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[CANCKR RESEARCH 53. 3465-3467. August 1. ITO]
Advances in Brief
Accumulation
of Wild Type p53 Protein in Human Astrocytomas1
Mari-Paz Rubio, Andreas von Deimling, David W. Yandell, Otmar D. Wiestler, James F. Gusella, and David N. Louis2
Molecular Neuro-Oncolo^v iMbortilory, Ni'iirosurgical Sen-ice IM-P. /?.. D. N. L f, Department of Pathology tNeitropatholoRy) ¡D. M L. }. and Molecular Neitrof>enetÃ-cx
Laboratory. Neurology Sen-ice ¡J.F. G.I. Massachusetts General Hospital and Hazard Medical School. Howe Laboratory of Ophthalmology, Massachusetts Eye and Ear
Infirmary and Hamird Medical School ¡D.W. Y.]. Boston. Massachusetts 02114: Institute for Neun>puthiil<>i>\,University of Bonn. Bonn, Germany ¡A.V. D., O. D. W.¡
Abstract
Materials and Methods
We have previously described 10 astrocytomas with accumulation of
p53 protein but no mutations in p53 exons 5-8, and we have suggested that
Cases. All tumors were cerebral hemispheric gliomas from adults that were
classified by a neuropathologist (D. N. L.) according to the WHO criteria.
Cases 114 and 308 were astrocytoma, WHO grade II; cases 10, 12 and 82 were
anaplastic astrocytoma, WHO grade III; cases 150 and 238 were anaplastia
oligoastrocytoma, WHO grade III; and cases 18, 228, and 294 were glioblas-
they might represent overexpression of wild type protein or mutations in
less conserved regions of the gene. To investigate these possibilities further,
we studied the tumors with immunohistochemistry
for wild type and
mutant p53 protein and showed that all cases stained with the wild type
l'Vii 1801 antibody but only one case stained with the mutant-specific PAb
240 antibody. To support the hypothesis that the accumulated p53 protein
is wild type in most cases, we used single-strand conformation polymor
phism analysis and DNA sequencing to evaluate p53 exons 4,9, and 10 and
did not detect mutations at these loci. Although the product of the MDM2
oncogene binds wild type p53 and may account for p53 accumulation,
slot-blot analysis of these astrocytomas did not detect MDM2 gene ampli
fication. Thus, evidence suggests that some astrocytomas may accumulate
wild type p53 protein but not as a result of MDM 2 gene amplification.
Furthermore, PAb 1801 immunohistochemistry
may not be an adequate
method of screening astrocytomas for p53 mutations.
sections were then exposed to biotinylated horse anti-mouse IgG (Vector Labs.
Burlingame. CA) and Streptavidin-HRP (Jackson Immuno Research. West
Grove. PA), followed by a diaminoben/.idine solution and methyl green coun-
Mutations of the p53 tumor suppressor gene are common in astro
cytomas and may be associated with elevated levels of p53 protein (1,
2). Recent studies of other human cancers, however, have suggested
that p53 gene alterations are not necessarily associated with immunohistochemically detectable p53 protein and that high levels of p53
protein may exist without mutations in exons 5-8 (3-5). We recently
studied 34 astrocytomas using immunohistochemistry on fixed, em
bedded tissues with the monoclonal antibody PAb 1801 to demon
strate p53 protein accumulation, and SSCP' and DNA sequence anal
ysis of exons 5-8 to reveal mutations in the p53 gene (2). Ten tumors
had p53 protein accumulation but no mutations by SSCP. We postu
lated that these cases might have elevated levels of wild type p53
protein or p53 mutations outside of the conserved exons. To determine
whether the accumulated protein was wild type or mutant, we used
immunohistochemistry on frozen tumor tissues with antibodies PAb
240, which is mutant specific, and PAb 1801, which reacts with both
wild type and mutant proteins. To confirm that these tumors did not
harbor mutations in the less-conserved regions of the p53 gene, we
performed SSCP on exons 4,9, and 10, since rare mutations have been
reported in these exons. Finally, because the product of the AÕDA/2
oncogene binds to p53 protein, MDM2 gene amplification could lead
to increased levels of MDM2 product and, in turn, wild type p53
protein (6, 7). We therefore used slot-blot analysis of tumor and
normal DNA to assay these tumors for MDM2 gene amplification.
Received 4/29/93; accepted 6/17/93.
The costs of publication of this article were defrayed in part hy the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by American Cancer Society Grant CB-31A (D. N. L.) and NIH Grant CA
Laboratory. CNY6. Massachusetts General Hospital. Charlestown. MA 02129.
1The abbreviations used are: SSCP. single strand conformation polymorphism: WHO.
World Health Organization: PCR. polymerase chain reaction.
114. 150. 228. 238, 194, and 308).
Immunohistochemistry.
Eight-/nm sections of frozen tumors were cut on
a cryostat and stored at -80°C. Sections were fixed in cold acetone, blocked
with normal horse serum, and incubated with either PAb 1801 (diluted 1:4000)
or PAb 240 (diluted 1:100) (Oncogene Science, Manhasset, NY) for 18 h at
4°C.Tissues were pretreated in 0.05% saponin before PAb 240 incubation. The
Introduction
57683 (J. F. G.. D. N. L.I.
2 To whom requests for reprints should be addressed, at Molecular Neuro-Oncology
toma multiforme. WHO grade IV. All cases were from primary resection
specimens before the patients had been treated with either radiation or che
motherapy. These 10 tumors are a subset of those included in a previous study
(2). Frozen tumor tissue was available for immunohistochemistry on seven of
10 cases (cases 10, 12, 18, 82, 228, 238. and 308). Tumor and blood DNA was
available for SSCP and slot-blot analysis on nine of 10 cases (cases 10. 12. 18.
terstain. Controls were performed by omitting the primary antibody and by
staining the human breast cancer cell line HTB26 (mutant p53) and human
bladder cancer cell line HTB5 (wild type p53) (American Type Culture Col
lection. Rockville. MD) as positive and negative controls, respectively. In
addition, normal human cerebellum was used as a negative tissue control. The
slides were evaluated by two observers (M-P. R.. D. N. L.) for the presence and
proportion of positively stained cells and the subcellular localization of the
reaction product.
SSCP Analysis and DNA Sequencing. SSCP was performed as described
(2. 8), with minor modifications. Exons 4. 9. and 10 of the ¡>53gene were
amplified from tumor DNA using PCR conditions and oligonucleotides similar
to those of Toguchida et al. (9). with the following exceptions. Forexon 4. the
primers used were 5'-ACTTCCTGAAAACAACGTTCT-3'
and .V-GAATCCCAAAGTTCCAAACA-3'.
These primers amplify a 479-base pair amplicon
which, after digestion with Ms/> I. results in 216-. 158-, and 105-base pair
fragments. The amplification products were separated on 67c nondenaturing
polyacrylamide gels with 10% glycerol overnight at 3 W. SSCP results were
confirmed by a second identical assay. Cases with mobility shifts on SSCP
were directly sequenced with Vent«(exo~) DNA polymerase and the CircuniVent Thermal Cycle kit (New England BioLabs. Beverly,
primers.
Slot-Blot Analysis. To generate a probe for MDM2.
licon from the 5' end of the published sequence (7) was
with the primers 5'-GTCTGTACCTACTGATGGTG-.V
TCAAAAGCAATGG-.V
and an annealing temperature
MA), using the SSCP
a 96-base pair amp
amplified using PCR
and .V-AATAACTof 52°C.The PCR
product was extracted with phenol:chloroform. precipitated, and run on a 4%
NuSieve agarose gel (American Bioanalytical. Natick. MA). The product was
isolated and cleaned with the Spin-Bind DNA extraction unit (FMC Bioproducts, Rockland, ME) and labeled with |a-'-P)dATP and (a-'2P]dCTP using the
random priming method. Tumor DNA (300, 30. and 10 ng) and blood DNA
(300 ng) were blotted onto nitrocellulose membranes (Schleicher and Schnell.
Keene. NH) using the Minifold II Slot-blot System (Schleicher and Schnell).
The membranes were prehybridized with salmon sperm DNA for 2 h. followed
by hybridization with the labeled probe for 18 h at 59°C.Membranes were
washed briefly at room temperature and X-OMAT film was exposed for 2 h at
-80°C. Amplification was assessed by comparing signal intensity between
tumor and blood DNA.
3465
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WILD TYPE P5Õ PROTEIN
IN HUMAN
ASTROCYTOMAS
10 228 238 294 308 +
Results and Discussion
To determine whether the 10 astrocytomas with p53 protein accu
mulation and no conserved region mutations had wild type or mutant
protein, we studied these tumors with immunohistochemistry using
two antibodies, PAb 1801 and PAb 240. PAb 1801 is a human-specific
monoclonal antibody that recognizes an epitope near the NH2 termi
nus of both wild type and mutant p53 protein (10). PAb 1801, how
ever, does not recognize most nonsense mutations (2). In this study,
immunohistochemistry on frozen sections showed that all seven avail
able cases were positive with the PAb 1801 antibody (Fig. 1, left),
confirming our previous results with this antibody on formalin-fixed,
paraffin-embedded tissues (2). In most cases, approximately one-third
of cells stained with PAb 1801. Positive reaction product was ob
served only in nuclei, not in cytoplasm, and varied in intensity, with
some nuclei staining more strongly than others. PAb 240 is a mono
clonal antibody that reacts with an epitope in the middle of only
mutant forms of mouse and human p53 protein (11). Immunohis
tochemistry with PAb 240 showed positive staining in only one case
(case 18), with moderately strong nuclear staining in scattered cells
(Fig. 1, right). No cytoplasmic staining was noted with PAb 240. The
positive control cell line HTB26 showed moderate to strong nuclear
staining with both PAb 1801 and PAb 240 antibodies. The normal
cerebellum, the negative control cell line HTB5, and omission of the
primary antibody all resulted in no immunohistochemical reaction.
The results suggest that the accumulated p53 protein may be wild type
in six of the seven studied astrocytomas.
To confirm that these cases did not have p53 mutations outside of
exons 5-8, we performed SSCP analysis on exons 4, 9, and 10.
Previous studies of astrocytomas that have included exons 2-11 have
shown that p53 mutations are exceedingly rare outside of exons 5-8,
with only one exon 4 mutation, one exon 9 mutation, and no mutations
in other nonconserved exons in 128 astrocytomas (1, 12-14). In other
human cancers, mutations outside of exons 5-8 are uncommon but
have also been noted in exons 4 and 9 (9, 15). Four of the nine
available astrocytomas (cases 12, 18, 150, and 228) had SSCP migra
tion shifts in exon 4, but sequencing showed that these four shifts were
caused by the common codon 72 polymorphism (CCC/CGC). No
other migration alterations were noted (Fig. 2), making it unlikely that
these astrocytomas have mutations in p53 coding regions. Intronic
mutations cannot be excluded in these cases but have been noted only
Fig. I. Immunohistochemistry
on frozen tissues of astrocytomas. PAb 1801 (left)
demonstrates both strongly and lightly (e.g., elongated cells in upper left comer) stained
cells. PAb 240 (right) reveals single positive cell in case #18.
Fig. 2. SSCP for p53 exon 10. No migration shifts are noted except in the positive
control. Top, case numbers; +, positive control.
rarely in astrocytomas (1, 16) and other human tumors (17). It is
possible that our one case (case 18) with positive PAb 240 staining has
an intronic mutation. For the remaining cases, however, these data
provide further support that the accumulated p53 protein may be wild
typeCases of lung (4), breast (18, 19), and ovarian carcinoma (20) have
been reported that have p53 protein accumulation, as detected by PAb
1801 immunohistochemistry, but no mutations in exons 2-11. PAb
240 immunohistochemistry, however, was not performed on these
cases to distinguish wild type from mutant protein. Davidoff et al. (21)
have reported overexpression of a stable wild type p53 protein in four
of five human neuroblastoma cell lines. The p53 protein in these cell
lines was detected by immunoprecipitation with both PAb 1801 and
PAb 421, which react with both the wild type and mutant forms of
p53, but could not be detected with the mutant-specific PAb 240. The
cell lines had no mutations in the conserved regions of the p53 gene.
Furthermore, Barnes et al. (22) have documented accumulation of
wild type p53 protein in the normal cells of two members of a
cancer-prone family. Although high levels of wild type p53 would be
assumed to have a tumor suppressor function, abnormally bound p53
may be inactive, as is hypothesized for normal p53 bound to viral
oncoproteins. Furthermore, accumulated wild type p53 protein may be
sequestered in the cytoplasm, away from its nuclear targets (23),
although we did not observe distinct cytoplasmic staining in our cases.
Tuck and Crawford (24) have shown that overexpression of wild type
p53 in normal fibroblasts may enhance the tumorigenic potential of
these cells. High levels of wild type p53 protein may also accumulate
in rapidly proliferating nonneoplastic cells, and it is possible that
similar high levels may arise in dividing tumor cells independent of
p53 mutation (3, 25). Finally, nuclear levels of wild type p53 protein
may increase after various types of DNA damage, and such accumu
lation may represent a common response to DNA injury (26). In
summary, increasing evidence argues that wild type p53 protein levels
may be elevated in the absence of gene mutations in some cases. At
the same time, it remains possible that elevated wild type p53 protein
may be associated with mutations in intronic sequences that are not
examined by current molecular genetic techniques.
Wild type p53 protein has a short half-life and is generally not
detectable by immunohistochemistry (27), whereas mutant p53 pro
teins often have extended half-lives (28) and can be detected immunohistochemically. In our cases, therefore, the wild type protein could
have an extended half-life. One possible explanation is that wild type
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WILD TYPE P53 PROTEIN
300 ng
N
30 ng
IN HUMAN
10 ng
5.
r
6.
238
7.
8.
N
10
9.
Fig. 3. Slot-blot analysis of MDM2 gene in blood (N) and tumor (7) DNA from cases
10 and 238. MDM2 gene dosage is the same for equal amounts (300 ng) of blood and
tumor DNA in the first column. Dilutions of the tumor DNA appear in the second and third
columns.
10.
11.
p53 is bound by another protein which elongates its half-life. A can
didate for such a protein is the p90 product of the MDM2 oncogene.
p90 binds to both wild type and mutant p53 protein and has trans
forming properties (6). MDM2 amplification has been noted in ap
proximately one-third of human sarcomas (7). For these reasons, we
evaluated our cases for MDM2 gene amplification. Slot-blot analysis
demonstrated equal amounts of MDM2 gene in tumor and blood DNA
in the nine astrocytomas studied (Fig. 3). Another study has recently
reported a similar lack ofMDM2 amplification in human astrocytomas
(29). Other mechanisms must therefore exist to produce accumulation
of wild type p53 protein in a subset of human astrocytomas. Recent
data have suggested that increased protein stability is a more likely
mechanism for wild type p53 protein accumulation than p53 overexpression (26). Further elucidation, however, awaits the identification
of other human proteins that bind p53 and clarification of factors that
control p53 expression (30).
The present findings provide further evidence that immunohistochemistry using the PAb 1801 antibody does not correlate well with
p53 gene analysis in human astrocytic tumors. We have previously
shown that PAb 1801 does not stain astrocytoma cells with nonsense
mutations (2). In turn, the present findings indicate that PAb 1801 may
not reliably distinguish wild type from mutant protein in human
astrocytomas. Jaros et al. (31) recently demonstrated that PAb 1801
immunopositivity correlates with poor clinical survival in astrocytoma
patients and attributed the immunopositivity to presumed mutations in
the p53 gene. An alternative explanation may be that PAb 1801
immunopositivity reflects accumulated wild type p53 protein and that
tumors with high levels of normal p53 protein are less susceptible to
the DNA-damaging effects of radiation and chemotherapy than those
tumors with p53 gene mutations (30). The biological basis of clinical
correlations with PAb 1801 immunohistochemistry
may therefore
need to be reevaluated in light of the present data.
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3467
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Accumulation of Wild Type p53 Protein in Human Astrocytomas
Mari-Paz Rubio, Andreas von Deimling, David W. Yandell, et al.
Cancer Res 1993;53:3465-3467.
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