Download Novel p53 mutants selected in BRCA

Oncogene (1999) 18, 2451 ± 2459
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Novel p53 mutants selected in BRCA-associated tumours which dissociate
transformation suppression from other wild-type p53 functions
Paul D Smith1, Susan Crossland1, Gillian Parker2, Peter Osin1, Louise Brooks3, Joanne Waller4,
Elizabeth Philp1, Mark R Crompton1, Barry A Gusterson4, Martin J Allday2 and Tim Crook*,4
1
Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, UK; 2Ludwig Institute for
Cancer Research and Section of Virology and Cell Biology, Imperial College School of Medicine, St Mary's Campus, Norfolk
Place, London W2 1PG, UK; 3Division of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street,
London W1, UK; 4Institute of Cancer Research, The Toby Robins Breast Cancer Research Centre, 237 Fulham Road, London
SW3 6JB, UK
Inheritance of germ-line mutant alleles of BRCA1 and
BRCA2 confers a markedly increased risk of breast
cancer and we have previously reported a higher
incidence of p53 mutations in these tumours than in
grade matched sporadic tumours. We have now characterized these p53 mutants. The results of these studies
identify a novel class of p53 mutants previously
undescribed in human cancer yet with multiple occurrences in BRCA-associated tumours which retain a
pro®le of p53-dependent activities in terms of transactivation, growth suppression and apoptosis induction which
is close or equal to wild-type. However, these mutants
fail to suppress transformation and exhibit gain of
function transforming activity in rat embryo ®broblasts.
These mutants therefore fall into a novel category of p53
mutants which dissociate transformation suppression
from other wild-type functions. The rarity of these
mutants in human cancer and their multiple occurrence
in BRCA-associated breast tumours suggests that these
novel p53 mutants are selected during malignant
progression in the unique genetic background of BRCA1and BRCA2-associated tumours.
Keywords: p53; novel; mutation; breast; BRCA-1;
BRCA-2
Introduction
The tumour suppressor function of p53 is widely held
to be exerted either through induction of G1 arrest via
transcriptional upregulation of the cyclin dependent
kinase inhibitor (CDK1) p21Waf1, or by activation of
apoptosis, following DNA damage (El-Deiry et al.,
1994; Ko and Prives, 1996). Induction of apoptosis
appears to be associated, at least in some cell types,
with expression of another p53-regulated gene Bax
(Miyashita and Reed, 1995). A third p53-inducible
gene, Mdm2, physically associates with p53 and via
this interaction promotes the ubiquitin-dependent
proteolysis of p53, thereby exerting a key role in
determining the stability of p53 protein (Haupt et al.,
1997; Kubbutat et al., 1997). Yet another p53-induced
gene, PIG3, was recently described (Polyak et al.,
*Correspondence: T Crook
Received 2 September 1998; revised 22 October 1998; accepted 3
November 1998
1997). PIG3 is related to a plant gene TED2,
implicated in the apoptotic process associated with
the formation of meristems, and the mammalian
NADPH-quinone oxidoreductase which is a potent
inducer of reactive oxygen species (ROS) which are
powerful inducers of apoptosis. The importance of
transactivation in mediating the biological e€ects of
p53 has been illustrated by studies of human tumour
associated p53 mutations the vast majority of which
occur in evolutionarily conserved regions in the DNA
binding domain and also by the study of rare, human
tumour-derived mutants which selectively transactivate
target p53 promoters. For example p53175P, originally
described in metastatic cervical carcinoma (Crook et
al., 1994), retains essentially wild-type ability to
suppress proliferation, consistent with transactivation
of p21Waf1. However, the same mutant not only fails to
suppress transformation of primary rat embryo
®broblasts (REFs), perhaps by virtue of compromised
ability to transactivate Bax or other p53 target genes,
but also demonstrates a modest gain of function
transforming phenotype (Crook et al., 1994; Rowan
et al., 1996). Interestingly, mice null for Bax retain a
normal p53-dependent apoptotic programme (Knudson
et al., 1995). In the present study we have investigated
the properties of a series of p53 mutants identi®ed in
BRCA1- and BRCA2-associated carcinomas.
Numerous lines of evidence implicate a role for p53
in the aetiology of BRCA-associated breast cancer.
Inheritance of mutant alleles of the breast cancer
susceptibility genes BRCA1 and BRCA2 is associated
with a substantial risk of developing breast, ovarian
and some other cancers (Miki et al., 1994; Wooster et
al., 1996). Tumorigenesis in carriers of germ-line
mutations in BRCA1 and BRCA2 is frequently
accompanied by loss of the wild-type allele (Smith et
al., 1992; Neuhausen and Marshall, 1994; Collins et al.,
1995) implying that these proteins are tumour
suppressors, and they have been proposed to have a
`caretaker' role in the maintenance of genomic stability,
rather than a `gatekeeper' function which operates in
pathways regulating cellular proliferation (Kinzler and
Vogelstein, 1997; Brugarolas and Jacks, 1997). BRCA1
and BRCA2 are essential for embryonic cell proliferation, embryos homozygously deleted for either gene
being inviable, undergoing a p21Waf1-mediated growth
arrest at around day 5 ± 6 (Hakem et al., 1996; Suzuki
et al., 1997). This lethality is partially reversed by
breeding into a p53 or p21Waf1 null background (Hakem
et al., 1997; Ludwig et al., 1997) suggesting that loss of
Novel p53 mutations in familial breast cancer
PD Smith et al
2452
function in either gene can partially reverse the cell
cycle arrest consequent to loss of BRCA-encoded
functions. Interestingly, analysis of ®broblasts derived
from mice homozygous for a truncating mutation in
BRCA2 has revealed a proliferation defect associated
with induction of p21Waf1 expression, and a DNA repair
defect (Connor et al., 1997; Patel et al., 1998).
However, it is not known whether either or both of
these defects occur in tumours arising in carriers of
germline mutant alleles of BRCA1 or BRCA2.
Nevertheless, these observations taken together imply
that loss of p53 function may be an important genetic
event in tumorigenesis in a mutant BRCA1 or 2
background. Consistent with this hypothesis, analysis
of a series of high grade breast and ovarian tumours
arising in carriers of germ-line BRCA1 and BRCA-2
mutations revealed frequent p53 mutation (Crook et
al., 1997, 1998b). We report here the thorough
characterization of these mutants identi®ed in BRCAassociated tumours and discuss their implications for
p53 biology and the mechanisms of BRCA-associated
carcinogenesis.
promoter: 199R shows wild-type activity, 181C and
202S are close to wild-type, 150I and 219H are
approximately 40% of wild-type and 158H and 240R
exhibit activities about 25% of wild-type.
To con®rm that the p53 mutants could also
transactivate the endogenous p21Waf1 allele, Saos-2
cells were transiently transfected with the p53 mutants
and processed for immunohistochemical detection of
p21Waf1 protein. as predicted by the reporter assays,
181C, 202S, 199R and 150I showed similar numbers of
positively staining cells as the wild-type p53. 215C was
approximately 40% of the wild-type whilst 219H and
158H showed very low numbers of p21Waf1 expressing
cells. Data from a typical experiment are shown in
Table 1. No positive cells were detected following
transfection of 240R, empty vector pCB6+ or a
transactivation dead mutant, 248W. These data
indicate that where a mutant shows close to wild-type
activity in the reporter assay, this re¯ects capacity to
transactivate the endogenous p21Waf1 allele. The two
assays di€er with mutants which have relatively weak
activity in the reporter assay e.g. 240R, 158H and
Results
Novel p53 mutations in BRCA1 and BRCA2-associated
tumours
p53 mutations were identi®ed in genomic DNA from a
high proportion of BRCA1- and BRCA2-associated
tumours (Crook et al., 1998b). Having completed
sequence analysis of the BRCA1- and BRCA2associated tumours, the incidence of the mutations in
human cancer was determined from the IARC human
p53 mutation database. Strikingly, three mutants, 150I,
199R and 202S, have not been previously described in
sporadic human cancers and many of the other
mutants were extremely rare in human cancer (Crook
et al., 1998b).
Most BRCA-tumour associated p53 mutants
transactivate the p21Waf1 promoter
Since studies on mice homozygously deleted for
BRCA1 and BRCA2 indicate that early embryonic
lethality is, at least in part, accounted for by a p53dependent induction of p21Waf1, we tested the ability of
p53 mutants associated with BRCA-tumours to
transactivate the human p21Waf1 promoter. Each
mutant was therefore constructed by in vitro mutagenesis, subcloned into a eukaryotic expression vector and
transactivating activity determined against the p21Waf1
promoter in transient transfection assays. Of the 13
BRCA-associated mutants tested, ®ve (144P, 163N,
168Y, 234C and 248W) had completely lost the ability
to activate transcription over a range of input DNA
concentrations up to 500 ng (data not shown). Those
showing evidence of ability to transactivate the p21Waf1
promoter were then subjected to more extensive
analysis. Each of these mutants were assayed in
triplicate at 1, 10, 50 and 500 ng of input DNA.
Results of analyses performed with 50 ng of input
DNA which is optimal for wild-type DNA are shown
in Figure 1. The eight transactivating mutants
demonstrate a range of activities against the p21Waf1
Figure 1 Transactivating activity of p53 mutants from BRCA1and BRCA2-associated tumours against the p21Waf1 promoter.
Saos-2 cells were transfected with 1 mg of each reporter plasmid,
together with the indicated amounts of each expression plasmid
and 250 ng of pCMV b-gal. Lysates were assayed for luciferase
and b-galactosidase activity as described in Materials and
methods. The data are expressed as relative to wild-type p53
transactivating capacity
Table 1 Induction of expression of endogenous p21waf1 in Saos-2
cells by transfection of p53 mutants
p53 plasmid
1
2
3
pCB6+
Wild-type
181C
202S
150I
219H
158H
240R
248W
0
26
21
17
21
1
1
0
0
0
92
64
89
88
24
6
4
0
0
94
39
68
67
13
1
1
0
Data shown are numbers of nuclei positive for expression of p21Waf1.
Results from three typical experiments are shown
Novel p53 mutations in familial breast cancer
PD Smith et al
219H, in this case, the ability to induce the p21Waf1
protein is low or zero.
Mutants retain p53-dependent suppression of
proliferation
The proliferative defect observed in BRCA7/7
embryos and in ®broblasts homozygous for a
truncating mutation in BRCA2 (Hakem et al., 1997;
Ludwig et al., 1997; Connor et al., 1997) is
hypothesized to be p53-dependent. As such, it was
surprising to observe retention of p21Waf1 transactivating activity in numerous p53 mutants from BRCA1and BRCA2-associated tumours, and it was clearly
important to directly assess whether the mutants were
compromised for p53-dependent inhibition of proliferation. We therefore utilized the well-validated stable
transfection assay of p53-dependent growth arrest in
Saos-2 (p537/7) cells. Of the 13 mutants tested, only
four (163N, 168Y, 234C and 248W) were completely
negative for growth arrest whereas the remaining nine
mutants exhibited reproducible growth suppressor
activity (Figure 2). Mutants with wild-type or close
to wild-type ability to transactivate the p21Waf1
promoter (181C, 199R, 202S and 215C) suppressed
Saos-2 growth with close to wild-type eciency. There
is a good correlation between p21Waf1 transactivating
activity and Saos-2 growth suppression. Interestingly
however, 144P suppressed growth by 40 ± 50% despite
being null for p21Waf1 transactivation.
Di€erential transactivating activity of p53 mutants
The retention of transactivating activity against the
p21Waf1 promoter by many of the BRCA-associated p53
mutants raised the question of whether the basis for
their selection in BRCA-associated tumours re¯ected
loss of activity against other p53 target promoters,
rather than p21Waf1. Each mutant was therefore assayed
against four other p53 responsive promoters: mdm2,
Bax, IGF BP-3 and the recently described human PIG3
(Figure 3). Experiments were performed as described
previously for the p21Waf1 promoter. As for the p21Waf1
promoter, 144P, 163N, 168Y, 234C and 248W failed to
transactivate any of the promoters over a range of
input DNA concentrations (data not shown). Analyses
of the remaining eight mutants, which transactivated
p21Waf1, are shown in Figure 3. These segregated
broadly into two groups. Group 1 comprises those
mutants (181C, 219H, 158H and 240R) which
transactivate p21Waf1 and mdm2 (181C and 219H also
transactivate PIG3) but not Bax or IGF BP-3. Group
2 consists of those mutants (150I, 199R, 202S and
215C) which transactivate all ®ve tested promoters.
Since 202S, 199R and 150I mutants transactivate all
®ve promoters tested at close to or more than wild-type
eciency and do so over a range of DNA concentrations (data not shown), we conclude that they are fully
transcriptionally competent. Some striking individual
variations are worthy of note. For example, 202S
reproducibly showed activity against IGF BP-3 which
was at least twofold higher than wild-type. Similarly,
150I transactivated PIG3 signi®cantly more eciently
than wild-type.
Apoptosis-inducing activity of p53 mutants
A number of studies have led to the proposal that the
capacity to induce apoptosis is a critical determinant of
the ability of p53 proteins to mediate transformation
suppression (Lowe et al., 1994). In order to investigate
whether the p53 mutants from BRCA1- and BRCA2associated tumours retained apoptosis-inducing activity, each was analysed in an in vitro assay of p53dependent apoptosis (Crook et al., 1998a). Plasmids
encoding each mutant were transfected into Saos-2
(p537/7) cells. Sixty hours after transfection, cells
were ®xed and stained for p53 with mAb DO-1 and for
DNA content with propidium iodide. p53-positive and
-negative fractions were identi®fed by ¯ow cytometry
and subjected to cell cycle analysis (Figure 4). Mutants
144P and 168Y were completely negative for the
induction of apoptosis. 163N, 219H, 234C and 240R
displayed about 10% of wild-type activity, comparable
with a previously characterized mutant 234H (Parker et
al., 1996) (Figure 4). In contrast, a number of the
BRCA-associated mutants retained signi®cant capacity
to induce apoptosis (Figure 4). 150I, 199R and 202S
displayed close to wild-type apoptosis-inducing activity, 181C and 215C were 30 ± 40% of wild-type, whilst
158H exhibited 15% of wild-type activity.
Apoptosis-competent p53 mutants are undescribed in
sporadic tumours
Figure 2 Analysis of growth suppressor functions of p53
mutants. Suppression of Saos-2 growth. Each dish of Saos-2
cells received 2 mg of pCB6+ p53 plasmid or vector alone. Data
shown are the mean of at least four separate experiments and are
expressed as survival relative to cells receiving pCB6+ vector
alone
The demonstration that some of the BRCA-associated
p53 mutants were competent not only for transactivation and growth suppression, but also, most strikingly,
for induction of apoptosis, was unexpected since the
vast majority of human tumour-derived p53 mutants
have lost these properties (Ko and Prives, 1996). It was
of interest, therefore, to correlate this activity with the
incidence of the mutants in sporadic human tumours.
2453
Novel p53 mutations in familial breast cancer
PD Smith et al
2454
Figure 3 Transactivating activity of p53 mutants from BRCA1- and BRCA2-associated tumours against Mdm2, Bax, PIG3 and IGF BP-3
promoters. Saos-2 cells were transfected with 1 mg of each reporter plasmid, together with the indicated amounts of each expression plasmid
and 250 ng of pCMV b-gal. Lysates were assayed for luciferase and b-galactosidase activity as described in Materials and methods. The data
are expressed as relative to wild-type p53 transactivating capacity
Those mutants which were totally compromised for
p53-mediated transactivation, growth suppression and
apoptosis (144P, 163N, 168Y, 234C and 248W) have
been commonly described in the IARC database,
whereas mutants 150I, 199R and 202S, which retained
close to wild-type levels of transactivating, growth
suppression and apoptosis-inducing activity, are previously undescribed in sporadic tumours.
Each p53 mutant fails to suppress transformation of
REFs
Expression of human wild-type p53 inhibits the
oncogenic transformation of early passage primary
rat embryo ®broblasts (REFs) by nuclear acting
oncoproteins such as that encoded by human
papillomavirus type 16 E7 (HPV16E7) and EJ-ras
(Crook et al., 1991). The majority of p53 mutants
derived from human tumours do not have this
property (Ko and Prives, 1996; Levine et al., 1994).
Moreover, some mutant p53 proteins exhibit gain of
function allowing them to cooperate with ras to
transform REFs. To determine the phenotype of the
p53 proteins from BRCA1- and BRCA2-associated
breast tumours, each mutant was introduced into
REFs either in combination with HPV16E7 and an
activated ras gene or with ras alone. The mutants 144P,
158H, 163N, 168Y, 234C and 248W not only failed to
suppress transformation by HPV16E7 and ras, but
markedly enhanced transformation (Figure 5a). Moreover, each clone was clearly capable of co-operating
alone with ras to eciently transform REFs. Indeed,
comparison with 173L (a p53 mutant with potent
transforming function (Crook et al., 1994)) revealed
that each clone was at least as potent as 173L (Figure
5b). Analysis of mutants 150I, 199R and 202S which
possessed an otherwise wild-type phenotype revealed,
surprisingly, that each was compromised in its ability
to suppress the transforming activity of HPV16E7 and
ras (Figure 5a). Furthermore, the same clones, when
transfected with ras alone, were capable of generating
transformed colonies above the background of vector
Novel p53 mutations in familial breast cancer
PD Smith et al
controls, although colony numbers were clearly below
those of the highly transforming complete loss of
function mutants (Figure 5b). Other mutants, 181C
and 215C, which are substantially wild-type also
exhibited both loss of transformation suppression and
independent gain of transforming function. To verify
expression of the transfected mutant p53 sequences,
transformed colonies were propagated as cell lines and
Figure 4 Flow cytometric analysis of p53-induced apoptosis in Saos-2 cells. In the graphs showing the total cell population, the Xaxis (FL2A) represents DNA content and the Y-axis FITC. The p53+ population is gated in the top right-hand box. In the graphs
showing p53+ve and 7ve populations separately, the X-axis (FL2A) represents DNA content and the Y-axis the number of cells.
Apoptosis is measured as the accumulation of cells with a sub-G1 DNA content (M1) which appears to the left of the G1/G0 peaks
(M2). (a) Mutants with apoptotic activity are compared with wild-type p53 and a previously characterized mutant which retains the
ability to induce apoptosis, 283H (Crook et al., 1988a); (b) Mutants negative for apoptosis are compared with a previously described
highly transforming mutant 234H (Parker et al., 1996); (c) Summary of at least three separate experiments; the average level of
apoptosis induced by each mutant is expressed as a percentage of wild-type
2455
Novel p53 mutations in familial breast cancer
PD Smith et al
2456
Figure 6 Expression of mutant p53 proteins in transformed REF
cell lines. Lysates of cell lines transformed by mutant p53 and ras
were subjected to Western blotting and p53 protein was detected
by the DO-1 antibody. The identity of the p53 mutants in each
cell line are: Lane 1: untransfected primary REFs; Lane 2: 240R;
Lane 3: 181C; Lane 4: 248W; Lane 5: 163N; Lane 6: 234C; Lane
7: 158H; Lane 8: 168Y; Lane 9: 144P; Lanes 10 and 13: Wild-type
p53; Lane 11: 150I; Lane 12: 202S
transfected, wild-type p53 was not expresed (Figure
6). These data are clearly consistent with suppression
of transformation by wild-type p53 and loss of this
property by mutants 150I, 199R and 202S, despite
retention of ecient apoptosis-inducing activity.
Discussion
Figure 5 Transformation suppression and gain of function
transformation properties p53 mutants from BRCA1- and
BRCA2-associated tumours. (a) Transformation of primary
REFs by p53 mutants co-transfected with pEJ6.6. (b) E€ect of
p53 mutants on transformation of primary REFs by HPV16E7
(pCB6+16E7) co-transfected with pEJ6.6. Data are the total
number of transformed colonies per plate. The error bars
represent standard error of the mean
p53 expression was then analysed by Western blotting
using the human ± speci®c DO-1 antibody. Expression
of each mutant was readily detectable in transformed
cell lines (Figure 6), although the level of expression of
mutants with close to wild-type transactivating
phenotypes was lower than that of mutants negative
for transactivation. Interestingly, analysis of the
occasional transformed colonies arising on plates
which received wild-type p53 revealed that the
The ®ndings in this paper have important implications
for our understanding of both BRCA-associated breast
cancer and p53 biology. Our recent ®ndings that p53
mutation is more frequent in BRCA-associated breast
tumours than in a grade matched set of sporadic breast
tumours (Crook et al., 1997, 1998b) was initially
consistent with the hypothesis that p53 mutation
might occur in such tumours in order to abrogate
p53-dependent induction of p21Waf1 expression. This
model was suggested by studies of embryos homozygously deleted for BRCA1 or BRCA2 which revealed
a proliferation defect that was partially relieved by
crossing into a p53 null or p21Waf1 null background
(Hakem et al., 1997; Ludwig et al., 1997). Furthermore,
analysis of murine ®broblasts homozygous for a
truncated BRCA2 which revealed a proliferation
defect associated with a modest increase in steadystate levels of p53 protein, suggested the possibility that
the proliferation defect might be, at least in part, p53dependent (Connor et al., 1997). Taken together, these
studies implied that abrogation of p21Waf1 induction via
loss of function in p53 might, at least in part, relieve
the proliferation defect, and provided a molecular
rationale for the selection of p53 mutations. As a
corollary of this, we had anticipated that loss of p21Waf1
transactivation would be a property common to
mutants from BRCA-associated tumours. However,
we now show the converse, namely that the majority of
the p53 mutants we have identi®ed and analysed retain
signi®cant ability to transactivate the p21Waf1 promoter.
Since the large majority of tumour-derived p53
mutants have lost this activity, this was a most
unexpected result and prompted additional studies to
determine whether other properties of wild-type p53
are intact in these p53 mutants. Analysis of the ability
of each mutant to suppress the proliferation of the
Saos-2 cell line revealed that this property too was
retained by many of the mutants. Previous studies have
demonstrated that ecient suppression of Saos-2
proliferation is often a function of the ability of p53
to transactivate expression of p21Waf1 (Crook et al.,
Novel p53 mutations in familial breast cancer
PD Smith et al
1994). The retention of this property by many of the
mutants in the present series is consistent with their
ability to transactivate p21Waf1. Taken together with the
analysis of p21Waf1 transactivation, these observations
demonstrate that ability to activate a p21Waf1 dependent
G1 checkpoint is substantially retained by these p53
mutants. We conclude, therefore, that the higher
frequency of p53 mutation in BRCA-associated
tumours does not re¯ect an absolute requirement
either for loss of p53-dependent p21Waf1 induction or
for abrogation of a predicted p53-mediated proliferation block.
Aside from their utility in addressing the role of
p53 mutation in BRCA-associated tumorigenesis,
study of these mutants has a€orded signi®cant insight
into general mechanisms of tumour suppression by
p53. We demonstrate a close correlation between
apoptosis-inducing activity of the p53 mutants in this
series and ability to transactivate PIG3. To our
knowledge, this is the ®rst detailed analysis of
transactivation of PIG3 by human tumour-derived
p53 mutants. The correlation between apoptosis and
transactivation of PIG3 is intriguing since Polyak and
colleagues, in describing the cloning and preliminary
functional characterization of the cDNA, showed that
expression of PIG3 was not per se sucient to induce
apoptosis (Polyak et al., 1997). The properties of 181C
and 215C clearly support this observation, since each
transactivates PIG3 more eciently than wild-type
p53, yet has lost about 2/3 of the apoptotic activity of
the wild-type protein, implying that transactivation of
additional genes is required. The close relationship we
observe between apoptosis and induction of PIG3
suggests, therefore, that PIG3 may be necessary,
although clearly not sucient, for implementation of
p53-dependent apoptosis. These observations then
raise the question of which additional gene(s) are
required for p53-mediated apoptosis. An attractive
possibility is Bax, since previous analysis of p53
mutants 175P and 181L demonstrated that both
mutants, although competent to suppress proliferation through transactivation of p21Waf1 (Crook et al.,
1994) lacked apoptosis-inducing activity, a defect
associated with inability to transactivate Bax (Rowan
et al., 1996). Comparison of the transactivating
abilities of 199R and 215C suggests, however, that
Bax is unlikely to represent a strong candidate for a
co-operating pro-apoptotic gene. Although both 199R
and 215C transactivate PIG3 as well as wild-type and
both mutants exhibit comparable activity against the
Bax promoter, 199R is essentially wild-type for
apoptosis induction whereas 215C retains only 30%
activity. Furthermore, 181C which possesses 100%
wild-type activity against PIG3 retains 30% wild-type
apoptotic activity yet is negative for transactivation
against Bax. The observation that mice homozygously
deleted for Bax remain competent to e€ect the p53dependent apoptotic programme further argues
against a critical role for transactivatioan of Bax
expression (Knudson et al., 1995). These results
strongly suggest, but do not prove, that activation
of expression of genes other than Bax must occur for
full implementation of the p53-dependent apoptotic
programme. One conclusion which can be drawn from
these studies is that the in vitro pro-apoptotic
functions of p53 correlate with comprehensive
sequence speci®c transactivation of all the promoters
tested particularly Bax, IGF-BP3 and PIG3. It has
been reported for some p53 mutants that both the
growth suppression and apoptotic functions of p53
have components which do not depend entirely upon
sequence speci®c transcription (Walker and Levine,
1996; Haupt et al., 1995) although these observations
were made with in vitro generated mutations. It has
also been reported that p53-dependent apoptosis can
occur in the presence of inhibitors of transcription
(Caelles et al., 1994; Wagner et al., 1994). Of note
therefore, in the present study is 144P which is null
for transactivation but retains some capacity to
suppress the growth of Saos-2 cells, the same mutant
is however null for in vitro apoptosis and transformation suppression. Therefore if 144P retains nontransactivating functions of p53 they are clearly
insucient to induce in vitro apoptosis or suppress
transformation.
The most striking conclusion from our studies is the
identi®cation of a group of p53 mutants occurring
uniquely in BRCA1- and BRCA2-associated tumours,
which are null for transformation suppression and have
acquired transforming ability despite retaining a
phenotype close to that of the wild-type protein in
terms of transactivation, growth suppression and
apoptosis-inducing functions. In previous studies, the
transforming ability of mutants 175P and 181L was
shown to correlate with absent or severely compromised apoptosis-inducing activity. Accompanying the
diminished apoptotic activity was loss of ability to
transactivate Bax. As such, possession of close to wildtype apoptosis-inducing activity and transactivating
activity against Bax by mutants 150I and 202S, and of
signi®cant activity by 199R and 215C, clearly
di€erentiates these mutants from 175P and 181L.
Similarly, whereas 175P lacks detectable activity
against IGF-BP3, 150I, 199R, 202S and 215C all
possess signi®cant transactivating activity towards this
promoter further distinguishing them from 175P and
181L. To summarize, since 202S, 199R and 150I
mutants transactivate all 5 promoters tested at close
to or more than wild-type eciency and do so over a
range of DNA concentrations (data not shown), we
can ®nd no evidence that they are transcriptionally
defective. However, we cannot formally exclude the
possibility that these mutants fail to transactivate other
p53 responsive genes and that this underlies their
failure to suppress transformation. It is a tantalising
possibility therefore that these mutants represent the
minimal loss of function sucient to lose p53
dependent tumour suppression, as such they would
make an invaluable tool with which to uncover the key
p53 dependent events which mediate tumour suppression.
Taken together, the results of these analyses place
these p53 mutants into a novel category. Their multiple
occurrence in BRCA-associated tumours, loss of
transformation suppression and gain of transforming
function indicate that they are genuine oncogenic
mutants despite the retention of an otherwise close to
wild-type pro®le of biological properties. Retention of
such wild-type properties, particularly ecient p53dependent apoptotic activity, is exceedingly unusual in
sporadic human tumour-derived p53 mutants (Ko and
Prives, 1996) and it is therefore remarkable to observe
2457
Novel p53 mutations in familial breast cancer
PD Smith et al
2458
this property in several of the analysed mutants in the
present series. The only described p53 mutant with a
similar phenotype is 283H which also retains wild-type
activities such as transactivation and apoptosis
inducing capacity (Crook et al., 1988a). However, the
mutants described here di€er from 283H because they
are retained in the nucleus (Paul Smith, unpublished
observations) whereas 283H is constitutively activated
for nuclear export (Crook et al., 1988a). Consistent
with the rarity of retention of wild-type p53-dependent
activities, analysis of the IARC database reveals that
150I, 199R and 202S which exhibited activity most
comparable to wild-type have never been described
previously in sporadic human cancer. Whilst the
compromised transformation suppression functions of
these mutants provides an obvious biological explanation for their presence in tumours, the basis for the
selection of these mutants rather than the full loss of
function mutants commonly seen in sporadic breast
cancer such as 175H and 273C is of interest. The
occurrence of these mutants uniquely in BRCAassociated tumours implies that the genetic background provided by speci®c tumour-associated
BRCA1 and BRCA2 mutations underlies the selection
of these mutants. The genetic background of the
tumours may confer tolerance to or abrogate the
transactivating and/or apoptotic functions of p53
which are retained by 150I, 199R and 202S and in so
doing allows expression of the transforming properties.
The demonstration (Zhang et al., 1998) that mutant
BRCA1 proteins inhibit the ability of p53 to
transactivate the p21Waf1 and Bax promoters provides
strong evidence to support this hypothesis. A second
possibility is that the transactivating ability of these
mutants is tolerated as a result of loss of function in
other components of the p53 pathway. The recent
observation that p19ARF is involved in activation of p53
function is clearly of interest in this respect (de
Stanchina et al., 1998; Zindy et al., 1998). Moreover,
an inverse correlation between expression of the human
p14ARF and p53 function has been described (Stott et
al., 1998). Alternatively, it is possible that survival of
cells within certain genetic backgrounds in conjunction
with the mutant BRCA-allele, is dependent upon
retention of some wild-type p53 functions. Further
investigation into the role of p53 mutation in the
development and progression of BRCA1 and BRCA2
associated tumours will have to include the e€ects of
BRCA-mutant alleles on p53 dependent activities and a
thorough characterization of the molecular pathology
and immuno-phenotype of the tumours.
Materials and methods
Tumours
Paran-embedded specimens were removed from pathology archives and the diagnosis of each tumour con®rmed
by examination of haematoxylin and eosin-stained tissue
sections by two pathologists (PO and BAG). The p53
mutations described in this series were identi®ed by SingleStrand Conformation Polymorphism analysis (SSCP) and
DNA sequencing in tumours arising in carriers of germ-line
mutant alleles of BRCA1 and BRCA2 as described
previously (Crook et al., 1997, 1998b).
In vitro mutagenesis and plasmids
Mutant p53 clones were constructed from pALTER1p53
using the `Altered Sites' system (Promega) as previously
described (Crook et al., 1994). Mutants were identi®ed by
sequencing, then subcloned into pCB6+ for eukaryotic
expression. Construction of pCB6+16E7 was described
previously (Parker et al., 1996).
Transactivation assays
The ability of the p53 mutants to transactivate p53
responsive promoters was assayed in Saos-2 (p537/7)
human osteosarcoma cells essentially as previously
described (Crook et al., 1998a) with minor modi®cations.
Brie¯y, 1.256105 cells were transfected by the calcium
phosphate co-precipitation technique, with 1 mg of luciferase reporter vector and 1 ± 500 ng of the pCB6 + p53
expression plasmid. Each assay included 250 ng of
pCMVb-gal as an internal transfection control. Twentyfour hours post transfection, cells were lysed and processed
for luciferase activity measurements according to the
manufacturers' instructions (Promega). b-galactosidase
assays were performed using b-D-galactopyranoside
(Boehringer Mannheim) as a substrate and activity was
measured using a micro-titre plate reader (Dynex
Technologies). Activation of expression of endogenous
p53 target genes was con®rmed by immuno¯uorescence
staining of aliquots of transfected cells with the anti-p21 Waf1
antibody SX21 (Fredersdorf et al., 1996).
Saos-2 proliferation assay
Assays for the p53-dependent suppression of proliferation
were performed essentially as previously described (Crook
et al., 1998a). Brie¯y, 56105 Saos-2 cells were transfected
with ®xed amounts of pCB6+ p53 expression plasmids and
grown in the presence of 500 mg/ml G418 for 3 ± 4 weeks,
®xed in formol saline and stained in Giemsa.
Rat embryo ®broblast transformation assays
REF transformation assays were performed as described
previously (Crook et al., 1994, 1998a; Parker et al., 1996).
Following transfection, cells were grown in the presence of
500 mg/ml G418 sulphate. Transformed colonies were
counted after 3 ± 4 weeks. Expression of p53 in transformed cell lines was con®rmed by Western blotting of cell
lysates using the DO-1 antibody.
Analysis of apoptosis by ¯ow cytometry
Sixty hours after transfection, the assay for apoptosis was
performed essentially as described previously (Crook et al.,
1998a). After ®xing and staining for p53 with mAb Do1
and DNA with propidium iodide (PI), analysis was
performed with a FACSort (Becton Dickinson). Cells
were measured for their FITC ¯uorescence intensity
(green channel) and PI intensity (red channel). Total
populations were gated to remove doublets and very small
debris and the p53+ve population was gated for high
FITC compard to control transfected cells. M1 represents
the sub-G1 apoptotic population, M2 the G1/G0 peak, M3
represents S phase and M4 is G2/M.
Acknowledgements
We thank K Vousden for pCB6+173L, R Weinberg for
pEJ6.6, B Vogelstein for Waf1 luciferase and PIG3
luciferase, J Reid for Bax luciferase, L Buckbinder for
IGF BP3 Box A luciferase, Moshe Oren for MDM2
luciferase and Xin Lu for the anti-p21Waf1 antibody.
Novel p53 mutations in familial breast cancer
PD Smith et al
T Crook is a Leopold Muller Fellow. J Waller is funded by
US Army grant number DAMD17-94-4066, L Brooks is
supported by the Medical Research Council, P Osin by the
Gilbert Fellowship and G Parker by the Wellcome Trust.
Various parts of the work were supported by the Cancer
Research Campaign and Breakthrough Breast Cancer.
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