Investigations on a clinically and functionally unusual and novel germline p53 mutation.

Abstract

This report describes an individual with a rare choroid plexus papilloma in adulthood (age 29) after earlier having an osteosarcoma (age 22). The results from this study, and others, suggest that it may be advisable to consider the possibility of a germline p53 mutation in adults presenting with choroid plexus tumours. In the current study automated DNA sequencing of genomic DNA detected a novel germline 7 base pair insertion in exon 5 of the p53 gene in this patient. The alteration in frame would produce amino acid substitutions beginning with alanine to glycine at position 161 and a stop codon at position 182 in the mutated protein. Surprisingly two assays of p53 function gave apparently wild-type results on peripheral blood lymphocytes from this individual. These results led us to carry out more detailed functional tests on the mutant protein. The mutant allele was expressed either at very low levels or not at all in phytohaemagglutinin stimulated lymphocytes. Further, the mutant protein was completely non-functional in terms of its ability to transactivate a series of p53-responsive genes (p21(WAF1), bax, PIG3), to transrepress a target gene and to inhibit colony growth in transfected Saos-2 cells. However, surprisingly, data from irradiated peripheral blood lymphocytes and transfected Saos-2 cells, suggested that this truncated, mutant protein retains significant ability to induce apoptosis.

Full text

Page 1

Investigations on a clinically and functionally unusual and novel
germline p53 mutation
J Rutherford1, CE Chu2, PM Duddy1, RS Charlton2, P Chumas3, GR Taylor2, X Lu4, DM Barnes5and
RS Camplejohn*,1
1Richard Dimbleby Department Cancer Research, Guy’s, King’s and St Thomas’ School of Medicine, St Thomas’ Hospital, London SE1 7EH, UK;2St James’s
University Hospital, Beckett Street, Leeds LS9 7TF, UK;3The General Infirmary, Leeds LS1 3EX, UK;4Ludwig Institute, St Mary’s Hospital, Norfolk Place,
London W2 1PG, UK;5ICRF Clinical Oncology Unit, Guy’s Hospital, London SE1 9RT, UK
This report describes an individual with a rare choroid plexus papilloma in adulthood (age 29) after earlier having an
osteosarcoma (age 22). The results from this study, and others, suggest that it may be advisable to consider the possibility of a
germline p53 mutation in adults presenting with choroid plexus tumours. In the current study automated DNA sequencing of
genomic DNA detected a novel germline 7 base pair insertion in exon 5 of the p53 gene in this patient. The alteration in
frame would produce amino acid substitutions beginning with alanine to glycine at position 161 and a stop codon at position
182 in the mutated protein. Surprisingly two assays of p53 function gave apparently wild-type results on peripheral blood
lymphocytes from this individual. These results led us to carry out more detailed functional tests on the mutant protein. The
mutant allele was expressed either at very low levels or not at all in phytohaemagglutinin stimulated lymphocytes. Further, the
mutant protein was completely non-functional in terms of its ability to transactivate a series of p53-responsive genes (p21WAF1,
bax, PIG3), to transrepress a target gene and to inhibit colony growth in transfected Saos-2 cells. However, surprisingly, data
from irradiated peripheral blood lymphocytes and transfected Saos-2 cells, suggested that this truncated, mutant protein
retains significant ability to induce apoptosis.
British Journal of Cancer (2002) 86, 1592–1596. DOI: 10.1038/sj/bjc/6600269
ª 2002 Cancer Research UK
www.bjcancer.com
Keywords: p53; choroid plexus; Li Fraumeni
The tumour suppressor gene p53 was discovered in 1979 because of
its ability to bind to large T antigen (Lane and Crawford, 1979).
p53 is a nuclear phosphoprotein that plays a central role in the
cellular response to DNA damage by inducing either G1arrest or
apoptosis. It functions mainly through its ability to transactivate
or repress target genes. Inactivation of wild-type p53 by mutation
or interaction with cellular or viral proteins has been found to
occur in over 50% of human cancers (Levine, 1997). Li-Fraumeni
Syndrome (LFS), which is a rare dominantly inherited cancer
predisposition syndrome, is associated with germline p53 muta-
tions (Malkin et al, 1990). Individuals with LFS are at increased
risk of developing a large spectrum of cancers, often with a very
early onset and multiple primary tumours are common. Primary
choroid plexus tumours are rare and usually occur in early child-
hood. However, several families with LFS have been described, in
which there are individuals with choroid plexus tumours, some-
times in young adults (Garber et al, 1990). There have been four
reports of germline p53 mutations in families with LFS and child-
hood choroid plexus tumours (Frebourg et al, 1995; Jolly et al,
1994; Sedlacek et al, 1998; Vital et al, 1998). In this report we
describe an adult patient with a novel germline 7 base pair inser-
tion in the p53 gene who presented with an osteosarcoma of the
femur at the age of 22 years. This tumour was treated successfully
by surgery and chemotherapy. At 29 years, she presented with a
choroid plexus tumour which was shown on histology to be an
atypical choroid plexus papilloma. There was no unusual family
history of cancer (Figure 1) and both parents were alive and well
in their 50s. Unexpectedly in routine screening this mutation
appeared functional in two assays of p53 function (FASAY and
apoptotic assay). The FASAY (Flaman et al, 1995) is a yeast based
assay that looks at the transactivational ability of p53. This assay
has been shown to reliably identify both germline (Lomax et al,
1997) and sporadic mutations (Duddy et al, 2000). The apoptotic
assay (Camplejohn et al, 1995, 2000), can detect germline p53
mutations by measuring a reduction in the radiation-induced
apoptoticresponseofperipheral
compared with that seen in cells from normal individuals. Due
to the unexpected results in these two assays, further functional
studies were carried out on this mutation, including an investiga-
tion of its ability to induce apoptosis in mammalian cells, to
transrepress a target gene and to suppress colony growth.
bloodlymphocytes(PBL)
MATERIALS AND METHODS
Sequencing of DNA
Genomic DNA was analysed from peripheral blood leukocytes by
direct sequencing of double-stranded PCR products using the
Big-Dye terminator kit (PE Applied Biosystems) for exons 5–9.
Samples were analysed using a 377 sequencer with 36 cm plates.
Sequences were read in both directions. Sequencing of plasmid
Genetics and Genomics
Received 24 September 2001; revised 19 February 2002; accepted 1 March
2002
*Correspondence: RS Camplejohn; E-mail: r.camplejohn@cancer.org.uk
British Journal of Cancer (2002) 86, 1592–1596
ª 2002 Cancer Research UKAll rights reserved 0007–0920/02 $25.00
www.bjcancer.com

Page 2

and cDNA samples was carried out on a ABI 310 sequencer (PE
Applied Biosystems).
Construction of the vectors
For the transfection experiments, the different p53 mutants (7 base
pair insertion, 337C and 344P) were synthesized from wild-type
p53 using the Quikchange site directed mutagenesis kit (Stratagene)
and were cloned into the mammalian expression plasmid pC53-
SN3. The synthesised mutations were verified using automated
sequencing. The 344P (Arg?Pro) mutation is functionally dead,
whilst 337C (Arg?Cys) retains partial function and along with
wild-type p53 these two mutants were used as controls in the
various assays. The FASAY vectors were kindly provided by Dr R
Iggo, Dr JM Flaman and Dr T Frebourg.
Cell culture and transfection
Saos-2 cells were cultured in DMEM (ICRF Cell Services), supple-
mented with 4 mM L-glutamine (ICRF Cell Services) and 20% FCS
(PAA Laboratories GmbH). Transfections were carried out in
10 cm dishes seeded with 86105cells, using 10 mg DNA and the
Profection Mammalian Transfection System-calcium phosphate
kit (Promega), according to the manufacturer’s instructions.
Functional analysis for the separation of alleles in yeast
(FASAY)
The FASAY was carried out on wild-type p53, 337C, 344P and the 7
base pair insertion containing plasmids as described by Lomax et al
(1997). Briefly, the yeast strain yIG397 contains the vector pLS210
which has the selection gene Ade2 driven by a minimal promoter,
CYC1. In the initial FASAY experiments the vector used has three
copies of the p53 consensus binding sequence from the ribosomal
gene cluster (RGC) immediately upstream of this minimal promoter.
Subsequently, yeast containing similar vectors with the p21, bax and
PIG3 p53 promoter binding sites respectively were used (Flaman et
al, 1998). If the yeast cells are transformed with a wild-type p53 gene
(thus producing wild-type protein) the p53 protein binds to the
RGC (or where relevant the p21WAF1, bax or PIG3) consensus
sequence, transactivating the Ade2 gene. If, however the p53 gene
has a mutation the resulting p53 protein cannot bind to the relevant
consensus sequence and there is no transactivation of the Ade2 gene.
In this case the yeast cells accumulate a red coloured intermediate of
the adenine biosynthesis pathway.
Apoptotic assay
This assay was carried out as described by Camplejohn et al (1995).
Briefly, PBL were separated from whole blood and cultured for 3
days, at which point half of the PBL were exposed to 4 Gy of radia-
tion. Control and irradiated cells were cultured for a further 24 h,
when they were fixed in 70% ethanol. Prior to analysis the samples
were acid denatured in 0.1 M HCl (BDH) and stained with propi-
dium iodide (Sigma). The amount of apoptosis was measured by
the size of the sub-G1peak by FACS (FACSCalibur, Becton Dick-
inson).
Western blotting
For Western blotting, Saos-2 cells were transfected as described and
were lysed using NP40 lysis buffer (BDH) after 24 h. The protein
concentration was measured using the Bio-Rad DC assay (Bio-
Rad Laboratories) and 15 mg of protein denatured in the presence
of SDS was loaded onto a 12% SDS–PAGE gel. Proteins were
transferred onto a nitrocellulose membrane (Amersham Life
Science) and were detected using DO-1 antibody (Santa Cruz
Biotechnology). The secondary antibody used was a rabbit anti
mouse HRP (DAKO) and this was in turn detected using the
ECL Western blotting detection reagent (Amersham Pharmacia
Biotechnology).
Induction of apoptosis in Saos-2 cells
Cells were transfected as described using the calcium phosphate
profection kit (Promega). Cells were washed after 16 h and cultured
for a further 72 h. The Saos-2 cells were then fixed in 70% ethanol
and were stained with propidium iodide (Sigma) and with anti-p53
monoclonal antibody DO-1 (Santa Cruz Biotechnology) for FACS
analysis. Apoptosis was determined for both the p53-positive and
p53-negative sub-populations by the sub-G1 peak size and all
samples were compared to the wild-type control.
Transrepression
56105Saos-2 cells were transfected in 60 mm dishes with 1 mg
pC53-SN3 (containing wild-type or mutant p53) and 5 mg SV40
b-gal. Cells were washed after 16 h and then cultured for 24 h
and washed in PBS. 1X reporter lysis buffer (Promega) was used
to lyse the cells. We added 20 ml lysate to 150 ml chlorophenolred
b-D-galactopyranoside (Boehringer Mannheim) in a 96-well plate,
and this was incubated at 378C. Measurements of absorbance were
recorded using a plate reader at 570 nm every 15 min.
Suppression of colony growth
16105Saos-2 cells were transfected in 60 mm dishes with 7 mg
pC53-SN3 (containing wild-type or mutant p53). Cells were
washed after 16 h and were cultured for 48 h. G418 (Calbiochem)
at 400 mg ml71was then added to the media on each dish, and
cells were cultured for 4 weeks. Cells were washed in PBS, fixed
by adding 70% ethanol, and stained using 0.1% methylene blue.
The number of colonies on each plate was counted.
RESULTS
Genomic sequencing of DNA
Genomic sequencing of DNA from PBL was carried out in both
directions in two independent laboratories from exon 5 through
Genetics and Genomics
3
5
Figure 1
The proband is denoted by the arrow and the numbers (3 and 5) denote
the number of unaffected siblings in the respective branches of the family
tree.
Patients family tree showing no excessive history of cancer.
Functional studies on a novel germline p53 mutation
J Rutherford et al
1593
ª 2002 Cancer Research UKBritish Journal of Cancer (2002) 86(10), 1592–1596

Page 3

to exon 9. A frameshift mutation, the result of a 7 base pair inser-
tion, was discovered in exon 5 of the p53 gene, after the first base
of codon 161 (Figure 2). The alteration in frame would produce
amino acid substitutions beginning with alanine to glycine at posi-
tion 161 and a stop codon at position 182 in the mutated protein.
The insertion, GCCATGG, was found to be a direct repeat of the 7
base pair sequence immediately upstream in the p53 gene.
One of the proband’s parents elected to have predictive testing
and was found not to have the mutation. The other parent
declined testing.
FASAY results
The FASAY is a yeast based functional assay, designed to investi-
gate the ability of p53 to transactivate a target gene by binding
to a consensus sequence from the ribosomal gene cluster. Wild-
type p53 will result in white colonies in the FASAY and mutant
p53 will give red colonies. mRNA from the patient’s PBL was
subjected to RT–PCR and the resulting cDNA was tested in the
FASAY. The results show that only 3% red colonies were produced
in this experiment, indicating a wild-type result (any value under
10% is considered wild-type). Wild-type p53 gave 2% red colonies,
a non-functional mutant (344P) gave a result of 100% red colonies,
and a partially functional mutant (337C) resulted in 98% pink
colonies; all these results were as expected (Figure 3). The 7 base
pair insertion mutation was then synthesised by site-directed muta-
genesis and was put back into the FASAY. Red colonies (100%)
were obtained, which indicated that this mutant p53 could not
transactivate the Ade2 gene by binding to the consensus site from
the RGC. The difference between the two FASAY results (from PBL
and site-directed mutagenesis) could be explained if the patient was
expressing the mutant allele either not at all or at only very low
levels. In this case the white colonies in the original FASAY using
mRNA from the patient’s PBL would result exclusively from the
wild-type p53 allele. The engineered mutated cDNA was then put
into three more FASAYs, using yeast engineered with different
p53 binding sites from the bax, p21WAF1and PIG3 promoters
(Flaman et al, 1998). The 100% red colonies were obtained with
all three binding sites (data not shown). The mutated p53 could
not therefore transactivate bax, p21WAF1or PIG3 in the FASAY.
Apoptotic assay
The apoptotic assay (Camplejohn et al, 1995) was carried out on
PBL obtained from the patient. The assay investigates the ability
of these cells to apoptose in response to radiation and measures
the increase in apoptosis after exposure to 4 Gy radiation. Unex-
pectedly, the results showed a normal response with an increase
in apoptosis of 46%.
Western blotting
The manufactured 7 base pair insertion mutation was cloned into a
mammalian expression plasmid and was transfected into Saos-2
cells. Cell lysates were made of these cells and of Saos-2 cells trans-
fected with wild-type p53. PHA stimulated lymphocytes from the
patient were also lysed and a Western blot was run using all three
lysates (Figure 4). The blot showed wild-type p53 with the expected
size of 53 kDa (lane 1). The lysate from the PHA stimulated
lymphocytes also showed a band at 53 kDa (lane 2). However,
the Saos-2 cells transfected with the 7 base pair mutation showed
the presence of a truncated protein with a size of about 27 kDa.
This corresponds to the predicted size of the mutated protein with
a stop codon at position 182. The patient’s lysate did not contain a
band of this size, which again indicated that either the patient was
not expressing detectable levels of the mutant allele or that PHA
stimulating the lymphocytes favoured the wild-type allele.
mRNA from PHA stimulated lymphocytes was subjected to
RT–PCR and the resulting cDNA was sequenced to try to detect
the 7 base pair insertion. However, only wild-type sequence was
obtained (data not shown).
Induction of apoptosis
Apoptosis in Saos-2 cells can be induced by the expression of wild-
type p53. Saos-2 cells were transfected with the mammalian expres-
sion plasmid pC53-SN3, which contained the 7 base pair insertion
mutation of p53. Controls used included wild-type p53, 337C p53
(a semi-functional mutant) and 344P p53. The results showed that
the 7 base pair insertion mutation retains 65% of the ability to
induce apoptosis compared with wild-type (Figure 5). The 344P
mutation only retained about 20% of the activity of wild-type
p53 and the 337C mutant retained about 55%. Statistical analysis
using both parametric (paired t-test) and non-parametric tests
(Fisher’s exact test) on these data showed that the 7 base pair inser-
tion mutant was significantly different for apoptosis from both
wild-type p53 and the 344P mutant, (P50.02). The 7 base pair
mutation was not significantly different from the 337C mutant.
Transrepression
This method was carried out on the mutants described previously
and involved co-transfection of the mammalian expression plasmid
pC53-SN3 with a reporter construct consisting of an SV40 promo-
ter upstream of the b-galactosidase gene. Wild-type p53 will repress
the SV40 promoter and therefore will prevent the expression of b-
galactosidase. Addition of a b-galactosidase substrate should
produce no colour change. The results obtained in this assay,
showed that the 7 base pair insertion mutant and the 344P mutant
could not effectively repress the SV40 promoter (Figure 6). The
337C mutant retained about 50% of the ability of wild-type to
repress the SV40 promoter.
Suppression of growth
Wild-type p53 can inhibit the growth of Saos-2 colonies and so a
colony forming assay was carried out to determine the ability of
the 7 base pair insertion to inhibit growth. Saos-2 cells were again
transfected with the pC53-SN3 expression plasmid, in order to
observe which of the mutants could suppress the growth of colo-
nies. The results showed that the 7 base pair insertion and the
344P mutants could not suppress the growth of colonies, compared
to wild-type p53 (Figure 7).
DISCUSSION
The 7 base pair insertion is a novel mutation and is not recorded
on the p53 database. The mutation is suspected to be de novo, due
Genetics and Genomics
CACCC GCGT CCGCGCCATGG N C T T NNC C TC T ACCACACANACAN GAC
Figure 2
from peripheral blood lymphocytes by direct sequencing of double
stranded PCR products. A frameshift could be clearly seen starting after nu-
cleotide 13160 in exon 5 (denoted by ;). The insertion was found to be 7
base pairs in length and was a repeat of the 7 base pair sequence immedi-
ately before it. This frameshift would produce amino acid substitutions be-
ginning with alanine to glycine at position 161 and a stop at position 182.
Wild-type: CGCGCCATGGCCATCTA; Mutant: CGCGCCATGGGC-
CATGGCCATCTA
Genomic sequence. Analysis of genomic DNA was carried out
Functional studies on a novel germline p53 mutation
J Rutherford et al
1594
British Journal of Cancer (2002) 86(10), 1592–1596
ª 2002 Cancer Research UK

Page 4

to the lack of family cancer history and the lack of mutation in the
parent tested. The insertion being so large is unusual, as most
reported insertions are between one and three base pairs. Cooper
and Krawczak (1993) looked at 20 short insertions, nine of which
were single base insertions. Two mechanisms for these insertions
were proposed; they could be caused either by slipped mispair
mediated by direct repeats (trinucleotide expansions) or mediated
by inverted repeats (palindrome). The repeats stabilise a hairpin
loop structure. The present reported insertion is a duplication of
the previous 7 base pairs and occurs at a palindrome sequence
of 8 base pairs.
An association between choroid plexus tumours and LFS has
been suggested by others (Garber et al, 1990; Yuasa et al, 1993).
Choroid plexus tumours are rare and are more common in child-
hood (Dohrmann and Collias, 1975). There have been three
previous reports of adult onset choroid plexus tumours in families
with multiple malignancies. Two of these were choroid plexus
carcinomas, presenting at ages 16 and 27 (Li et al, 1988) and
Genetics and Genomics
FASAY results
120
100
80
60
40
20
0
% red colonies
wt
Patient mRNA
Manufactured
mutation
344P
Figure 3
only 3% red colonies when mRNA was extracted from the patients PHA-
stimulated leukocytes. In contrast the 7 base pair mutation synthesised by
site directed mutagenesis gave 100% red colonies. The 344P mutation,
which is an inactive mutant, also gave 100% red colonies and wild-type
p53 gave only 2% red colonies.
FASAY results. The FASAY gave a normal wild-type result with
1 2 3
53 kDa
27 kDa
Figure 4
mammalian expression plasmid pC53-SN3, which was transfected into
Saos-2 cells. A Western blot was carried out using lysates from these cells,
cells transfected with WT p53 and PHA stimulated lymphocytes from the
patient. The blot showed WT p53 with the expected size of 53 kDa (lane
1) and the p53 from the patient’s lymphocytes also at size 53 kDa (lane 2).
The cells transfected with the manufactured mutation showed a protein on
the blot at about size 27 kDa (lane 3).
Western blot. The 7 base pair mutation was cloned into the
Induction of apoptosis in S aos-2 cells
120
100
80
60
40
20
0
% Apoptosis (with wt taken as 100%)
wt
337C
344P
7 base pair
insertion
Figure 5
p53 insert of interest was transfected into Saos-2 cells, which were washed
after 16 h and reincubated for 72 h. The results show that the 7 base pair
mutation retained about 65% of the apoptotic ability of WT p53. 344P, the
non-functional mutant only retained about 20% apoptotic ability. Statistical
analysis showed that the 7 base pair mutation was significantly different
from both 344P and WT, with P50.02.
Induction of apoptosis. The pC53-SN3 plasmid containing the
Transrepression in S aos-2
110
100
90
80
70
60
50
40
30
20
Ability to transrepress (where wt is 100%)
wt
337C
344P
7 base pair
insertion
Figure 6
and pSV40-bgal. Cells were washed after 16 h and lysed after a further
24 h. The results show that the 7 base pair insertion is similar to 344P
(P=0.33), in that it cannot repress the SV40 promoter, whereas wild-type
p53 can.
Transrepression. Saos-2 cells were transfected, with pC53-SN3
Suppression of growth in S aos-2 cells
120
100
80
60
40
20
0
% Colonies (344P taken as 100%)
wt
337C
344P
7 base pair
insertion
Figure 7
pC53-SN3 as before, but were seeded at only 16105cells per 60 mm
dish. Cells were washed 16 h after transfection and were treated with
G418 at 400 mg ml71after 48 h. The cells were left for 4 weeks and
the number of colonies produced was then counted. The results show that
the two mutant proteins (344P and the 7 base pair insertion) produced the
most colonies in this experiment. Indeed, the number of colonies was iden-
tical for these two mutants. As the 344P mutant is known to be functionally
dead, this demonstrates a complete lack of suppression of growth by the 7
base pair insertion mutation.
Suppression of growth. Saos-2 cells were transfected with
Functional studies on a novel germline p53 mutation
J Rutherford et al
1595
ª 2002 Cancer Research UK British Journal of Cancer (2002) 86(10), 1592–1596

Page 5

one, a choroid plexus papilloma, at age 29 (Faber, 1934). There
have been only four previous reports of mutations in p53 found
in families with choroid plexus tumours (Jolly et al, 1994; Frebourg
et al, 1995; Sedlacek et al, 1998; Vital et al, 1998). However, none
of the previously reported mutations were insertions and the
patients were all children.
The results from the first two functional assays carried out
were normal (FASAY and Apoptotic assay). Further investigations
were therefore carried out in order to characterise this mutation.
The FASAY using the site-directed mutagenesis 7 base pair inser-
tion cDNA gave an abnormal result (100% red colonies). This
result most likely indicated that the mutant allele is expressed
at an undetectable level in the original FASAY carried out using
mRNA from PHA stimulated PBL from the patient. However,
the normal apoptotic response of the patient’s cells was surprising
as the presence of a non-expressed mutant allele would be
expected to result in an abnormal apoptotic response. It is possi-
ble that stimulation by PHA of the lymphocytes favoured
expression of the wild-type allele hence the wild-type result in
the original FASAY. The lymphocytes used in the apoptotic assay
are not treated with PHA and it is possible that both alleles were
expressed at the very low levels seen normally in unstimulated
PBL. This result would then be consistent with the ability of
the 7 base pair insertion to induce apoptosis in Saos-2 cells.
Western blotting of the patient’s PHA stimulated lymphocytes
gave a single band present at the expected size of 53 kDa. The
transfected Saos-2 cells showed a band at about 27 kDa, which
was the size expected for a truncated protein with a stop codon
at position 182. No truncated protein was seen in PBL from
the patient and sequencing of the mRNA showed no mutation.
The induction of apoptosis results showed that the 7 base pair
insertion mutation retained 65% of the ability of wild-type to
induce apoptosis in Saos-2 cells. However, the mechanism by
which a 182 amino acid truncated p53 protein can partially
induce apoptosis is not known. The studies on transactivation
and transrepression in this report show the 7 base pair insertion
mutation to be non-functional. The suppression of colony growth
assay also showed the 7 base pair insertion mutation to be clearly
inactive. However, in 1995 Haupt and colleagues reported a trun-
cated protein, containing only the first 214 amino-terminal
residues of murine p53, which was found to retain its ability to
induce apoptosis in HeLa cells, but was transactivationally non-
functional. These results are consistent with the findings described
here for the 7 base pair insertion mutation. Wild-type p53-
induced apoptosis may involve the processes of transactivation
or transrepression of target genes. However, an intriguing possibi-
lity has arisen following reports that p53 can play a pro-apoptotic
role by binding directly to the mitochondrial membrane and
interacting with protein members of the bcl-2 family (Marchenko
et al, 2000). It was suggested by these latter authors that such
binding might indeed be the mechanism by which the truncated
protein described by Haupt et al (1995) induces apoptosis and
the same could be true for the 7 bp insertion mutation.
In summary, the 7 base pair insertion mutation is a novel and
unusual mutation. The clinical details for the patient were
unusual, with the occurrence of a choroid plexus tumour at 29
years. The p53 mutation found in this individual is clearly func-
tionally abnormal in terms of transactivation and transrepression
of target genes and in suppression of colony growth. However,
the normal results obtained for apoptosis induction in lympho-
cytes and the partial ability of the mutant to induce apoptosis
in Saos-2 cells implies that a p53 protein with only half of the
DNA binding domain present and no oligomerisation domain
can retain significant ability to induce apoptosis. In terms of clin-
ical significance it may be advisable to consider the possibility of
germline p53 mutations in adults presenting with choroid plexus
tumours since it may influence decisions regarding treatment
and imaging.
REFERENCES
Camplejohn RS, Perry P, Hodgson SV, Turner G, Williams A, Upton C,
MacGeoch C, Mohammed S, Barnes DM (1995) A possible screening test
for inherited p53-related defects based on the apoptotic response of
peripheral blood lymphocytes to DNA damage. Br J Cancer 72: 654–662
Camplejohn RS, Sodha N, Gilchrist R, Lomax ME, Duddy PM, Miner C,
Alarcon-Gonzalez P, Barnes DM, Eeles RA (2000) The value of rapid func-
tional assays of germline p53 status in LFS and LFL families. Br J Cancer
82: 1145–1148
Cooper DN, Krawczak M (1993) Human Gene Mutation, Oxford, UK: BIOS
Scientific Publishers
Dohrmann GJ, Collias JC (1975) Choroid plexus carcinoma. J Neurosurg 43:
225–232
Duddy PM, Hanby AM, Barnes DM, Camplejohn RS (2000) Improving the
detection of p53 mutations in breast cancer by use of the FASAY, a func-
tional assay. J Mol Diagnost (Am J Pathol, Part B) 2: 139–144
Faber V (1934) Fall von carcinomatos entartetem Papillom des Seitenventri-
kels. Frankfurt Z Pathol 47: 168–172
Flaman JM, Frebourg T, Moreau V, Charbonnier F, Martin C, Chappuis P,
Sappino AP, Limacher J-M, Bron L, Benhattar J, Tada M, Van Meir EG,
Estreicher A, Iggo RD (1995) A simple p53 functional assay for screening
cell lines, blood and tumours. PNAS 92: 3963–3967
Flaman JM, Robert V, Lenglet S, Moreau V, Iggo R, Frebourg T (1998) Iden-
tification of human p53 mutations with differential effects on the bax and
p21 promoters using functional assays in yeast. Oncogene 16: 1369–1372
Frebourg T, Barbier N, Yan YX, Garber JE, Dreyfus M, Fraumeni J, Li FP,
Friend SH (1995) Germ-line p53 mutations in 15 families with Li Fraume-
ni syndrome. Am J Hum Genet 56: 608–615
Garber JE, Burke EM, Lavally BL, Billett AL, Sallan SE, Scott RM, Kupsky W,
Li FP (1990) Choroid plexus tumours in the breast cancer-sarcoma
syndrome. Cancer 66: 2658–2660
Haupt Y, Rowan S, Shaulian E, Vousden KH, Oren M (1995) Induction of
apoptosis in HeLa cells by trans-activation-deficient p53. Genes Dev 9:
2170–2183
Jolly KW, Malkin D, Douglass EC, Brown TF, Sinclair AE, Look AT (1994)
Splice-site mutation of the p53 gene in a family with hereditary breast-
ovarian cancer. Oncogene 9: 97–102
Lane DP, Crawford LV (1979) T antigen is bound to a host protein in SV40-
transformed cells. Nature 278: 261–263
Levine AJ (1997) p53, the cellular gatekeeper for growth and division. Cell 88:
323–331
Li FP, Fraumeni JF, Mulvihill JJ, Blattner WA, Dreyfus MG, Tucker MA, Mill-
er RW (1988) A cancer family syndrome in twenty-four kindreds. Cancer
Res 48: 5358–5362
Lomax ME, Barnes DM, Gilchrist R, Picksley SM, Varley JM, Camplejohn RS
(1997) Two functional assays employed to detect an unusual mutation in
the oligomerisation domain of p53 in a Li-Fraumeni like family. Oncogene
14: 1869–1874
Malkin D, Li FP, Strong LC, Fraumeni JF, Nelson CE, Kim DH, Kassel J,
Gryka MA, Bischoff FZ, Tainsky MA, Friend SH (1990) Germline p53
mutations in a familial syndrome of breast cancer, sarcomas and other
neoplasms. Science 250: 1233–1238
Marchenko ND, Zaika A, Moll UM (2000) Death signal-induced localisation
of p53 protein to mitochondria. J Biol Chem 275: 16202–16212
Sedlacek Z, Kodet R, Kriz V, Seemanova E, Vodvarka P, Wilgenbus P, Mares
J, Poustka A, Goetz P (1998) Two Li-Fraumeni syndrome families with
novel germline p53 mutations: loss of the wild-type p53 allele in only
50% of tumours. Br J Cancer 77: 1034–1039
Vital A, Bringuier P-P, Huang H, Galli FS, Rivel J, Ansoborlo S, Cazauran J-
M, Taillandier L, Kleihues P, Ohgaki H (1998) Astrocytomas and choroid
plexus tumours in two families with identical p53 germline mutations. J
Neuropathol Exp Neurol 57: 1061–1069
Yuasa H, Tokito S, Tokunaga M (1993) Primary carcinoma of the choroid
plexus in Li-Fraumeni syndrome: case report. Neurosurgery 32: 131–134
Genetics and Genomics
Functional studies on a novel germline p53 mutation
J Rutherford et al
1596
British Journal of Cancer (2002) 86(10), 1592–1596
ª 2002 Cancer Research UK