Introduction
Non-melanoma skin cancer (NMSC) is the most common cancer in adult fair-skinned populations
[1]. Ultraviolet light (UV) is a key risk factor for the NMSC [2]–[4]. In addition,
it appears that infectious agent(s) may favor skin carcinogenesis. This is suggested
by the fact that immuno-compromised organ transplant recipients (OTRs) have a 50–100-fold
higher risk of developing NMSC compared to the general population [5], [6] [6] [7]
[8]. A sub-group of cutaneous human papillomavirus (HPV) types, belonging to the genus
beta of the HPV phylogenetic tree, are putative etiological factors of NMSC [9] [10].
These HPV types were first isolated in individuals with an autosomal recessive disorder,
termed epidermodysplasia verruciformis (EV). EV individuals are susceptible to infection
by beta HPV types and have a propensity to develop confluent flat warts, which, in
approximately 30% of the cases, progress to squamous cell carcinomas (SCC) on sun-exposed
areas [9] [10]. Accordingly, DNA from several beta HPV types was found in a high percentage
of precursor lesions, actinic keratoses, and SCC from OTRs [11] [12] [13]. More recent
studies indicate that beta HPV types are also involved in skin carcinogenesis in the
normal population. Detection of antibodies against the major capsid protein L1 showed
an increased seroreactivity to beta HPV types in patients with cutaneous SCC in comparison
to healthy individuals [14] [15] [16] [17] [18] [19].
Functional studies have provided further evidence of an association of beta HPV types
with NMSC. Since previous studies demonstrated the key role in cellular transformation
of E6 and E7 oncoproteins from cervical cancer-associated mucosal high-risk (HR) HPV
types, functional investigations on beta HPV types focused on the characterization
of E6 and E7 biological properties. These studies showed that E6 and E7 from beta
HPV types also displayed transforming capability in in vitro and in vivo experimental
models [10]. E6 from beta HPV types associates with the pro-apoptotic protein Bak,
a member of the Bcl-2 family, promoting its proteasomal degradation and preventing
apoptosis in response to genomic stress [20] [21]. Studies from our group have shown
that E6 and E7 from beta HPV38 are able to immortalize primary human keratinocytes
[22] [23], similarly to E6 and E7 from the mucosal HR HPV types. Accordingly, we observed
that HPV38 E6 and E7 expression in these cells leads to the accumulation of ΔNp73α,
which in turn alters the p53 transcriptional functions [24].
Tg mouse lines expressing the entire early region of beta HPV8 (E6, E7, E1 E2 and
E4 genes) or the E6 gene alone driven by the K14 promoter, spontaneously developed
multifocal skin tumours and, in approximately 6% of the cases, SCC [25] [26]. In addition,
a single dose of UV rapidly promoted papillomas and SCC formation [26]. In another
study, Tg mouse models for the beta HPV20 and the benign cutaneous HPV27 were generated,
in which E6 and E7 oncoproteins were expressed as single polycistronic transcript
under the control of the K10 promoter that is active in the supra-basal differentiated
layer of the skin epidermis [27]. Both Tg models developed skin lesions, including
SCC after exposure to UV irradiation. However, no significant difference in the skin
tumour incidence was observed between HPV 20 and 27 Tg animals [27]. Based on our
in vitro data [22] [23] [24], we generated K10 HPV38 E6/E7 Tg mice [28]. These animals
displayed hyperplastic and dysplastic patches in the skin epidermis, but no spontaneous
development of skin cancer was observed during their life span. Application of the
two-stage skin carcinogenesis protocol led to a strong increase in skin tumour incidence
[28]. However, chronic UV irradiation of K10 HPV38 E6/E7 Tg did not lead to development
of any type of skin lesions (Dong et al. unpublished data). The failure of HPV38 E6
and E7 to cooperate with UV irradiation in skin tumour development in this animal
model could be explained by the fact that the viral genes were expressed in the suprabasal
layers of the epidermis, while in humans beta HPV types infect and initiate the transcription
of the early genes in the basal layer. To explore this hypothesis, we developed a
novel transgenic mouse model for HPV38 with K14 promoter-driven expression of E6 and
E7 in the basal and proliferative rather then the differentiated compartment of skin
epidermis. Here, we show that ectopic HPV38 E6 and E7 expression in this location
strongly enhances the susceptibility to chemical- and UV-induced carcinogenesis. Most
importantly, chronic UV irradiation of K14 HPV38 E6/E7 Tg mice results in the development
of actinic keratosis-like lesions and SCC, closely resembling the scenario observed
in humans.
Results
Generation and characterization of K14 HPV38 E6/E7-Tg mice
To evaluate the transforming properties of E6 and E7 from HPV38 in the proliferative
compartment of skin epidermis, we generated Tg mouse lines expressing the two viral
oncogenes under the control of the human keratin 14 promoter that is active in the
basal layer of the epidermis [29]. A schematic representation of the transgene construct
used is shown in Figure 1A. Transgene-positive offspring were identified by PCR of
tail DNA using HPV38 specific primers. Two independent Tg mouse lines (183 and 187)
were identified and bred successfully. Viral oncogene expression was determined by
RT-qPCR in different epithelia, i.e. ear, dorsal skin, tongue, and esophagus. Line
183 expressed higher HPV38 E6/E7 levels than line 187 in all four examined epithelia
(Figure 1B). In each Tg line, HPV38 E6/E7 expression also differed in the four epithelia,
being highest in the dorsal skin and the ear, and comparably low in the tongue and
esophagus (Figure 1B). As expected, no viral oncogene expression was observed in liver
tissue that was included as a negative control (Figure 1B). No HPV38 E6 and E7 expression
was detected in the same tissues of the wild-type animals (data not shown).
10.1371/journal.ppat.1002125.g001
Figure 1
HPV38 E6 and E7 expression in Tg mice.
(A) Schematic representation of the K14-HPV38 E6/E7 construct. (B) HPV38 E6 and E7
transcripts are differentially expressed in the epithelia of the two hemizygous Tg
mouse lines 183, and 187. Total RNA was extracted from the ear, the skin, tongue,
esophagus, and liver. After preparation of cDNA, E6 and E7 expression was determined
by RT-qPCR and normalized towards the expression level of GAPDH. The data shown in
the Figures are the means ±SD of three independent experiments. In each experiment
the 187 ear data is set to 1 and the other values are consequently resized.
HPV38 E6 and E7 induce cellular proliferation in the epidermis of Tg mice
Next, we examined whether HPV38 E6/E7 expression induced morphological alterations
in the epithelia analyzing HE-stained sections of skin, ear, tongue and esophagus
of FBN/V and K14 HPV38 E6/E7-Tg mice. Epidermal hyperplasia in the ear skin was observed
in approximately 5% of 6–8 week-old mice from both Tg lines, as representatively shown
in Figure 2A. These alterations, although also detected, were much less evident in
dorsal skin (Figure 2A). No significant morphological changes were observed in epithelia
of the esophagus and tongue of both Tg lines (data not shown).
10.1371/journal.ppat.1002125.g002
Figure 2
Histological analysis of skin specimens from wild-type FVB/N and Tg mouse lines.
Representative pictures (original magnification 40×) of HE-stained sections of paraffin-embedded
tissues are shown: (A) ear (left panel) and dorsal skin (right panel) of wild-type
FVB/N and Tg mice of the lines 183, and 187. (B) Dysplastic ear skin of K14 HPV38
E6/E7-Tg mice.
The morphological alterations observed in ear skin were even more severe in older
animals of lines 183 and 187. Approximately 10–15% of 12-month-old mice from both
lines presented dysplasia and, hyperkeratosis. A representative section is shown in
Figure 2B, where severe dysplastic keratinocytes, hyperkeratosis, endophytic papillomatous
epidermis and a pronounced inflammation could be observed.
To determine whether the expression of HPV38 E6 and E7 oncoproteins resulted in a
deregulation of cellular proliferation, we next analysed the expression of the proliferation
marker Ki-67 by immunohistochemistry. A significant increase of Ki-67 positivity was
observed in the ear (line 187 Vs FVB/N p<0,001, line 183 Vs FVB/N p<0.05) and dorsal
skin (line 187 Vs FVB/N p<0.001, line 183 Vs FVB/N p<0.01) epidermis of the two Tg
mouse lines (Figures 3A and 3B). Although morphological changes were not observed
in tongue and esophagus up to the age of 12 months, an increased Ki-67 index was detected
in the epithelia of these tissues at similar levels to those observed in the ear and
dorsal skin (data not shown). To corroborate these data, we determined the levels
of the positive cell cycle regulator cyclin A in protein extracts from dorsal skin
of wild-type and Tg mice by immunoblotting. Cyclin A levels were higher in the two
Tg mouse lines as compared to the control animals (data not shown).
10.1371/journal.ppat.1002125.g003
Figure 3
Analysis of cellular proliferation in the ear and dorsal skin of wild-type and transgenic
mice.
(A and B, top panels) Representative pictures of Ki-67 immunostained sections of paraffin-embedded
ear skin and dorsal skin from wild-type (FVB/N) and Tg animals (lines 183 and 187).
(A and B, lower panels) Quantification of Ki-67-positive cells (brownish signal) in
wild-type and Tg epidermis was done by counting 400 hematoxylin-stained cells under
40× magnification in 4 different fields of epidermis. Differences between the Ki-67-positive
cells in the HPV38 E6/E7 Tg mice (lines 183 and 187) versus the FVB/N mice were statistically
significant as determined by Student's t-test with Welch correction for unequal standard
deviation.
Together, these data show that ectopic overexpression of HPV38 E6 and E7 oncogenes
in the basal layer of the mouse epithelia significantly increased cellular proliferation.
Enhanced formation of SCC in skin of K14 HPV38 E6/E7 transgenic mice upon DMBA/TPA
treatment
Previous studies reported an increased incidence of papillomas and SCC in Tg mouse
models expressing E6 and E7 from human or animal papillomaviruses [28] [30] [31] when
exposed to chemical carcinogens. Therefore, we compared tumour susceptibility of wild-type
and K14 HPV38 E6/E7 Tg mice in the multi-stage skin carcinogenesis protocol using
DMBA as initiator and TPA as tumour promoter. Wild-type and Tg animals were exposed
to a single treatment of DMBA followed by repeated TPA treatments for 20 weeks and
subsequent examination for a further five weeks (Figure 4A). Seven weeks after initiation,
100% of Tg animals of both lines had developed tumours, while at the same time wild-type
animals showed no skin lesions and became 100% tumour positive three weeks later (Figure
4B). At week 10, the DMBA/TPA-treated back skin of Tg mice was entirely covered by
tumours, in contrast to the wild-type animals that developed only a small number of
skin lesions (Figure 4C). Due to the high number of skin lesions including large and
multiple SCC per mouse, all Tg animals of lines 183 and 187 were sacrificed at week
12 and 14, respectively, while this event was delayed until week 24 in the wild-type
group (Figure 4D). The number of tumours per animal was significantly higher in the
Tg cohort as compared to the wild-type cohort (Figures 4E). After completion of the
experiment at week 24, histological examination of skin lesions from all control and
Tg mice was performed. Accordingly, Tg mice of both lines developed SCC more rapidly
(Figure 4F) and at higher incidence than the wild-type mice (Figure 4F). Representative
sections of the tumours at week 10 from control and Tg animals are shown in Figure
4G. The shown tumour of the control animal was at an early stage and disruption of
the basement membrane and extension of tumour islands into the underlying dermis started
to be visible (Figure 4G). The representative sections from Tg animals evidenced SCC
characterized by tumour cells with prominent intercellular bridges, abundant eosinophilic
cytoplasm and a large and vesicular nucleus, plus aberrant accumulations of keratin
(keratin pearls) (Figure 4G, line 183), as well as by irregularly elongated rete pegs
with atypia, defined as vacuolization and nuclear abnormalities of cells of the stratified
squamous epithelium invading the connective tissue (Figure 4G, line 187).
10.1371/journal.ppat.1002125.g004
Figure 4
Tumour burden in wild-type and transgenic mice after DMBA/TPA treatment.
(A) Schematic diagram of the initiation and promotion protocol using DMBA as initiator
and TPA as promoter of the two-stage skin carcinogenesis approach. (B) Tumour incidence.
Percentage of animals with skin tumours in the group of FVB/N wild-type and Tg cohorts
of lines 183 and 187. Skin tumour formation was recorded each week until the end of
the experiment in week 24 after the beginning of promotion. The difference between
the curves of control and transgenic mice is statistically significant (p<0.0001 determined
by logrank test for group data). (C) Representative pictures of dorsal skin from wild-type
FVB/N and Tg mice 10 weeks after the beginning of tumour promotion. (D) Survival curves
for DMBA/TPA-treated wild-type FVB/N and Tg animal cohorts of lines 183 and 187. Mice
were sacrificed when putative SCC skin lesions reached the size of 15 mm in diameter.
The difference between the curves of control and transgenic mice is statistically
significant (p<0.0001 determined by logrank test for group data). (E) Tumour multiplicity.
Maximum number of tumours per animal in the groups of wild-type and Tg lines. The
number of tumours was recorded every week. Differences between the tumour multiplicity
in the group of wild-type and Tg lines are statistically significant (control versus
line 183, p<0.001; control versus line 187, p<0.001 as determined by Wilcoxon rank
sum test). (F) Incidence of cutaneous SCC in the group of wild-type and transgenic
mice. SCC in the sacrificed wild-type and Tg animals were confirmed by histological
analyses. The difference between the curves of control and transgenic mice is statistically
significant (p<0.0001 determined by logrank test for group data). (G) Representative
pictures of HE-stained skin lesions from wild-type (FVB/N) and K14 HPV38 E6/E7-Tg
mice (lines 183 and 187) collected after 10 weeks of chemical carcinogens treatment
(original magnification 5×). Magnified areas are shown in the right panels.
In summary, these results show that ectopic expression of HPV38 E6 and E7 in the proliferative
compartment of skin epidermis significantly increases the tumour burden including
papillomas and SCCs in a DMBA/TPA multi-step skin carcinogenesis approach.
Reduced UVB-induced cell-arrest and enhanced UVB carcinogenicity in K14 HPV38 E6/E7
Tg mice
UV irradiation is a key risk factor for NMSC in humans. Therefore, we next determined
whether K14 HPV38 E6/E7-Tg mice had an enhanced susceptibility to UVB irradiation.
Induction of DNA damage by UV irradiation normally leads to the activation of cellular
defense mechanisms, mainly mediated by p53 activation that in turn induces cell cycle
arrest prior to the S phase or apoptosis. The cell cycle block is primarily mediated
by accumulation of the cyclin-dependent kinase inhibitor (CDK) p21WAF1, whose gene
is positively regulated by p53. Short-term UVB irradiations of the skin of wild-type
mice resulted in an accumulation of p21WAF1 in keratinocytes of the basal layer of
the epidermis (Figure 5A). In contrast, this phenomenon was significantly less evident
in the skin of Tg animals (Figure 5A, right panel, P<0,001 after the third UVB irradiation).
In agreement with the p21WAF1 expression levels, staining of the Ki-67 proliferative
marker was stronger in the skin of the Tg mice in comparison to the wild-type animals,
even after several doses of UV irradiation (Figure 5B). These data show that HPV38
E6 and E7 have the ability to interfere with the regulation of cellular checkpoints
activated by genomic stress, such as UV-induced DNA damage. Thus, it is likely that
HPV38 enhances the carcinogenicity of UV irradiation.
10.1371/journal.ppat.1002125.g005
Figure 5
p21WAF1 and Ki-67 levels in the skin of wild-type and K14 HPV38 E6/E7-Tg mice after
UVB irradiation.
Wild-type and Tg animals were irradiated up to 5 times as described in Materials and
Methods. 24 hours after the last irradiation, mice were sacrificed and skin tissue
was analyzed by immuno-histochemistry. (A) Representative Ki-67 and p21WAF1 immunostainings
of skin from wild-type and Tg mice non-exposed (0×) or four time (4×) exposed to UVB.
(B) Quantification of p21WAF1 and Ki-67-positive cells in skin of wild-type and Tg
mice before and after UVB irradiation. The percentage of p21WAF1 and Ki-67-positive
cells in the epidermis was determined as described in the legend of Figure 3. The
differences between the percentages of p21WAF1 or Ki-67-positive cells in the HPV38
E6/E7 Tg mice (lines 183 and 187) versus the FVB/N non-Tg mice are statistically significant
(* = p<0.05, ** = p<0,001) as determined by Student's t-test.
To evaluate this hypothesis, long-term UVB experiments, in which FVB/N and Tg mice
of lines 183 and 187 were exposed to multiple and increasing doses of UVB, were carried
out (Figure 6A). After 20–25 weeks of treatment, the majority of the Tg mice from
both lines showed thick, scaly, crusty and reddish patches in dorsal skin exposed
to the UVB light, while no lesions were observed in wild-type animals (Figure 6B).
Histological analyses revealed that these lesions resemble the precancerous condition
of actinic keratosis. The representative sections in Figure 6 C show downward prolongations
and slight atypia of the rete pegs without stromal invasion (top panels) and parakeratosis,
acanthosis and broadened elongated rete ridges with atypia (bottom panels), which
are all features of the SCC precursor, actinic keratosis. In later weeks (25–30),
SCC become visible on the UV-irradiated skin of Tg mice from both lines, while still
no lesions were detected in the control mice (Figure 6D). Histological analysis of
skin sections revealed that more than 80% of the Tg animals from line 183 developed
SCC during the 30 weeks of UV irradiation (Figure 6E). SSC were also detected in the
dorsal skin of line 187 Tg animals, but after a longer latency period than in line
183 (Figure 6E). The different incidence of SCC in the two Tg lines tightly correlates
with the HPV38 E6 and E7 expression levels (Figure 1B). Representative images of SSC
lesions observed in K14 HPV38 E6/E7 Tg mice after 30 weeks of treatment are shown
in Figures 6F. These lesions show the presence of tumour cells with atypia, horn formation
(Figure 6F, top panels) and tumour cell invasion deep into the dermis (Figure 6F,
bottom panels). In contrast, histological examination of dorsal skin sections from
FVB/N animals at weeks 30 evidenced only irritation and slight atypia in the epidermis
(Figure 6G).
10.1371/journal.ppat.1002125.g006
Figure 6
Tumour burden in wild-type and K14 HPV38 E6/E7-Tg animals upon UVB irradiation.
(A) Schematic diagram of the experimental procedure of long-term UVB irradiation.
(B) Representative pictures of dorsal skin from wild-type FVB/N and HPV-Tg mice exposed
to UVB light for 24 weeks. (C) Representative pictures of HE-stained actinic keratosis
affected epidermis (AK) from K14 HPV38 E6/E7-Tg mouse lines 183 and 187 after 24 weeks
of irradiation (original magnification 10×). Magnified areas are shown in the right
panels. (D) Representative pictures of dorsal skin from wild-type FVB/N and HPV-Tg
mice exposed to UVB light for 29 weeks. (E) Percentage of animals with skin SCC in
wild-type and Tg cohorts of lines 183 and 187. Tumour formation was monitored each
week until the end of the experiment in week 30 after start of treatment, and confirmed
by histological analyses after sacrifice of the animals. The difference between the
curves of control and transgenic mice is statistically significant (p<0,0001 determined
by logrank test for group data). (F) Representative pictures of HE-stained of SCC
sections (SCC) from K14 HPV38 E6/E7-Tg mouse lines 183 and 187 after 30 weeks of UVB
irradiation (original magnification 5×). Magnified areas are shown in the right panels.
(G) Representative pictures of HE-stained epidermis from wild-type FVB/N mice after
30 weeks of UVB irradiation (original magnification 10×). Magnified area is shown
in the right panel.
Together, these data show that HPV38 oncoproteins and UVB irradiation cooperate in
the development of actinic keratosis and SCC.
Discussion
The role of beta HPV types in NMSC is still not conclusively fully established. Functional
studies revealed that beta HPV E6 and E7 proteins, similarly to their homologues from
the mucosal HR HPV types, have the ability to deregulate fundamental cellular events,
such as cell cycle, apoptosis and senescence [20] [21] [22] [23] [24] [32]. However,
despite the functional similarities between E6 and E7 from beta and mucosal HR HPV
types, the two subgroups of HPV types may induce cancer development by two distinct
mechanisms. It is well demonstrated that the mucosal HR HPV types play a key role
in cancer initiation and maintenance. In fact, their genomes and E6 and E7 expression
are detected in all cervical cancer cells, and inhibition of the expression of these
viral oncogenes in those cells resulted in a rapid induction of apoptosis and/or senescence
[33]. In contrast, analyses of skin lesions suggest that beta HPV types may be involved
only at early stage of skin carcinogenesis. This hypothesis is mainly based on two
findings: (i) higher viral load was found in the SCC-precursor lesion AK than in SCC
and (ii) not all cancer cells resulted positive for beta HPV DNA [34]. Taking into
consideration also the properties of beta HPV oncoproteins to interfere with the regulation
of cell cycle and apoptosis, it is conceivable to hypothesize that beta HPV E6 and
E7 enhance the carcinogenicity of sunlight, facilitating the accumulation of DNA damages
induced by UV and consequently cancer development. In normal cells, DNA damage induced
by UV irradiation activates cellular defense processes leading to p53 activation,
which in turn induces cell-cycle arrest or apoptosis to allow repair or elimination
of the damaged cells, respectively. In contrast in beta HPV infected cells, E6 and
E7 expression circumvent the activation of the cellular defense processes by the UV
irradiation, maintaining the cells in a proliferative state and allowing efficient
viral DNA replication. As a side-effect, these events favour the accumulation of UV-induced
DNA damages and cellular transformation. Due to the irreversible nature of the UV-induced
damages, e.g. mutation of tumour suppressor genes, the maintenance of the transformed
phenotype may become independent of the viral gene expression.
Our data obtained with K14 HPV38 E6/E7 Tg mice support this model. Indeed, while in
normal mice UV irradiation led to accumulation of the cell-cycle inhibitor p21WAF1
and cell cycle arrest, in the Tg animals these UV-induced phenomena were strongly
inhibited. In addition, although HPV38 E6 and E7 expression per se did not lead to
significant morphological alterations of the epidermis, it strongly facilitated the
induction of SCC by chemical carcinogens or chronic UV irradiation. Most importantly,
SCC development upon chronic UV exposure of K14 HPV38 E6/E7 Tg mice irradiation was
preceded by lesions that closely resemble actinic keratosis, the precursor lesions
of SCC also observed in humans.
The different susceptibility of the K10 and K14 HPV38 E6/E7 Tg models to UV-mediated
carcinogenesis indicates that the expression of HPV38 E6 and E7 in the basal layer
of the epidermis is an essential event for the development of skin cancer induced
by chronic UV irradiation. This conclusion is supported by the fact that the natural
HPV infection and expression of the viral oncoproteins initiate in the cells of the
basal layer. However, it is also possible that the different behavior of the K10 and
K14 Tg mice after exposure to chronic UV irradiation is due to the different efficiency
of the two keratinocyte promoters, K10 and K14, in expressing the viral genes. Indeed,
K10 Tg animals express approximately 3–4 lower levels of HPV38 E6 and E7 than K14
Tg animals (Viariso et al, unpublished data). Thus, additional studies are required
to elucidate the different cancer susceptibility of Tg mice expressing the viral oncogenes
in the basal or supra-basal layers of the skin. To evaluate the importance of the
HPV38 E6 and E7 expression levels in UV-induced carcinogenesis, we are currently generating
a novel K14 Tg line that expresses similar levels to the K10 Tg animals.
Previous transgenic models used to study the role of beta HPV in cutaneous cancer
express the entire early region (ER) of HPV8 or E6 gene under control of the K14 promoter
[25] [26]. The HPV8 ER and HPV8 E6, showed a remarkable similarity in the development
of skin lesions. Indeed, a single dose of UV led to a rapid development of papillomas
and SCC in both Tg models [26], which also spontaneously developed benign tumours
and, in a small percentage, SCC [25] [26]. Although these data support the role of
HPV8 in skin carcinogenesis, the HPV8 animal models do not closely correspond to the
situation observed in humans, where beta HPV infection is normally asymptomatic and
SCC development appears to be strongly associated with chronic UV exposure. The difference
observed in K14 HPV8 E6 or HPV8 ER and K14 HPV38 E6/E7 Tg mice may simply reflect
the more aggressive properties of the oncoproteins from HPV8 in comparison to HPV38.
However, studies in in vitro experimental models do not support this hypothesis. In
fact, HPV8 E6 and E7 displayed lower in vitro transforming activities when compared
to the oncoproteins from the mucosal HR HPV types [35] [36] [37]. In contrast, HPV38
E6 and E7 were able to immortalize primary human keratinocytes [22] [23], to deregulate
p53 functions [24] and up-regulate the expression of the catalytic subunit of the
telomerase hTERT [23] [32], all features shared with E6 and E7 from the mucosal HR
HPV types. The different phenotype of the K14 HPV8 ER and K14 HPV38 E6/E7 Tg mice
could be explained by a different number of integrated copies of HPV DNA and expression
levels of the viral oncoprotein in the two Tg models. In addition, regarding the K14
HPV8 ER Tg mice, it is likely that the product of other early genes cooperate with
E6 and E7 in promoting cancer development. For instance, HPV8 E2 has been shown to
display transforming properties in in vitro and in vivo models [38] [39]. However,
as already described above, K14 HPV8 E6 and HPV8 ER Tg mice showed a very similar
phenotype, indicating that E2 may play a less important role than E6 in the induction
of skin lesions [25] [26]. Thus, it is not yet clear why these two animal models,
K14 HPV8 E6/E7 and K14 HPV38 E6/E7, showed different phenotypes.
Independently of these differences, both Tg animal models provided evidence for a
cooperation of the beta HPV types and UV irradiation in skin carcinogenesis. Interestingly,
the cooperation of infectious agents and environmental factors in carcinogenesis has
also been shown in previous studies on bovine papillomavirus type 4 (BPV4). In cows
grazed on grass or fed on hay, BPV4 infection leads to development of papillomas of
the upper gastrointestinal tract that are spontaneously rejected in a relative short
time, e.g. 12 months. In contrast, in cows kept on a diet of bracken fern, BPV4-induced
papillomas persist and progress to cancer. This phenomenon is explained by the fact
that bracken fern contains several molecules that induce immunosuppression or mutagenesis.
These immunosuppressants favour the persistence of the viral infection, while mutagenic
substances, e.g. quercetin, promote DNA damage, rendering the infected cell more susceptible
to transformation. Thus, studies on beta HPV types and BPV4 underline the importance
of environmental factors in virus-mediated carcinogenesis.
Methods
Plasmid construction and generation of Tg mice
The E6 and E7 ORFs of HPV38 were amplified by polymerase chain reaction (PCR) using
as template the entire HPV38 genome, and were cloned in a pGEM-3Z vector containing
the K14 promoter, a β-globin intron 2, and the K14 polyadenylation sequence (kindly
provided by Professor Herbert Pfister, University of Cologne). The complete insert
was isolated (see Figure 1A) and microinjected, at a concentration of 3 ng/µl, into
the pronuclei of fertilized eggs to generate Tg mice, as described previously [40].
HPV38 E6 and E7 positivity was determined by PCR using specific primers located in
the 5′ (5′-ATG GAA CTA CCA AAA CCT CA-3′) and 3′(5′-TTA TCG TCC GCC ATT GCG-3′) regions
of the E6 and E7 genes, respectively.
We identified two lines (183 and 187) of HPV38 E6/E7 Tg mice in a FVB/N genetic background.
Experiments were performed with K14 HPV38 E6/E7 transgenic lines 183tg/wt, and 187tg/wt
and wild-type FVB/N littermates. The animals were kept in the central animal unit
of the DKFZ, Heidelberg, Germany, under an artificial day/night rhythm and were fed
Kliba 3437 standard food pellets and water ad libitum if not stated otherwise.
Ethics statement
All experiments described in this study were performed in strict accordance to federal
law and the standard ethical guidelines (NIH, 1985; European Communities Directives,
1986 86/609/EEC) and approved by local government authorities (Regierungspräsidium
Karlsruhe, Germany) under license G162-08. Animal treatments, e.g. UV irradiation,
were performed under Sevorane anesthesia, and all efforts were made to minimize suffering.
Total RNA isolation and reverse transcription PCR analyses
Total RNA was isolated from dorsal skin, ear, esophagus, tongue, and liver of 6–8-week-old
Tg animals using the Qiagen RNeasy isolation kit (Quiagen, Hilden, Germany). cDNAs
were synthesized from 1 µg of total RNA using the M-MLV reverse transcriptase (Invitrogen,
Darmstadt, Germany), 18 bp length polydT were used as primers. Quantitative reverse
transcription PCRs (RT-qPCRs) were performed in a 25 µl mixture containing 1 µl of
1∶5 diluted cDNA and SYBR-green master mix (SA bioscience, Frederick, Maryland) with
specific HPV38 E6 primers (5′-TGC TTA TGC TTC TGC TCA ATA TG-3′ and 5′-GTC TGT TGC
TCC ACC TGT TC-3′) or mouse GAPDH primers to amplify a housekeeping gene as internal
control (5′ –AAG AAG GTG GTG AAG CAG GCA TC-3′ and 5′-CGA AGG TGG AAG AGT GGG AGT
TG-3′), using an Applied Biosystems 7300 machine (Applied Biosystems, Darmstadt, Germany).
The fluorescence threshold value was calculated using the SDS analysis software from
Applied Biosystems.
Histological and immunohistochemical analysis
Tissue samples from 6–8-week-old mice were fixed in 4% formaldehyde for 24 h at room
temperature, and embedded in paraffin. Five µm sections were either stained with hematoxylin/eosin
(HE) or used for immunostaining using anti Ki-67 (1∶200) (MM1, Novocastra, Wetzlar,
Germany) or p21WAFI antibody (1∶250) (556431, BD Pharmingen, Heidelberg, Germany).
Staining was performed using biotin-labeled goat anti-mouse immunoglobulin G and ABC
agent from M.O.M kit (Vector Peterborough, UK). The percentages of positive cells
were determined by counting 400 hematoxylin-stained cells under 40× magnification
in four different fields of the epithelium.
For histological analyses, tissue samples from wild-type and Tg mice were fixed in
4% formaldehyde for 24 h at room temperature, and embedded in paraffin and five µm
sections were stained with hematoxylin/eosin (HE).
UVB treatments
UVB irradiation was performed with a Bio-Spectra system (Vilber Lourmat, Marne La
Vallee, France) at a wavelength of 312 nm. Each animal was anesthetized with 3% Sevorane
(Abbott, Wiesbaden, Germany) in an inhalation anesthetizer (Provet, Lyssach, Switzerland)
and placed in a covered compartment with an upper square opening (3×2 cm) at a distance
of 40 cm from the UVB lamp. To determine the impact of viral proteins on UV-induced
checkpoints, 7-week-old mice where shaved on the dorsal skin, and irradiated up to
5 times in a row, maximum 2 times a day, with UVB at 450 mJ/cm2. Twenty-four hours
after the last dose, mice were sacrificed and skin sections stained as described above.
To study UV-induced carcinogenesis, groups of n = 30 7-week-old female FVB/N wild-type
or transgenic mice of lines 183tg/wt and 187tg/wt were shaved on the dorsal skin with
electric clippers and irradiated 3 times a week for 20 weeks with increasing doses
of UVB, starting from 120 mJ/cm2 to a final dose of 450 mJ/cm2, with a constant weekly
increase to allow skin thickening. For the following 10 weeks mice were irradiated
3 times a week with 450 mJ/cm2. The tumour incidence (tumour bearers/group) was recorded
weekly. Tumours were identified first macroscopically and by histological diagnosis.
After thirty weeks, or earlier if the tumour reached the ethically allowed maximal
size, the animals were sacrificed and HE-stained sections of dorsal skin served for
histological diagnosis.
Initiation-promotion experiments
Five weeks after birth, mice were shifted to Altromin 1324 diet. For epicutaneous
applications of initiator, the dorsal skin was shaved with electric clippers 7 days
before treatment. Experimental groups of n = 20 (acetone/acetone) or n = 23–27 (7,12-dimethylbenz-anthracene
(DMBA)/12-O-tetradecanoyl-phorbol-13-acetate (TPA)) 7-week-old female FVB/N wild-type
or transgenic mice of lines 183tg/wt and 187tg/wt were initiated either by a single
epicutaneous application of 0,2 ml acetone or 400 nmol DMBA in 0.2 ml acetone. Beginning
one week later, the mice were treated each twice weekly with 0.1 ml acetone or with
5 nmol TPA in 0.1 ml acetone for maximally 20 weeks. Papilloma and carcinoma development
was monitored up to week 24 without further treatment. Animals were monitored every
three days throughout the experiment. The tumour incidence (tumour bearers/survivors
in percent) and yield (number of tumours/survivors) were recorded weekly. Tumours
were first identified macroscopically and later on by histological diagnosis.
Statistical analysis
Percentages of positive cells in immunostained sections were compared between the
different lines with the Student's t-test and statistical analysis were performed
with GraphPad Prism (version 4.00, GraphPad Software Inc., La Jolla, CA, USA) Time
to first tumor, time to death and time to SCC were displayed with Kaplan-Meier plots
or 1- Kaplan-Meier plots. Animals sacrificed for analysis were considered censored.
Time to first tumor, time to death and time to SCC were compared between the different
groups with the logrank test. For the CoCa experiment, maximal tumor burden was determined
for every animal and groups were compared with the Wilcoxon rank sum test. Statistical
analyses were performed with R (version 2.12.0, Copyright (C) 2010 The R Foundation
for Statistical Computing) and SAS (Version 9.2, SAS Institute Inc., Cary, NC, USA).