Oncogene (1997) 15, 3113 ± 3119
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
p53 in polyoma virus transformed REF52 cells
Orna Mor, Moira Read and Mike Fried
Eukaryotic Gene Organisation and Expression Laboratory, Imperial Cancer Research Fund, PO Box 123, London WC2A 3PX,
UK
While cellular transformation by small DNA tumour
viruses usually involves targeting the product of the p53
tumour suppressor gene by a virally encoded protein,
none of the three polyoma virus (Py) speci®ed T antigens
have been observed to interact with p53. We show that
primary mouse embryo ®broblasts and REF52 cells,
which resemble primary cells in requiring co-operating
oncogenes for transformation, cannot be transformed by
the Py oncogene, middle T-antigen (PyMT), alone.
These cells can be transformed by the complete Py early
region, which encodes the Py large, middle and small Tantigens. We ®nd that PyMT can transform rodent cells
lacking a functional p53 protein (p53 null mouse embryo
®broblasts and DN-REF52 cells which contain a
dominant negative p53). In Py transformed REF52 cells
(Py-REF52) there is no signi®cant accumulation of p53
protein, as opposed to SV40 transformed REF52 cells
(SV-REF52) in which the amount of steady state p53
protein is elevated. However accumulation of p53 is
observed following exposure of Py-REF52 cells to u.v.
Treatment of Py-REF52 cells with X-rays results in a
rapid increase in the levels of the p53-induced proteins
p21/WAF1 and MDM2. In untransformed REF52 cells,
X-irradiation causes p53 activation, which results in
induction of both G1/S and G2/M blocks. In SV-REF52
and DN-REF52 cells, p53 abrogation results in the
absence of both the G1/S and G2/M blocks. Only the
absence of a G1/S block is observed in Py-REF52 cells
exposed to X-irradiation. Together these results indicate
that in contrast to most other DNA tumour viruses, Py
does not appear to interefere with the DNA damage
induced transactivation activities of the p53 protein but
absence of a functional p53 protein can mediate
transformation by the PyMT oncogene in the absence
of other co-operating oncogenes. Possible modes of
transformation by Py are discussed.
Keywords: p53; polyoma virus; cell cycle; DNA
damage; p21; MDM2
Introduction
Inactivation of tumour suppressor genes is one of the
hallmarks of carcinogenesis. The Rb and p53 tumour
suppressor genes, which are both involved in monitoring cell cycle progression, are major targets for
inactivation by the small DNA tumour viruses. The
papovaviruses (polyoma virus (Py) and SV40 (SV)
large T antigens (LT), the adenovirus E1A protein and
Correspondence: M Fried
Received 27 June 1997; revised 26 August 1997; accepted 5
September 1997
the `high risk' human papillomavirus (HPV) E7
proteins all bind and inactivate the Rb tumour
suppressor protein (see Ko and Prives, 1996).
Inactivation of p53 function can occur via di€erent
mechanisms by the di€erent DNA tumour viruses. The
SVLT and adenovirus E1B 55 kd proteins form stable
inactive complexes with the p53 protein (Debbas and
White, 1993; Lane and Crawford, 1979) while the HPV
E6 proteins target p53 for ubiquitin-mediated degradation (Sche€ner et al., 1990).
The Py early region encodes three proteins, large,
middle and small T-antigens. Direct binding of these
Py proteins to p53 has not been observed (Wang et al.,
1989; Dilworth, 1990). At present it is unclear whether
p53 remains functionally active in Py transformed cells.
Transformation of primary rodent cells by Py requires
the coordinated functions of the three early T antigens
(Cuzin, 1984). The nuclear PyLT, which intereacts with
the Rb protein, can immortalise primary ®broblasts
and is involved in viral DNA synthesis. The
cytoplasmic middle T-antigen (PyMT), which is the
Py oncogene interacts with cellular membranes, has an
associated tyrosine-speci®c protein kinase activity and
is capable of transforming most established rodent cell
lines. The e€ect of the small T-antigen (PyST) on
cellular attachment has been well documented but its
role in transformation is unclear (Mumby, 1995).
The p53 protein is important in maintaining
genomic stability and homeostasis following genotoxic
stress (see Ko and Prives, 1996). Being a transcription
factor it can speci®cally a€ect the expression of genes
containing a p53 consensus binding site (Barak et al.,
1993; El-Deiry et al., 1993; Zauberman et al., 1995). In
normal cells p53 levels are low due to its short half-life
(Oren et al., 1981). Its activation by a wide variety of
DNA-damaging agents results in an increase in its
speci®c DNA binding activity and in most cases in the
accumulation of the protein (Ko and Prives, 1996).
Functional p53 is required for the activation of a G1
arrest following irradiation at least partly as a result of
the transcriptional activation of the p21/WAF1/CIP1/
SDI1 protein which acts as an inhibitor of cyclin
dependent kinases that target the Rb proteins (Di
Leonardo et al., 1994; Slebos et al., 1994; Smith et al.,
1994). Antiproliferative activity of p53 has also been
shown to be involved in the G2/M part of the cell cycle
in some cells (Agarwal et al., 1995; Stewart et al.,
1995). In other cell types p53 activation following
DNA damage results in apoptosis (Lowe et al., 1993)
by a mechanism which is as yet unclear. Thus it has
been suggested that the p53 might not be required for
normal cell function but may become activated to
prevent damage. The p53 gene is frequently altered in
transformed cells and transfection of wild type p53 into
tumour cells can result in a decrease in their
tumourigenicity. Mice de®cient for the p53 gene
p53 in polyoma virus transformed REF52 cells
O Mor et al
3114
develop normally but are highly susceptible to tumour
formation (Donehower et al., 1992). The contribution
of p53 to tumour prevention is therefore evident but
the mechanisms underlying this p53 function are not
fully clear at this time.
REF52 is a rat embryo ®broblasts cell line which,
like primary cells, is resistant to transformation by
single oncogenes but can be successfully transformed
by co-operating oncogenes (Hirakawa and Ruley,
1988). Introduction of activated H-ras alone results in
growth arrest/senescence and subsequent lethality of
REF52 (Serrano et al., 1997) while cotransfection of
this oncogene together with Ad5 E1A or SV40 large T
antigen causes transformation of REF52 (Hirakawa
and Ruley, 1988; Hicks et al., 1991). Introduction of a
dominant negative p53 can also overcome the negative
growth regulation induced by H-ras, implying that the
endogenous p53 in REF52 cells, is functionally wild
type (Stewart et al., 1995). REF52 cells do not respond
to wild type p53 overexpression by apoptosis, but show
a cell cycle arrest at both the G1/S and G2/M
boundaries (Stewart et al., 1995).
REF52 therefore appears to be a good model system
for the study of transformation by co-operating
oncogenes in cells containing wild type p53. In this
work we report that PyMT alone is not capable of
transforming either primary mouse embryo ®broblasts
or REF52 cells. However, the complete Py early region
which encodes the three Py T-antigens can transform
both primary mouse embryo ®broblasts and REF52
cells. Furthermore p53 null mouse embryo ®broblasts
and REF52 cells containing a dominant negative p53
can be transformed by PyMT alone. In addition we
show that neither the p53 steady state levels nor the
transcriptional transactivation properties of p53
following both U.V. and X-irradiation are altered in
Py transformed REF52 cells.
Results
The role of p53 in the transformation of primary mouse
embryo ®broblasts and REF52 cells by Py middle Tantigen (PyMT)
Both ras and the Py oncogene, middle T-antigen
(PyMT), require co-operating oncogenes to stably
transform primary cells (Land et al., 1983; Ruley,
1983). We initially assessed the role of p53 in PyMT
transformation by comparing the abilities of PyMT
and the complete Py early region, which encodes the
three Py T-antigens, to transform mouse embryo
®broblasts (MEFs) from wild type and p53 null mice.
Whereas the complete Py early region induced
Table 1 Transformation of p53+/+ or 7/7 mouse embryo
®broblasts (MEFs) and various rat cell lines (Rat-1, REF52, DNREF52) by either the polyoma virus (Py) early region or the polyoma
virus middle T-antigen (PyMT) oncogene
p53+/+ MEF
p537/7 MEF
Rat-1 cells
REF52 cells
DN-REF52 cells
Py early region
PyMT
8
10
298
32
18
0
14
483
0
25
transformation in both primary wild type and p53
null MEFs, PyMT was only capable of transforming
p53 null MEFs (Table 1).
In contrast to primary cells which require cooperating oncogenes for stable transformation most
continuous cell lines, like Rat-1, can be transformed by
single oncogenes. In a similar fashion to primary cells,
stable transformation of cells of the continuous cell line
REF52 by the ras oncogene requires a co-operating
oncogene (Franza et al., 1986; Hirakawa and Ruley,
1988). We investigated whether PyMT also required
co-operating oncogenes for the stable transformation
of REF52 cells. Cells from the established rat cell line
Rat-1 (highly sensitive to DNA transfection) and
REF52 cells (less sensitive to DNA transfection) were
assessed for their ability to be transformed by DNA
constructs containing either PyMT alone or the
complete Py early region. While foci of transformed
cells were generated from both cell lines after
transfection with the Py early region construct, the
PyMT construct was only capable of inducing
transformed foci in the Rat-1 cells (Table 1).
Furthermore, cotransfection of REF52 cells with
PyMT and a neo resistance gene resulted in many
fewer drug resistant colonies than transfection with the
neo gene alone (data not shown). The few neo-resistant
colonies isolated from the cotransfection were not
found to be expressing PyMT. These results indicate
that in the absence of a co-operating oncogene PyMT
has a cytostatic or cytocidal e€ect on REF52 cells.
Thus PyMT appears to be similar to ras in requiring
co-operating oncogenes for the transformation of
REF52 cells as well as for the transformation of
primary cells.
We were interested in determining whether the p53
protein could also play a role in the ability of the cooperating Py early region genes to transform REF52
cells. We assessed the ability of PyMT and the Py early
region to transform DN-REF52 cells in which the
endogenous p53 is inactivated by a dominant negative
p53 mutant. PyMT was capable of transforming DNREF52 cells (Table 1) indicating that, in a similar
fashion to the transformation of primary cells, PyMT
did not require a co-operating oncogene in the absence
of a functional p53 gene to induce transformation of
REF52 cells.
The state of the p53 protein in Py-REF52 cells
We next wanted to evaluate the state of the p53 protein
in the Py-REF52 cells. The SV40 large T-antigen
(SVLT) can complex to the p53 protein and increase its
stability resulting in an elevation in its steady state
levels. Therefore we compared the steady state of the
p53 protein in a series of independently isolated Py
transformed REF52 (Py-REF52) cell clones relative to
REF52 and to SV40 transformed REF52 cells (SVREF52). The steady state p53 levels are relatively low
in REF52 cells and no signi®cant di€erences in p53
levels were detected in the Py-REF52 clones (Figure 1)
indicating that transformation with Py does not result
in stabilisation of the p53 protein. In contrast, p53
steady state protein levels were signi®cantly elevated in
SV-REF52 cells (Figure 1). Unless otherwise stated the
data presented below for Py-REF52 cells were
generated with the PyE clone. In all cases similar
p53 in polyoma virus transformed REF52 cells
O Mor et al
SV-REF52
Pyl-REF52
PyH-REF52
PyE-REF52
PyD-REF52
REF52
3115
a
p53 —
Figure 1 Expression of p53 protein in REF52 and derivative cell
lines. The steady state protein level of p53 protein was analysed in
REF52 and in four independently isolated Py-REF52 cell lines
(PyD-REF52 to PyI-REF52). SV40 transformed REF52 (SVREF52) served as a positive control for p53 detection. 50 mg of
total protein extracts were resolved in a 9% SDS gel, transferred
to nitrocellulose, probed with Pab240 monoclonal antibody
against p53 and detected by ECL. The upper band is a crossreacting band with Pab240 in rat cells
results were also found with the other Py-REF52
clones.
Cell cycle analysis following DNA damage by treatment
with X-rays
The activity of the p53 protein in Py transformed cells
was tested by analysing Py-REF52 cells for their cell
cycle distribution after exposure to X-irradiation.
Exposure of REF52 cells to 4 Gy resulted in a
signi®cant decrease of the S phase cell population
and both a G1 and G2 arrest. On the other hand, PyREF52 cells exposed to the same radiation dose did
not arrest in G1 and no reduction in S phase cells was
noted (Figure 2a). The G2 block typically observed in
REF52 cells was retained in Py-REF52 cells.
The e€ect of p53 on the cell cycle distribution
following DNA damage of REF52, Py-REF52, DNREF52 and SV-REF52 cells was compared following
exposure to 4 Gy. Both the DN-REF52 and SVREF52 cells responded in a similar fashion to the PyREF52 cells following irradiation with the absence of a
G1 arrest (Figure 2a). In all these REF52 derivative
cell lines (Py-REF52, DN-REF52 and SV-REF52) the
G1/S ratio was unchanged following radiation while a
dramatic increase in the G1/S ratio was observed in
REF52 following the DNA damage (Figure 2b).
Unlike Py-REF52, both DN-REF52 and SV-REF52
cells lost the G2/M cell cycle block following
irradiation (Figure 2a).
b
Expression of p53, p21/WAF1 and MDM2 proteins
following X-irradiation or U.V. exposure
We next sought to determine whether the transactivation properties of the p53 protein in Py-REF52 cells
were altered after X-ray or U.V. treatment. The
transcription of the p21/WAF1 gene is thought to be
regulated, at least in part, by p53 and p21/WAF1 is a
key e€ector of p53-dependent G1/S growth arrest.
REF52 and Py-REF52 cells were exposed to 4 Gy and
the p21/WAF1 protein levels were assessed 2 and 4 h
after treatment. As shown in Figure 3 (lower panel) the
parental REF52 cells and the PyREF52 cells both
show a similar increase in p21/WAF1 protein levels
after X-rays.
Figure 2 Cell cycle progression by ¯ow cytometry following
exposure to X-irradiation. REF52 cells, Py-REF52 (Py transformed REF52 clone E, see Figure 1) DN-REF52 (REF52
expressing pBpuro-p53302 ± 390) and SV-REF52 (expressing SV 40
T antigen) were exposed to 0 or 4 Gy X-irradiation and analysed
for BrdUrd incorporation 17 h later. (a) Cell cycle progression
depicted by ¯ow cytometric dot plot. DNA content and the
BrdUrd incorporation are represented along the X and Y axis
respectively. (b) Quantitation of the G1 arrest (increases in G1/S
ratio)
p53 in polyoma virus transformed REF52 cells
O Mor et al
3116
protein band (90/85 kd) is elevated in Py-REF52 cells
and that MDM2 protein levels are further increased
following X-irradiation. To test whether this upregulation is typical of Py transformation of REF52 cells,
several independent Py-REF52 clones were tested for
their MDM2 steady state level. Compared to REF52,
all species of MDM2 protein (except the smallest
57 kd) were upregulated in the di€erent Py-REF52 cell
clones studied by Western analysis (Figure 5).
The MDM2 protein can bind to p53 and abrogate
its function (Chen et al., 1994; Wu et al., 1993). Thus it
is possible that a Py encoded or induced protein
upregulates MDM2 which interferes with p53 activity.
However, we observed that elevation in the MDM2
protein level can occur in untransformed REF52 cells
after long periods of time in culture. Thus, at this time,
we can not attribute the high levels of MDM2 in the
Upregulation of p21/WAF1 can also occur in a p53
independent manner (Macleod et al., 1995; Michieli et
al., 1994). Thus we examined the levels of the MDM2
protein whose activation is also under the control of the
p53 gene (Barak et al., 1993). Similar to p21/WAF1,
MDM2 proteins levels were observed to rise in both
REF52 and Py-REF52 cells following 4 Gy (Figure 3,
top panel). In addition to the major 90/85 kd MDM2
band, 2 smaller MDM2 bands (76/74 kd and 62 kd) were
also dramatically upregulated following a 4 Gy. These
species represent di€erent splice variants of the MDM2
protein (Olson et al., 1993) which are speci®cally
regulated by DNA damage in REF52 cells. No
alteration in the levels of the smallest MDM2 species
(57 kd) was observed following irradiation. The peak of
MDM2 expression occurred 2 h following the radiation
in both the REF52 and Py-REF52 cell lines. Upregulation of both p21/WAF1 and MDM2 following X-rays
indicates that p53 retains its transcriptional transactivation activity in Py-REF52 cells.
No increase in the p53 protein was detected after Xray treatment (data not shown). However, accumulation of the p53 protein was observed in both REF52
and Py-REF52 cells 18 h following U.V. treatment
(Figure 4). Indeed, the p53 response has been shown to
be slower but more pronounced after exposure to U.V.
in comparison to treatment with ionising radiation (Lu
and Lane, 1993). Taken together, these results imply
that the p53 DNA damage response in Py-REF52 cells
is intact.
Py-REF52
REF52
C
C
UV
p53 —
Figure 4 Induction of p53 steady state protein levels following
U.V. irradiation. REF52 and Py-REF52 cells were exposed to 0
or 25 J/m2 U.V. irradiation (C and U.V. lanes respectively) 18 h
after plating 2.56105 cells in 5 cm2 dishes. Total cellular proteins
were extracted 18 h following U.V. exposure. Similar protein
amounts were resolved in a 9% SDS gel and the blots were
probed with anti-p53 Pab240 and detected by ECL
MDM2 expression in Py-REF52 cells
Comparison of the MDM2 protein levels between
REF52 and Py-REF52 (Figure 3) shows that the
steady state basal level of at least the major MDM2
REF52
Py-REF52
2h
–
4h
+
UV
–
2h
+
–
—
p90/85
—
—
P76/74
—
—
p62
—
—
p57
—
—
p21
—
4h
+
–
+
Figure 3 Induction of MDM2 and p21/WAF1 steady state protein levels following X-irradiation. REF52 and Py-REF52 cells Py
transformed REF52 clone E, see Figure 1) were exposed to X-irradiation (0 or 4 Gy, 7 and + lanes respectively). Total cellular
proteins were extracted 2 and 4 h following X-irradiation 18 h after plating 2.56105 cells in 5 cm dishes. Similar protein amounts
were resolved in a 9% SDS gel for the detection of MDM2 (upper panel) and in a 15% SDS gel for the detection of p21 (lower
panel). Following transfer to nitrocellulose, the blots were probed with Pab 2A10 (against MDM2) and an anti-p21 antibody and
detected by ECL. The increase of p21 steady state levels in the unirradiated REF52 cells at 4 h (and to a lesser extent in the 4 h
unirradiated Py-REF52 cells) results from a p53 induction due to changes in pH and temperature of the cells when they were
transported from the incubator to and from the X-ray machine
PyB-REF52
Pyl-REF52
PyH-REF52
PyE-REF52
PyD-REF52
REF52
p53 in polyoma virus transformed REF52 cells
O Mor et al
— p90/85
— p76/74
— p62
— p57
Figure 5 Detection of MDM2 protein steady state level in
REF52 and Py-REF52 transformed cells. Total cellular proteins
(50 mg) extracted from logarithmically growing REF52 and
several Py-REF52 independently isolated cell lines were
separated in 9% SDS gels. The proteins were transferred to
nitrocellulose ®lter probed with Pab210 (against MDM2) and
detected by ECL
di€erent Py-REF52 cells solely to expression of Py T
antigens.
Discussion
We have attempted to assess the role of the p53 protein
in Py transformation of rodent cells. The polyoma
virus oncogene PyMT is insucient to transform either
wild type primary mouse embryo ®broblasts or the
established rat cell line REF52 by itself, but can
achieve successful transformation of these cell types in
the context of the other Py T-antigens. In contrast,
PyMT alone can transform primary p53 null mouse
embryo ®broblasts and DN-REF52 cells in which the
endogenous rat p53 is inactivated by a dominant
negative mutant p53. Thus in the absence of a
functional p53 gene, PyMT does not require a cooperating oncogene to induce transformation of
primary mouse embryo ®broblasts and REF52 cells.
In Py-REF52 transformed cells the p53 protein was
found to be nearly undetectable which is similar to the
p53 steady state level in untransformed REF52 cells.
Following U.V. irradiation the p53 protein levels rise
in both these cell types. This suggests that in PyREF52 cells the p53 protein retains its ability to be
induced as a result of DNA damage. Increases in both
the p21/WAF1 and MDM2 proteins following Xirradiation show that the p53 transcriptional transactivation activity is also retained in Py-REF52 cells. Thus,
in all these aspects, Py-REF52 cells resemble the
REF52 parental cells which have a functional p53
protein (Stewart et al., 1995). In a similar fashion, EBV
e€ectively immortalises human B cells without altering
the ability to induce p53 functions following DNA
damage (Allday et al., 1995). These ®ndings indicate
that not all DNA tumour viruses inhibit the ability of
p53 to be activated after irradiation.
However, it could be that some p53 functions not
associated with DNA damage are inactivated in Py
transformed cells. It has been suggested that the Nterminal region of p53 may be involved in transcriptional transactivation independent growth suppression.
Deletion of this proline rich region was shown to
severely impair the ability of p53 to suppress tumour
cell growth in culture without a€ecting the p53 damage
response (Ko and Prives, 1996). Py transformation may
speci®cally abrogate the transcriptional repression
function of p53 (Ginsberg et al., 1991; Kern et al.,
1992; Mack et al., 1993; Seto et al., 1992) or may a€ect
proteins which are involved in tumour suppression and
function downstream of p53 without a€ecting p53 itself.
We found no detectable upregulation of p53 protein
following X-irradiation while a rapid activation of
expression of p53 responsive genes was observed. On
the other hand, the accumulation of p53 protein was
seen following U.V. Single strand breaks are generated
by X-irradiation while U.V. creates thymidine dimers
in the DNA (Nelson and Kastan, 1994). These di€erent
forms of DNA damage may result in di€erent modes
of induction of the p53 protein. It has been reported
that p53 accumulation is enhanced in response to U.V.
irradiation as compared to ionising radiation (Lu and
Lane, 1993). Discordance between accumulation of p53
protein and its transcriptional activity has also been
described (Lu et al., 1996) and may suggest that, like
the p53 protein in insect cells, mammalian p53 protein
is expressed in both active and latent forms (Hupp and
Lane, 1995) and may become active without detectable
protein accumulation.
We show that REF52 cells arrest in both G1 and G2
phases of the cell cycle following X-irradiation. It has
been reported that REF52 cells expressing the
p53val135 temperature sensitive mutant at the nonpermissive temperature also arrest in G1 and G2
(Stewart et al., 1995) indicating that p53 is involved in
both these checkpoints. In this previous study the p53
dependent G2 arrest was attributed to lack of
apoptosis following over expression of the wild type
p53 at the permissive temperature in REF52 cells.
Indeed we found no evidence for apoptosis in REF52
after serum withdrawal (0.1% FCS for 24 days)
exposure to X-irradiation or following methotrexate
administration. It is interesting to note that the
expression of ras alone in REF52 cells also results in
cell cycle block and not apoptosis (Hirakawa and
Ruley, 1988; Serrano et al., 1997).
In contrast to the REF52 cells the Py-REF52 cells
show a lack of a G1 block following X-irradiation.
This result is seen even though there is an accumulation of p21/WAF1 following DNA damage. The p21/
WAF1 protein is considered to be a mediator of p53
G1 arrest. It can form complexes with and down
regulate the activity of several cdk-cyclin complexes.
The inactive complexes formed with cyclin D/CDKs
combinations result in hypophosphorylation of the Rb
protein which can lead to a G1 arrest. In Py-REF52
cells, the PyLT protein can bind to the Rb protein
resulting in loss of this G1 block. In these circumstances inhibition of cyclin D1/CDK4/6 complexes by
the p21/WAF1 can be ine€ective on Rb. Thus, even
though p21/WAF1 is upregulated following DNA
damage the Rb induced G1 block may be lost. At
this point we cannot attribute the lack of G1 block in
the Py-REF52 cells solely to the binding of PyLT to
Rb. It is possible that absence of the G1 arrest in PyREF52 is also due to the inability of the p53 protein to
suciently induce p21/WAF1 or other factors monitoring the G1 checkpoint. Furthermore at this time we
do not know the role of the PyST in the Py
transformation of REF52 cells. We are presently
attempting to determine the contributions of these Py
proteins in transformation of REF52 cells.
3117
p53 in polyoma virus transformed REF52 cells
O Mor et al
3118
The functional p53 detected after irradiation in Py
REF52 cells most likely plays a role in the observed G2
block. Lack of both G1 and G2 blocks following
irradiation in cells expressing inactivated p53 protein
(DN-REF52 and SV-REF52) also suggests that
functional p53 is necessary for cell cycle arrest in
both check points in REF52 cells. We conclude that
the DNA damage response found in Py-REF52 is
similar to REF52 and di€ers from that of the DNREF52 and SV-REF52 cells.
In summary we show that although the complete Py
early region is required for the transformation of
primary mouse embryo ®broblasts and REF52 cells,
PyMT alone can transform these rodent cells in the
absence of a functional p53 protein. There is no
apparent stabilisation of p53 in transformed Py-REF52
cells. The response to irradiation in terms of p53
stabilisation and induction of p53 responsive genes in
Py-REF52 cells is similar to that observed in
untransformed REF52 cells except for the absence of
a G1 block in the Py-REF52 cells.
It has recently been reported that either a loss of p53
or p16 function can complement ras to successfully
transform REF52 and primary mouse embryo fibroblasts (Serrano et al., 1997). It is possible that a similar
strategy exists for PyMT. Thus PyMT can transform
primary mouse embryo ®broblasts and REF52 cells
when p53 is inactive. In the case of transformation by
the complete early region the presence of the PyLT
inactivates Rb which in e€ect is functionally equivalent
to the loss of p16 function. Under this scheme there
would be no reason for p53 to be inactivated in the Py
transformed REF52 cells. This hypothesis would not
explain why other DNA tumour viruses which also
specify proteins that inactivate Rb also specify proteins
which target p53. We are presently attempting to
further elucidate the role of p16 and p53 in Py
transformation.
Materials and methods
Cell, transfection and plasmids
REF52 is an established rat embryo ®broblast cell line
(Franza et al., 1986) and early passage cells were obtained
from Cold Spring Harbor Labs. Rat-1 cells (Ford et al.,
1985), which can be transformed by a single oncogene were
used in the transformation experiments as control for the
transfection eciency. Mouse embryo ®broblasts (MEFs)
from p53 wild type and p53 null mice (Donehower et al.,
1992) were established from 11 ± 13 day old embryos and
passaged once before use. All transfections were carried
out using 5 mg plasmid DNA and 5 mg carrier DNA
(salmon sperm DNA) transfected into 5 cm dish using
either CaCl2 or lipofectamine (Gibco-BRL). The Py early
region construct contained the complete Py genome cloned
at the BamHI site in pAT153 and the construct containing
the Py middle T-antigen cDNA was previously described
(Zhu et al., 1984). Transformation of REF52, wild type
and p53 null MEFs and/or RAT-1 cells was assessed 21
days after transfection by scoring transformed foci using
Lieshman staining. Some foci were picked for further
analysis and Py-REF52 cell lines were established. SVREF52 was established by transformation of REF52 cells
using the pSE-SV construct, which contains the SV40 early
region (a gift from PS Jat). DN-REF52 was established by
transfection of pBpuro-p53302 ± 390 a dominant negative p53
c-terminal fragment (gift from T Littlewood) and selection
with 1.25 mg/ml puromycin.
All cells were cultured in Dulbecco's modi®ed Eagle
medium (DMEM) supplemented with 10% fetal calf serum
(FCS), 100 mg/ml penicillin and 100 mg/ml streptomycin.
Puromycin or G418 were added as indicated. Expression of
the transfected proteins in all these cell lines was
corroborated by Western analysis.
Radiation treatment
Exponentially growing cells (80% con¯uence) were exposed
to either X-rays (4 Gy, Pantax HS320) or 25 J/m2 of U.V.
radiation. Medium was removed and replaced by PBS
before the U.V. treatment. After the radiation exposure,
cells were grown for 17 h before ¯ow cytometry analysis or
for certain periods of time for the protein analysis.
Cell cycle analysis
Cells exposed to X-rays or U.V. irradiation were pulse
labelled with 10 mM BrdUrd for 4 h at the indicated time
after irradiation and ®xed with 70% ethanol. Subsequently
they were analysed simultaneously for DNA synthesis with
a ¯uorescein isothiocyanate (FITC)-conjugated antiBrdUrd antibody and for DNA content with propidium
iodide.
Protein analysis
Cells were lysed directly in a protein loading bu€er for
SDS gels (Maniatis, et al., 1982). Protein concentration
was estimated using a Biorad kit and 25 ± 50 mg/ml soluble
cellular protein was loaded on SDS-polyacrylamide gels.
After separation, the proteins were transferred onto
nitrocellulose paper, blocked with 5% marvel milk powder
for 1 h at room temperature. The membranes were probed
with monoclonal antibody Pab 240 (ICRF) against p53,
Pab210 (gift from A Levine) against MDM2, an anti p21
(Santa Cruz) against p21/WAF1 or C1 polyclonal antibody
against the Py T antigens (ICRF). The antibodies were
detected with peroxidase-conjugated rabbit anti mouse (for
MDM2 and p53 detection) or an anti rabbit (for p21/
WAF1). Detection was performed using ECL (Amersham)
according to the manufacturer's protocol.
Acknowledgements
We would like to thank Gordon Peters, Lindsey Allan and
Jon Gilley for their help in the preparation of this
manuscript and Derek Davies for his help with the FACS
analyses.
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