Behrens A, Jochum W, Sibilia M, Wagner EF.. Oncogenic transformation by ras and fos is mediated by c-Jun N-terminal phosphorylation. Oncogene 19: 2657-2663

Abstract

The nuclear phosphoprotein c-Jun is a major component of the AP-1 transcription factor, whose activity is augmented by many oncogenes. An important mechanism to stimulate AP-1 function is N-terminal phosphorylation of c-Jun at the serine residues 63 and 73 by the c-JunN-terminal kinases (JNKs). Mice and cells harboring a mutant allele of c-jun, which has the JNK phosphoacceptor serines changed to alanines (junAA), were used to determine the function of c-Jun N-terminal phosphorylation (JNP) during oncogenic transformation in vitro and in vivo. JunAA immortalized fibroblasts expressing v-ras and v-fos showed reduced tumorigenicity in nude mice, but the efficiency of v-src transformation was unaffected by the lack of JNP. To assess the significance of JNP in tumour development in vivo, two transgenic mouse tumour models were employed. Skin tumour development caused by constitutive activation of the ras pathway by K5-SOS-F expression and c-fos-induced osteosarcoma formation were impaired in mice lacking JNP. Inhibition of JNP may, therefore, be a novel therapeutic strategy to inhibit tumour growth in vivo. Oncogene (2000).

Full text

Oncogenic transformation by ras and fos is mediated by c-Jun N-terminal
phosphorylation
Axel Behrens
1
, Wolfram Jochum
1
, Maria Sibilia
1
and Erwin F Wagner*
,1
1
Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, A-1030 Vienna, Austria
The nuclear phosphoprotein c-Jun is a major component
of the AP-1 transcription factor, whose activity is
augmented by many oncogenes. An important mechanism
to stimulate AP-1 function is N-terminal phosphorylation
of c-Jun at the serine residues 63 and 73 by the c-Jun
N-terminal kinases (JNKs). Mice and cells harboring a
mutant allele of c-jun, which has the JNK phosphoac-
ceptor serines changed to alanines (junAA), were used to
determine the function of c-Jun N-terminal phosphoryla-
tion (JNP) during oncogenic transformation in vitro and
in vivo.JunAA immortalized ®broblasts expressing v-ras
and v-fos showed reduced tumorigenicity in nude mice,
but the eciency of v-src transformation was unaected
by the lack of JNP. To assess the signi®cance of JNP in
tumour development in vivo, two transgenic mouse
tumour models were employed. Skin tumour development
caused by constitutive activation of the ras pathway by
K5-SOS-F expression and c-fos-induced osteosarcoma
formation were impaired in mice lacking JNP. Inhibition
of JNP may, therefore, be a novel therapeutic strategy
to inhibit tumour growth in vivo.Oncogene (2000) 19,
2657 ± 2663.
Keywords: C-Jun N-terminal phosphorylation; tumor-
igenesis; Ras; Fos; Src
Introduction
AP-1 is a sequence-speci®c DNA binding transcription
factor, which is composed of the fos and jun gene
products (Angel and Karin, 1991). The founding
members of these two gene families, v-fos and v-jun,
have originally been identi®ed as the transforming
activities of the FBJ and FBR murine sarcoma virus
(v-fos) (Curran et al., 1983; Van Beveren et al., 1983)
and avian sarcoma virus 17 (v-jun) (Bos et al., 1988),
respectively. The oncogenic activity of c-jun has been
demonstrated in several experimental systems. Deregu-
lated expression of c-Jun, or its viral counterpart v-Jun,
can trigger transformation of avian and mammalian
®broblasts (Vogt, 1992). c-jun expression in transgenic
mice from an ubiquitous promoter (H2-c-junLTR) does
not result in any apparent phenotype, but over-
expression of v-jun causes wounding-induced ®brosar-
comas (Schuh et al., 1990; Wang et al., 1995). c-jun
eciently cooperates with oncogenic ras in cellular
transformation (Binetruy et al., 1991; Smeal et al.,
1991) and c-jun has been shown to be an important
target of ras-mediated oncogenesis as ras-induced
transformation is greatly reduced in the absence of
c-jun (Johnson et al., 1996). In addition, c-jun is also
capable of cooperating with c-fos since osteosarcoma
formation caused by c-fos overexpression (H2-c-
fosLTR) was enhanced in H2-c-fosLTR/H2-c-junLTR
double transgenic mice (Wang et al., 1995).
A number of stimuli including growth factors,
tumour promoters and transforming oncoproteins
augment AP-1 activity by stimulating mitogen-acti-
vated protein kinases (MAPKs) (Angel and Karin,
1991; Karin, 1996). The c-Jun protein is phosphory-
lated at N-terminal serine residues 63 and 73 within the
transactivation domain by the c-Jun N-terminal kinases
(JNKs) (Derijard et al., 1994; Kyriakis et al., 1994).
C-Jun N-terminal phosphorylation (JNP) is believed to
strongly augment c-Jun activity by recruiting the
coactivator proteins CBP and p300 to target gene
promoters resulting in increased gene transcription
(Arias et al., 1994; Bannister et al., 1995). Ras and
several other oncoproteins stimulate phosphorylation
of serines 63 and 73 (Derijard et al., 1994; Smeal et al.,
1992; Smeal et al., 1991), however, the function of JNP
during oncogenic transformation has remained unclear.
We have previously reported the generation of mice
and cells carrying a mutant c-jun allele with the JNK
phosphoacceptor serines 63 and 73 changed to alanines
(junAA) (Behrens et al., 1999). Mice lacking JNP
undergo normal embryonic development, but show
defects in kainate-induced neuronal apoptosis (Behrens
et al., 1999). We have also shown that junAA primary
mouse embryonic ®broblasts (MEFs) proliferate
slowly, although the proliferation defect is not as
severe as observed in c-jun7/7MEFs (Behrens et al.,
1999; Johnson et al., 1993; Schreiber et al., 1999).
In this study we have tested the function of JNP
during oncogenic transformation in vitro and in vivo.
JNP was required for ecient transformation of
immortalized ®broblasts by v-ras and v-fos, but not
by v-src. To analyse the role of JNP in ras-mediated
tumour formation in vivo, mice expressing a dominant
form of the guanine nucleotide exchange factor SOS
(Aronheim et al., 1994; Chardin et al., 1993) in the
basal cells of the epidermis were employed (K5-SOS-F;
Sibilia et al., 2000). These mice develop skin
papillomas with 100% penetrance in a wild-type
background (Sibilia et al., 2000), whereas in a junAA
homozygous genetic background SOS-F-mediated tu-
mour formation was delayed suggesting a function of
JNP as a target of the ras pathway in keratinocytes. In
addition, absence of JNP also resulted in decreased
osteosarcoma formation in H2-c-fosLTR transgenic
mice. These results provide genetic evidence that the
absence of JNP reduces cellular transformation by the
Oncogene (2000) 19, 2657 ± 2663
ã
2000 Macmillan Publishers Ltd All rights reserved 0950 ±9232/00 $15.00
www.nature.com/onc
*Correspondence: EF Wagner
Received 9 November 1999; revised 6 April 2000; accepted 6 April
2000

v-ras and v-fos oncogenes, but that the oncogenic
activity of v-src is independent of JNP.
Results
Reduced fibroblast transformation by v-ras and v-fos in
the absence of JNP
To study the function of JNP during oncogenic
transformation in vitro, immortalized ®broblasts were
derived from wild-type and junAA homozygous E12.5
fetuses. JunAA primary mouse embryonic ®broblasts
(MEFs) entered senescence at the same time as wild-
type control cells, and the duration of the crisis phase
before the appearance of immortalized cells was similar
to controls (data not shown). This is in contrast to
c-jun7/7MEFs, which undergo premature senescence
and exhibit an extremely prolonged crisis phase
(Schreiber et al., 1999). Immortalized 3T3 cell lines
lacking JNP did not show a proliferation defect as
junAA MEFs did (Behrens et al., 1999) (Figure 1a). As
immortalization occurs at least in part by mutation of
genes encoding cell cycle regulators, JNP might control
the activity of these proteins in MEFs, but this control
mechanism is relieved in established ®broblasts.
To investigate the role of JNP in oncogenic
transformation, two independent wild-type and two
junAA immortalized 3T3 ®broblast cell lines were
infected with recombinant retroviruses expressing v-
ras,v-src or v-fos (Boulter and Wagner, 1988; Keller et
al., 1985; Redmond et al., 1988; Wagner et al., 1985).
Whereas c-jun7/7®broblasts were resistant to
morphological alterations induced by oncogenic Ras
(Johnson et al., 1996), junAA cells exhibited all
characteristics of the transformed phenotype and were
morphologically indistinguishable from wild-type con-
trols in response to all three oncogenes tested (Figure
1b).
A functional assay for transformation is the ability
to form tumours when injected into immunosuppressed
mice. Whereas neo-expressing control and junAA cells
did not form tumours, all oncogene-expressing wild-
type 3T3 cells were tumorigenic (Figure 1c). Absence of
JNP considerably suppressed v-ras-induced tumour
growth, but had no eect on v-src-expressing cells
(Figure 1c). The requirement for JNP was most
dramatic in v-fos transformed cells. Nude mice injected
with v-fos-infected wild-type ®broblasts developed large
tumour masses and had to be sacri®ced 4 weeks after
injection, when no visible or palpable tumours could be
detected in v-fos-expressing junAA cells (Figure 1c).
Nevertheless, 16 weeks after injection these cells also
gave rise to tumours (data not shown). Consistent with
the results from tumour induction in nude mice, focus
formation in vitro required both activated ras expres-
sion and JNP, since the number of foci formed in
junAA cells was signi®cantly decreased, but v-src-
induced focus formation was normal (data not shown).
Consistent with previous observations, oncogenic ras
strongly stimulated JNP (Figure 2a). To a lesser extent
also v-src and v-fos expression increased JNP in wild-
type cells (Figure 2a). Antibodies directed against the
phosphorylated form of serine 63 of c-Jun failed to
give a signal in all junAA cell lines, con®rming the
absence of JNP. Moreover, there were no signi®cant
dierences in the steady-state levels of the c-Jun
protein among the various wild-type and junAA cell
lines (Figure 2a). To determine the contribution of JNP
to AP-1 transcriptional activity in the oncoprotein
expressing cells, AP-1 activity was measured using a
luciferase reporter gene construct containing ®ve AP-1
DNA binding sites (56TRE-Luc). AP-1 transcrip-
tional activity was comparable in wild-type and junAA
cells infected with the control virus (Figure 2b).
Oncogenic ras elevated AP-1 activity 5 ± 6-fold in
wild-type cells, but only 3 ± 4-fold in the absence of
Figure 1 Characterization of junAA/junAA immortalized ®bro-
blasts infected with oncogene-expressing retroviruses. (a) Wild-
type (white squares) and junAA/junAA (black squares) ®broblasts
were plated in DMEM with 10% FCS at 10
5
cells per 6-cm dish,
and cell numbers were counted every 24 h. (b) Phase-contrast
photographs of wild-type and junAA/junAA ®broblasts infected
with the indicated oncogene expressing retroviruses. Expression of
the oncoproteins in the infected cells was con®rmed by Western
blot analysis (data not shown). (c) Cells of the indicated
genotypes were injected subcutaneously into the scapular region
of nude mice (2610
5
cells per injection site). The tumour volume
was determined 4 weeks after injection. One bar represents one
injection site. One result representative of two independent
experiments is shown
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A Behrens et al
2658
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JNP. AP-1 induction by v-fos was slightly reduced in
junAA cells, whereas v-src-expressing wild-type and
junAA cells showed no signi®cant dierence in reporter
gene activity (Figure 2b). These results suggest that
JNP is required for ecient transformation by v-fos
and oncogenic ras.
JNP is required for efficient skin tumorigenesis caused by
overexpression of a dominant form of hSOS
To determine the function of JNP as a target of
cellular transformation by the Ras pathway in vivo, the
skin tumour prone K5-SOS-F transgenic mice were
bred into a homozygous junAA background (Sibilia et
al., 2000). C-jun is highly expressed in basal keratino-
cytes of the skin, the same cells that express the hSOS-
F transgene (Rutberg et al., 1996; Sibilia et al., 2000).
The ®rst skin abnormalities induced by K5-SOS-F were
visible already 2 weeks after birth in wild-type and
junAA/+ mice and progressed into highly disorganized
papillomatous structures, which in the tail region
became more than 10 mm thick (data not shown). In
addition, papillomas frequently occurred at the edges
of tail- and toe-biopsy sites, suggesting that wounding
was capable of contributing to tumour development
(data not shown). The absence of JNP resulted in a
delay of tumour formation.
Determination of the maximal tumour diameter at
the age of 2 months showed an average decrease in
tumour size by approximately 50% in a junAA
background (Figure 3a). However, wounding-induced
papillomas and tumour progression occurred with
unaltered frequency in these mice (data not shown).
With increasing age (4 ± 6 months) larger tumour
masses also developed in initially protected junAA
homozygous mice (data not shown). Despite a
signi®cant dierence in tumour size, analysis of
junAA/+ and junAA/junAA tumours revealed no
striking dierences in the histological appearance and
cellular composition (Figure 3b). Therefore, the
absence of JNP delays but does not abolish skin
tumour formation induced by the hSOS-F transgene.
Figure 2 Reduced AP-1 activity in oncogene-expressing junAA/
junAA ®broblasts. (a) Phospho-c-Jun, c-Jun and b-actin levels in
wild-type and junAA/junAA ®broblasts expressing the indicated
genes were determined by Western analysis. (b) AP-1 activity
assay of wild-type and junAA/junAA 3T3 ®broblasts infected with
retroviruses expressing the indicated genes. Each value represents
the mean+s.e.m. of two independent clones of each genotype
measured in duplicate
Figure 3 Reduced K5-SOS-F-induced skin tumour formation in
junAA/junAA mice. (a) The maximal diameter of K5-SOS-F-
induced skin tumours was determined at the age of eight weeks.
The average numbers of K5-SOS-F-induced skin tumours in a
junAA/+ (n=13) and junAA/junAA (n=18) genetic background
are given+s.e.m. (b) Histology of K5-SOS-F-induced skin
tumours in junAA/+ and junAA/junAA littermates at the age of
8 weeks
Oncogene
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2659

Inhibition of c-fos-induced bone tumour formation in
junAA homozygous mice
We next studied the eect of JNP on c-fos-induced
skeletal osteosacomas (Grigoriadis et al., 1993). H2-
c-fosLTR transgenic mice in a junAA/+ heterozygous
and in a junAA homozygous background were generated
and the spatial and temporal course of osteosarcoma
development was studied using skeletal X-ray analysis
(Figure 4a). Radiographic examination revealed that the
spatial distribution of tumours and the onset of tumour
formation were comparable in mice carrying the H2-
c-fosLTR transgene in a junAA homozygous and
heterozygous background (7.7+2.1 and 8.2+2.1 weeks,
respectively) (Figure 4b). Only at older age a modest
dierence in tumour onset became apparent. Whereas all
control mice had developed radiographically detectable
tumours by the age of 3 months (15/15), 4/18 (22%)
junAA homozygotes were still tumour-free at this time-
point (Figure 4b). To quantify the eect of JNP on
osteosarcoma formation, the number of tumours in H2-
c-fosLTR transgenic junAA/junAA and junAA/+ mice
was determined. The absence of JNP resulted in impaired
tumour development since the mean tumour numbers at
2, 3 and 4 months of age were reduced and the tumours
were on average smaller in size (Figure 4a,c).
Bone tumours of both junAA homozygous and
heterozygous H2-c-fosLTR transgenic mice (n=17
and n=18, respectively) showed the morphological
features of low-grade chondroblastic osteosarcomas.
The tumours were largely composed of mineralized
neoplastic bone lined by tumour cells with osteoblastic
dierentiation (Figure 4d,f). Areas of anaplastic
tumour morphology were not detected. Staining for
acid mucopolysaccharide (Alcian blue) indicated the
presence of areas of chondroblastic dierentiation,
which were comparable in junAA homozygous and
heterozygous tumours (data not shown). Furthermore,
histochemical staining for tartrate-resistant acid phos-
phatase (TRAP), a marker for osteoclasts, revealed
that comparable numbers of TRAP-positive multi-
nucleated cells lined the bone surfaces of junAA
homozygous and heterozygous tumours suggesting that
bone turnover was not aected (data not shown).
To determine whether absence of JNP had an eect
on the proliferation of the tumour cells, Bromodeox-
yuridine (BrdU) incorporation detected by immunohis-
tochemistry was used to measure the number of S
phase cells. Proliferating tumour cells were predomi-
nantly located in the vicinity of the tumour surface and
were hardly detected in the central part, which consists
primarily of mineralized bone matrix (Figure 4e,g).
Whereas 6.2% of the tumour cells in a junAA/+
background had gone through S phase within the 12 h
labelling period, the number of proliferating cells was
decreased by *30% in the absence of JNP (Figure 4h).
Therefore, it is tempting to speculate that the reduced
proliferation capacity of these cells might be respon-
sible for the decreased tumour number and size
observed in junAA homozygous mice.
Discussion
Although it has been noted previously that JNP is
stimulated by a number of oncogenes, the functional
signi®cance and contribution of JNP to the trans-
formed phenotype has been unclear. This study
demonstrates that absence of JNP does not aect the
oncogenic activity of v-src, but reduces transformation
by components of the Ras pathway and by oncogenic
fos both in vitro and in vivo although both ras and fos
are still capable of transforming junAA cells.
Ras and fos have dierent biological activities and
biochemical interactions with JNP. Ras activates the
JNKs and thereby JNP resulting in increased AP-1
activity. Since oncogenic ras activates multiple other
eector molecules including Raf and PI3-kinase
(Joneson and Bar-Sagi, 1997), our results suggest also
a role of the JNK pathway as a downstream target of
ras during oncogenesis. However, lack of JNP has a
milder eect on ras-induced transformation compared
to the complete absence of c-jun (Johnson et al., 1996).
c-jun7/7®broblasts are completely resistant to ras
transformation (Johnson et al., 1996), whereas the
JunAA protein is still capable of signi®cantly con-
tributing to ras-mediated oncogenesis. Therefore, JNP
contributes to but is not required for ecient
cooperation between ras and c-jun. One explanation
for this observation is that JNP is required for the
maximal transcriptional induction of target genes
involved in oncogenic transformation. The JunAA
protein might still stimulate transcription at a low
level, whereas complete absence of c-Jun would abolish
gene induction. Alternatively, c-Jun and phospho-c-Jun
may regulate dierent sets of target genes which both
contribute to ras-induced oncogenesis. N-terminal
phosphorylation of c-Jun could result in the recruit-
ment of dierent coactivator proteins thereby altering
the activity and target gene speci®city of c-Jun. It was
reported that expression of a junAA allele in ras
expressing c-jun7/7cells rescued soft-agar colony
formation, but not tumorigenicity, which may argue in
favour of the latter possibility (Johnson et al., 1996).
We have chosen skin tumorigenesis as a model
system to test the signi®cance of JNP as a target of Ras
in vivo. Since activating mutations of ras occur
frequently during human skin carcinogenesis, the
identi®cation of Ras downstream eectors in keratino-
cytes is of medical relevance (Yuspa, 1998). Using
chemically induced papillomas as a model of skin
carcinogenesis it was shown that the formation of
papillomas was unaected in c-fos knock-out mice, but
that benign-to-malignant progression was severely
impaired (Saez et al., 1995). In contrast, c-jun has
been implicated in papilloma formation since trans-
genic mice expressing a dominant negative allele of c-
jun (TAM67) in basal keratinocytes show greatly
decreased numbers of chemically induced papillomas
(Young et al., 1999). Similarly, in the K5-SOS-F
transgenic model, the absence of JNP delayed tumour
formation, but the eventual progression to large
tumour masses was not aected. These results indicate
that c-jun and c-fos may have dierent functions during
ras-induced skin cancerogenesis: c-jun and JNP appear
to be required for papilloma formation, a process that
is c-fos-independent. Only at later stages of tumour
development, when malignant progression occurs, c-fos
is essential. Whether these dierences are indicative of
dierent biological activities of c-fos and c-jun during
skin carcinogenesis or are due to the dierent tumour
models used requires further investigation.
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Figure 4 Impaired c-fos-induced osteosarcoma formation in the absence of JNP. (a) X-ray analysis of H2-c-fosLTR transgenic
mice in a junAA homozygous and heterozygous background, respectively. The X-ray was taken at the age of 4 months.
Radiographically detectable tumours are indicated by arrows. (b) Time course of tumour formation. The appearance of
osteosarcomas in junAA/+ and junAA/junAA mice carrying the H2-c-fosLTR transgene was monitored by skeletal X-ray analysis.
(c) The number of osteosarcomas at the indicated time-points was determined by skeletal X-ray analysis. (d,f) Histological
appearance of +/+ control (d) and junAA homozygous (f) osteosarcomas. (e,g) BrdU labelling of +/+ control (e) and junAA
homozygous (g) osteosarcomas. Some labelled nuclei are indicated by arrows. (h) Quanti®cation of BrdU-labelled tumour cells in
junAA homozygous, junAA/+ and +/+ osteosarcomas. The values are given+/7s.e.m.
Oncogene
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2661

In contrast to skin tumours, injury-induced papillo-
mas showed similar growth kinetics in junAA and
control mice. Wounds are known to activate a number
of growth factors and cytokines and thereby contribute
to tumour development (Rappolee et al., 1988). The
function of JNP in tumour induction, which results in
a delay of papilloma formation in K5-SOS-F trans-
genic mice, may be bypassed by a wounding-induced
event explaining normal papilloma formation at the
sites of injury in the absence of JNP.
Whereas Ras directly stimulates JNP, overexpres-
sion of oncogenic fos results in elevation of AP-1
activity by recruiting and binding to dimerization
partners, most likely Jun proteins, thereby forming a
functional AP-1 transcription factor complex (Kovary
and Bravo, 1991, 1992). It has been shown previously
by generating double transgenic lines that c-jun is
capable of cooperating with fos during oncogenesis
(Wang et al., 1995), our data demonstrate that
endogenous c-Jun is an important partner for
oncogenic Fos during cellular transformation. In
particular, N-terminal phosphorylation of c-Jun
appears to be required for the activity of c-Fos/c-
Jun heterodimeric complexes in transgenic osteoblasts.
The partial suppression of osteosarcoma formation in
the absence of JNP may be explained by a reduction
of tumour cell proliferation. c-Jun and JNP have been
shown to regulate cellular proliferation in several
types including ®broblasts and hepatocytes (Behrens et
al., 1999; Eferl et al., 1999; Johnson et al., 1993;
Schreiber et al., 1999; Wisdom et al., 1999). Moreover,
c-jun is highly expressed in c-fos transgenic osteoblasts
(Grigoriadis et al., 1993) and JNP appears to
contribute to c-Jun's function in controlling the
proliferation of these transformed cells.
In humans, osteosarcoma is the second most
common neoplasm of bone after myeloma, and
accounts for approximately 20% of all primary
malignant bone tumours. C-fos is ampli®ed in a
signi®cant number of osteosarcomas and alterations
of c-fos correlate with a bad prognosis, since they
occur more frequently in patients with recurrent or
metastatic disease (Pompetti et al., 1996). The ®nding
that JNP contributes to oncogenic transformation and
tumour growth suggests that inhibition of JNP could
be a novel therapeutic principle to interfere with
tumour growth and disease progression, particularly
in patients with osteosarcomas and skin tumours.
Materials and methods
Mice
Mice carrying a mutant c-jun allele, which has the JNK
phosphoacceptor serines 63 and 73 mutated to alanines, were
generated using homologous recombination in ES cells as
described (Behrens et al., 1999). The H2-c-fosLTR transgenic
line (Grigoriadis et al., 1993) and the K5-SOS-F line (Sibilia
et al., 2000) are described elsewhere.
Cells and viruses
Immortalized cell clines were derived from wild-type
(3T3AA25 and 3T3AA30) and junAA homozygous
(3T3AA28 and 3T3AA29) E12.5 fetuses according to the
3T3 protocol (Todaro et al., 1965). The following retroviruses
were used for transformation assays: C2-she-ras (LTR-Ha-v-
ras); SR1 (LTR-neo TK-v-src); C2-v-fos (LTR-neo TK-v-
fos); and N2/C2 (LTR-neo) (Boulter and Wagner, 1988;
Keller et al., 1985; Redmond et al., 1988; Wagner et al.,
1985). There was no dierence in the infection frequency
between wild-type and junAA cells (data not shown).
Focus formation assay
Wild-type and junAA/junAA immortalized ®broblasts were
plated at a density of 3610
5
cells per 10 cm dish and infected
with the above-described retroviruses the next day. Cells were
cultured in the same dishes without selection for 2 weeks.
Thereafter, the medium was removed, plates were washed
with PBS, ®xed with methanol and stained with 0.2%
methylene blue (v/v in methanol). The number of macro-
scopically visible foci was estimated by visual examination.
Tumorigenic assay
Wild-type and junAA/junAA immortalized ®broblasts were
plated at a density of 3610
5
cells per 10 cm dish, and
infected with the above-described retroviruses the next day.
Cells were cultured in the same dishes in the presence of
1 mg/ml G418 for 1 week until all non-infected cells had
died. 2610
5
subcon¯uent G418-resistant cells were subcuta-
neously injected into 3 ± 5 week old anaesthetized nude mice.
The mice were monitored weekly for the appearance of
tumours.
Histology and BrdU immunohistochemistry
Mice of various genotypes were injected intraperitoneally
with 100 mg of bromodeoxyuridine (BrdU) per gram of body
weight 12 h prior to sacri®ce. Tumour tissues were ®xed in
4% formaldehyde, dehydrated, embedded in paran and
sectioned (5 mm). Calci®ed tissues were decalci®ed in EDTA
(0.5 M, pH 7.6) for 5 ± 10 days prior to embedding. For
histological analysis sections were stained with Harris
haematoxylin and eosin (Sigma). For BrdU immunohisto-
chemistry sections pretreated with protease type XXIV and
2 M HCl were incubated with biotinylated mouse mono-
clonal anti-BrdU antibody (Caltac; 1 : 50 dilution) at 48C
overnight. The signal was ampli®ed with the Vectastain ABC
Kit (Vector Laboratories) and visualized with 3, 3'-
diaminobenzidine (Vector Laboratories); sections were coun-
terstained with Harris haematoxylin (Sigma).
Western blot analysis
Cell extracts from ®broblasts of various genotypes were
prepared as described (Behrens et al., 1999). Between 100 and
200 mg of cell extracts were used for Western blot analysis
according to standard procedures (Sabapathy et al., 1999).
Brie¯y, the extracts were separated on SDS polyacrylamide
gels, transferred onto Immobilon-P membrane (Millipore)
and probed with anti-phospho-c-Jun (from M Yaniv), anti-c-
Jun (Transduction Laboratories) and anti-bactin (Sigma)
antibodies. Immunoreactive bands were visualized using the
Enhanced Chemoilluminescence (ECL) kit (Boehringer
Mannheim) according to the manufacturer's recommenda-
tions.
Reporter gene assay
Wild-type (3T3AA25 and 3T3AA30) and junAA homozygous
(3T3AA28 and 3T3AA29) ®broblasts cells lines were
cotransfected with a luciferase reporter gene regulated by a
pentameric AP-1 site (56TRE-Luc) and CMV-LacZ (trans-
fection control). Cell extracts were harvested 16 h after
transfection. Each value represents the mean+s.e.m. of two
independent clones of each genotype measured in duplicate.
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Acknowledgments
We thank Svetlana Pekez for maintaining the mouse
colony. We are also grateful to Uta Moehle-Steinlein for
technical assistance, to Moshe Yaniv for antibodies, to
Gerhard Christofori, Thomas Jenuwein and Laura Stingl
for critical reading of the manuscript and to Hannes
Tkadletz for help with preparing the illustrations. Sup-
ported in part by the Austrian Federal Ministry of Science,
Transport and the Arts and the Austrian Industrial
Research Promotion Fund.
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