocyte apoptosis
and carcinogenesis in the liver, exhibited enhanced induction
upon treatment in both PML-expressing mouse strains. TRAIL
was clearly induced in treated WT;PML+/+ mice which showed
mild tumor growth after liver carcinogenesis. In contrast, TRAIL
expression was low in the PML-deficient mice, independent of
HCV (Fig. 7b). Furthermore, HCV-Tg expression also resulted in
reduced TRAIL expression levels, indicating that HCV suppresses
TRAIL expression in vivo. These findings were as well reflected on
the protein level (Fig. 7c). Since TRAIL-expression was increased
upon treatment in both PML-positive treatment groups, our
findings suggest that TRAIL-regulation requires functional PML
in this context. Furthermore, in accordance with our previous
findings  this suggests that HCV core may inactivate PML
partially and, thus, TRAIL-expression is compromised upon
expression of the HCV-Tg.
HCC is recognized as one of the most common and most
malignant cancers worldwide. The hepatitis C virus (HCV) counts
as a major risk factor for the development of HCC [20,21] and
there is increasing experimental evidence to suggest that the virus
plays a direct role in neoplastic transformation . However, the
molecular mechanisms causing this sequence of events are still
poorly understood. In this study, we describe HCC development
in HCV-Tg mice after depletion of the tumor suppressor PML.
Our results suggest that: (I) PML protects against hepatocarcin-
ogenesis, (II) PML-deficiency makes liver cells more susceptible
towards carcinogenic stimuli, (III) HCV promotes carcinogenesis
Figure 6. Development of liver cell tumors (HCC) in PML- HCV+ + mice. The Gomori staining (A) and H&E (B) of the specimens demonstrate
that no fibrotic changes have occurred during tumor development. Immunohistochemistry indicates a high expression of glutamine synthetase in
most HCCs, however only pericentral expression was observed in the peritumorous tissue area (C). PCNA staining revealed a high proliferation rate in
the HCC (D). The dashed border lines show the intersection between the HCC and the peritumorous tissue areas. The lower pictures were high
magnification of the squared areas in the upper pictures in the figure legends.
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in the liver (IV) the oncogenic potential of HCV is supported by
inactivation of PML.
The tumor suppressor PML is the essential structural organizer
of nuclear multiprotein structures termed PML-NBs . Studies
in knockout mice and cells unraveled an essential pleiotropic role
for PML in multiple p53-dependent and -independent apoptotic
pathways [19,24]. PML2/2 mice and cells are protected from
apoptosis triggered by a number of stimuli such as ionizing
radiation, interferon, ceramide, Fas and TNF . Recently we
could show that the HCV core interferes with p53-dependent
apoptosis in liver cells by interacting with PML and compromising
its function . Thus, we hypothesized that inactivity of PML
could be a crucial madiator in HCV-associated carcinogenesis.
Therefore, we generated HCV-transgenic PML-deficient mice by
mating a mouse strain expressing the HCV transgene in the liver
with PML2/2 mice. In order to achieve tumor development in
an appropriate time, the resulting mouse genotypes were treated
with DEN/PB for half a year .
In our model, HCV-Tg livers exhibited a decrease in
hepatocyte apoptosis rate, and an increase in proliferation and
liver size. In HCV;PML2/2 mice an enhanced proliferation rate
of hepatocytes was observed. However, the decrease of apoptosis
rate was not sufficient to compensate for the high grade of
proliferation in the liver. Serum markers of liver cell damage, AST
and ALT, were also significantly increased in PML-deficient mice
with even higher values in presence of the HCV-Tg. Decreased
body weight, increased liver size, high aminotransferase levels and
high proliferation rate coincided with impressively pronounced
hepatocarcinogenesis in HCV;PML2/2 mice. Those mice
displayed a considerably higher number of not only dysplastic
nodules but also HCCs. However, both PML-deficient mouse
strains developed more HCCs than the PML-expressing mouse
strains. Interestingly, in PML-positive livers less but larger lesions
were found, which were histologically characterized as mainly
unspecific fibrotic nodules or early dysplasias. This might be
explained by the fact that PML-expressing livers have a higher
apoptosis rate and the process of damage, apoptosis and
compensatory proliferation may lead to fibrotic changes due to
regeneration. In case of PML-deficiency, apoptosis is severely
compromised, proliferative processes prevail and carcinogenic
processes take their track.
We detected liver tumors of varying size in all mouse strains
after 7 months of treatment. Some liver tumors did not (yet) meet
the histological criteria of HCC. However, morphology on a
Figure 7. Expression of pro- and antiapoptotic genes. (A) mRNA expression analysis of several anti-apoptotic factors (Mcl-1, Survivin, Bcl-2,
XIAP, Bcl-xL) by quantitative RT-PCR in the four treated transgenic groups. Boxes represent the average expression level of control. All values are
normalized to SDHA mRNA expression. Standard deviation is indicated by error bars. (B) mRNA expression analysis of TRAIL by quantitative RT-PCR,
comparing the four treated mouse strains versus untreated controls. Boxes represent the average expression level of control. All values are
normalized to SDHA mRNA expression. Standard deviation is indicated by error bars. (C) Whole liver extracts derived from 7 months old mice were
analysed by immunoblot analysis for the protein expression of TRAIL as well as tubulin (loading control). A representative of five independent
experiments is shown.
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macroscopic and histological level of a number of tumors,
combined with the expression of glutamine synthetase, confirmed
them as HCC. The malignant character did not depend on the
size of the lesions. In summary, HCC were heterogenous as
corroborated by different patterns of morphology and immuno-
histochemistry. This argues against one particular molecular
pathway involved in HCC formation in HCV-dependent carci-
nogenesis. In contrast it favours the notion that compensatory
mechanisms underlie HCC formation in HCV-transgenic mice.
Under physiologic conditions, presence of viral proteins in the
liver can cause an inflammatory response. Studies involving the
use of transgenic mice have demonstrated that HCV and its
proteins have the ability to induce fibrosis, either through direct
interference with cell activation pathways or by indirect means, via
the induction of steatosis . The chronic inflammatory process
of HCV infection could lead to increased mutagenesis in the
regenerating hepatocytes, leading to a multi-step process of
mutations finally presenting as HCC . However, in auto-
immune hepatitis, the occurrence of HCC is rare , raising
doubts as to whether inflammation alone is able to lead to such a
high incidence of HCC in patients infected with HCV. In line with
that, in our model, we did not observe an increased invasion of
inflammatory cells in treated or untreated transgenic mice. This is
supported by unaltered IL-6 and TNF-alpha expression in
untreated HCV-Tg mice. Mice treated with DEN/PB, though,
have an increased expression of these inflammatory mediators
which is somewhat expected in the context of the hepatocarcin-
ogenesis protocol. But no differences between the different
transgenic mouse groups were observed. Thus, contribution of
inflammatory processes to the carcinogenic process may not be
relevant in this model.
Previous experimental evidence suggests that the pathogenesis
of HCC from HCV infection includes various viral protein–host
cell interactions, which may play a direct role in the development
of HCC [22,28]. Perturbations in the cell cycle, combined with
upregulation of oncogenes and loss of tumour-suppressor gene
functions, may contribute to HCC development. Notably, HCV
proteins have been shown to interact with these cellular pathways.
We previously demonstrated that HCV core compromises
apoptotic processes in liver cells by inhibition of PML .
Moreover, our herein presented data contribute the in vivo finding
that HCV-transgenic mice are more susceptible towards carcino-
genic stimuli. Thus, our results support a direct oncogenic
implication of the hepatitis C virus which is in accordance with
PML is a proapoptotic factor, and its deficiency favours an anti-
apoptotic and pro-proliferative state. However, the absence of
other anti-apoptotic factors may result in a hypoapoptotic
environment, finally resulting in the outgrowth of a malignant
cell population. The proapoptotic molecule TRAIL has gained
attention for its ability to induce apoptosis in liver cancer cells
without damaging normal liver cells [29,30]. It may play an
important role in preventing development and outgrowth of liver
tumors. Moreover, TRAIL is partially regulated by PML in
certain settings in the liver . Our finding that TRAIL-levels
are significantly reduced in PML-deficient animals suggests a
possible contribution in the development of HCC in our model.
While PML-positive livers do express TRAIL, the expression level
is considerably compromised in presence of HCV. This is in line
with our observation of HCV core compromising PML-function,
leading to an impaired expression of PML-dependent proapopto-
tic factors. In addition to that, we were able to previously show a
direct link between PML and TRAIL in HCC cell lines by RNAi
silencing of PML, resulting in down-regulation of TRAIL
expression in hepatoma cells. In addition, PML-deficient primary
human hepatocytes fail to upregulate TRAIL upon IFN-alpha-
treatment in contrast to their WT counterparts19. Furthermore,
TRAIL and its receptors have been shown to be upregulated in
the liver of HCV infected patients and PML is a molecule which,
as well, is upregulated in inflammatory settings as viral hepatitis.
The fact that TRAIL is also upregulated in an inflammatory
context underlines PML-dependent regulation of TRAIL and
supports a link between TRAIL and PML. One may speculate
that compromised expression of a death inducing molecule like
TRAIL contributes to a privileged setting for outgrowth of liver
Taken together, this study provides the first in vivo evidence that
deletion of the proapoptotic factor PML is accompanied by
severely enhanced sensitivity of the liver towards carcinogenic
stimuli and, as well, supports a direct oncogenic function of HCV
driving HCC formation.
We thank Stanley M. Lemon, Urzula Hibner and Herve Lerat for
providing FL-N/35 -mice and Hans Will for providing PML-deficient
mice. Sandra Weyer and Claudia Braun contributed excellent technical
assistance. We are grateful to Johannes Lotz for coordination of the clinical
chemical analysis. We are particularly endepted to Stephan Kanzler,
Markus Mo ¨hler, Torsten Maas, and Peter H Krammer for helpful
comments, critical reading of the manuscript and constant support.
This work forms part of the thesis of Anna Carbow.
Conceived and designed the experiments: KH TGH A. Canbay.
Performed the experiments: KH A. Carbow SS JPS SB. Analyzed the
data: KH SB PRG A. Canbay. Contributed reagents/materials/analysis
tools: SB TGH PRG GG. Wrote the paper: KH A. Canbay.
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