Download Selective Mutation of Codons 204 and 213 of the

[CANCER RESEARCH 52, 2995-2998, May 15, 1992]
Advances in Brief
Selective Mutation of Codons 204 and 213 of the p53 Gene in Rat Tumors Induced
by Alkylating W-Nitroso Compounds'
Hiroko Ohgaki, Gordon C. Hard, Norio Hirota, Akihiko Maekawa, Michihito Takahashi, and Paul Kleihues2
Institute of Neuropathology, University of Zurich, CH-8091 Zurich, Switzerland ¡H.O., P. K.¡;American Health Foundation, I'alhalla, New York 10595 [G. C. H.];
Department of Pathology, Jichi Medical School, Kawachi-gun, Tochigi 329-04 Japan [N. H.]; Department of Pathology, Sasaki Institute, Chiyoda-ku, Tokyo 101, Japan
[A. M.I; and Division of Pathology, National Institute of Health Sciences, Setagaya-ku, Tokyo 158, Japan ¡M.T.]
Abstract
Materials
Kidney and esophageal tumors induced by alkylating /V-nitroso com
pounds in rats contain a high incidence (75-100%) of G -> A transition
mutations in the p53 gene. These are almost selectively (89%) located in
the tirsi base of codon 204 and the second base of 213, leading to amino
acid substitutions Glu -»Lys and Arg -»Gin, respectively. In contrast
to human neoplasms, a considerable fraction of rat kidney and esophageal
tumors carries multiple p53 mutations. All nephroblastomas induced by
transplacental exposure to /V-nitrosoethylurea and 56% of esophageal
tumors induced by /V-nitrosomethylurea showed double mutations in
codons 204 and 213 of exon 6. The selective targeting of p53 codons by
alkylating nitrosamines may provide a basis for molecular epidemiológi
ca! studies on this class of chemical carcinogens.
Induction of Tumors. Renal mesenchymal tumors were induced in
Charles River Wistar rats treated at 5 weeks of age with a single i.p. 40
mg/kg dose of NDMA' (8). Carcinomas (epithelial tumors originating
Introduction
The pattern of point mutations in the p53 tumor suppressor
gene varies considerably in human neoplasms and appears to
be related to organ site. G:C to A:T transitions predominate in
colon, brain, and lymphoid malignancies, whereas G:C to T:A
transversions are most frequent in lung and liver cancers (1). In
addition, specific etiological agents may produce typical mutational hotspots. This was first shown for hepatocellular carci
nomas from high-incidence areas in South Africa and China,
which in 30-40% contain G —¿Â»
T transversion in codon 249 (2,
3). This mutation is assumed to result from the interaction of
aflatoxin B, with cellular DNA and is not observed in hepato
cellular carcinomas from patients with hepatitis B virus infec
tion (4). UV light typically induces pyrimidine dimers in skin
DNA, and a recent report by Brash et al. (5) showed that
squamous cell carcinomas of the skin contain in more than onehalf of cases CC to TT mutations, i.e., double-base changes
that are likely to be due to miscoding by UV light-induced
thymine dimers. /V-nitroso compounds constitute a group of
powerful chemical carcinogens which in experimental animals
induce a high incidence of tumors with a remarkable degree of
organ specificity (6). Although /V-nitroso compounds have been
implicated as contributors to the overall human tumor load, it
has proven difficult to identify human neoplasms specifically
induced by nitrosamines (7). We have, therefore, analyzed a
variety of tumors induced by /V-nitroso compounds in rats and
found a high incidence ofp53 mutation, clustered in two codons
of exon 6.
Received 2/20/92; accepted 4/14/92.
The costs of publication of this article were defrayed in part by 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.
1Supported by grants from the Swiss National Science Foundation.
2To whom requests for reprints should be addressed, at Department Pathol
ogie, UniversitätZurich, Schmelzbergstraße 12, 8091 Zurich, Switzerland.
and Methods
from renal tubles) were induced by the same treatment regimen, except
that the dose was 30 mg NDMA/kg, and the age of the rats at treatment
was 9-10 weeks (8). Nephroblastomas were induced in Nb hooded rats
by a single i.p. (transplacental) exposure to NEU on day 18 of gestation
at a dose of 60 mg/kg body weight (9). Spontaneous nephroblastomas
from a breeding colony of inbred WAB/Not rats were also examined.
These tumors occurred in 0.5% of breeders surviving up to 2 years of
age, representing 40% of the total spontaneous tumors in this rat strain
(10). Esophageal papillomas and carcinomas were induced in F344 rats
by exposure to NMD in the drinking water (200 ppm) for 42 weeks
(11). Adenocarcinomas in the glandular stomach were induced in F344
rats by NMU in the drinking water (200 ppm; 15 weeks) (12), by
intragastric (gavage) doses of NMU (4 mg/kg each), or in newborn
Brown Norway rats by a single dose of NMU (200 mg/kg body weight).
Oligodendrogliomas were induced in fetal brain transplants by two
doses of NEU (50 mg/kg each), using adult F344 rats as recipients
(13).
Single-Strand Conformation Polymorphism Analysis. DNA was ex
tracted from paraffin sections by the procedure described by Shibata et
al. (14) with slight modifications as follows. After comparison with
serial sections stained with hematoxylin and eosin, tumors were scraped
off the histológica!slide. Samples were deparaffinized with xylene and
washed with absolute ethanol. Dried samples were treated with 500 Mg/
ml of proteinase K (Boehringer Mannheim GmbH, Mannheim, Ger
many) in 40-80 n\ of digestion buffer (50 mivi Tris, pH 8.5: 1 HIM
EDTA; and 0.5% Tween 20) at 55°Cfor 3 h. After inactivation of
proteinase K by heating at 95°Cfor 10 min, samples were kept at
-20°Cuntil PCR reaction.
Pairs of 20-mer oligonucleotide primers corresponding to exons 58 of p53 gene were synthesized on a model 391 PCR-MATE DNA
synthesizer (Applied Biosystems, Inc.). The sequences of PCR primers
were 5'-ACTCAATTTCCCTCAATAAG
and 5'-CACCATCAGAGCACGCTCA for exon 5, S'-GGCCTGGCTCCTCCCCAACA
and
5'-GGTGGCTCATACGGTACCAC
for exon 6, 5'-GTCGGCTCCGACTATACCAC and 5'-GGAGTCTTCCAGCGTGATGA
for exon 7,
and 5'-TGGGAATCTTCTGGGACGGG
and 5'-TCTCTTTGCACTCCCTGGGG for exon 8. For prescreening the samples for mutations
in thep5^ gene, PCR-single-strand conformation polymorphism analy
sis was performed according to a slight modification of the method of
Onta et al. (15). Briefly, PCR was performed with 2 /jl of DNA solution,
2.5 pmol of each primer, 50 /JM of deoxynucleotide triphosphates, 1
^Ci of [a-32P)dCTP (Amersham; specific activity, 3000 Ci/mmol). 10
mM Tris (pH 8.8), 50 mM KCI, 1 mM MgCl2, and 0.5 units Taq
polymerase (Perkin-Elmer Cetus) in a final volume of 10 fi\. After
adding 10 ßlof mineral oil (Sigma), 40 cycles of denaturation (95°C)
for 50 s, annealing (58°Cfor exons 5, 62°Cfor exon 6, 6PC for exon
7, and 65°Cfor exon 8) for 50 s, and extension (72°C)for 70 s were
performed using an automated DNA thermal cycler (Perkin-Elmer
Cetus).
'The abbreviations used are: NDMA. /V-nitrosodimethylamine; NEU, Nnitrosoethylurea; NMU, jV-nitrosomethylurea; PCR. polymerase chain reaction.
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pS3 MUTATIONS
IN NITROSAMINE-INDUCED
The reaction mixture (1.5 M') was mixed with 2 M' of 0.1 M NaOH
and 9 »/I
of sequencing stop solution (USB). Samples were heated at
95°Cfor 10 min and immediately loaded onto a 6% polyacrylamide
nondenaturing gel containing 7.5% glycerol. Gels were run at 7 W for
13-15 h at room temperature. Gels were dried at 80°C,autoradiography
was performed with an intensifying screen for 5-48 h, and the pattern
of single-stranded DNA was checked for differences.
Direct DNA Sequencing of PCR Products. For exon 6, all samples
were analyzed by direct sequencing of PCR products. For exons 5, 7,
and 8, direct sequencing was only carried out on samples which had
scored positive in single-stranded conformation polymorphism analy
sis. PCR was performed in a total volume of 80 ^1, as described above.
After amplification, 70 M'of the PCR reaction mixture were separated
on a 6% polyacrylamide gel. Amplified bands were cut out; eluted in
0.5 M ammonium acetate, and 1 mM EDTA at 37°Covernight, and
precipitated with ethanol. Dried DNA was resuspended in 15 ^1 of TE
buffer ( 10 mM Tris-HCl, l mM EDTA, pH 8.0). Sanger dideoxynucleotide sequencing was performed using [«-'2P]dCTP and amplification
primers. The template-primer mixture (4 ^1 DNA and 10 pmol primer)
was heated at 95°Cfor 5 min and immediately placed in liquid nitrogen.
An aliquot containing 20 mM Tris-HCI (pH 7.5), 10 mM MgCl2, 25
mM NaCl, 0.1 M dithiothreitol,
10% dimethyl sulfoxide, and
5 nCi [a-32P]dCTP was added to each of the four termination mixtures
and incubated at 37°Cfor 10 min with 2 units of Sequenase, version
2.0 (USB). Samples were mixed with 4 ^1 stop solution, heated at 80°C
for 2 min, and immediately loaded onto a 6% polyacrylamide/7 M urea
gel. Gels were fixed with 10% acetic acid and 10% methanol, dried,
and autoradiographed for 5 h to 3 days. When mutations were detected,
they were further confirmed by sequencing the opposite strand. In each
reaction, normal rat kidney DNA was concurrently amplified and
sequenced as a negative control.
Results
Independent of the histopathologic tumor type and mode of
induction, all rat kidney tumors investigated contained a high
incidence (75-100%) of p53 mutations (Table 1). These were
heavily clustered in codons 204 and 213 of exon 6. Often, both
exons were mutated in the same neoplasm. This was most
prominent in nephroblastomas induced by transplacental ex
posure to NEU, all of which contained G —¿Â»
A transitions in
the first base of codon 204 and in the second base of 213,
leading to amino acid substitutions Glu —¿*
Lys and Arg —¿Â»
Gin,
TUMORS
respectively. A loss of the normal alÃ-elewas observed in only
one renal mesenchymal tumor (Fig. 1). Esophageal papillomas
and carcinomas induced by NMU also contained a high inci
dence of/753 point mutations in the same two hotspots of exon
6 (Table 1). In both renal and esophageal tumors, mutations
other than in exon 6 were rare (11%); among these, G -»A
transitions were most frequent (Table 1). Glandular stomach
carcinomas induced by NMU and oligodendrogliomas induced
by NEU did not contain miscoding p53 point mutations. One
stomach carcinoma was found to have a mutation in exon 5
(codon 144), but this was nonmiscoding (CAG —¿Â»
CAA; Glu —¿Â»
Glu).
Discussion
DNA sequence analyses were carried out on a variety of
tumors induced in rats by methylating or ethylating A'-nitroso
compounds, using formalin-fixed, paraffin-embedded tissue
samples (Fig. 1). We found a high incidence of miscoding p53
mutations (75-100%) in tumors of the kidney and esophagus
but not in neoplasms induced by the same carcinogens in
stomach and brain. This suggests that the involvement of p53
inactivation during carcinogenesis occurs with a high degree of
tissue specificity. It has previously been shown that NEUinduced gliomas of the central nervous system do not carry ras
mutations (16), and when we analyzed NMU-induced glandular
stomach carcinomas, we were also unable to detect mutations
in the H- and K-ras genes. In contrast, tumors which in the
present study were found to carry p53 mutations are also known
to have a high incidence of K-ras (kidney) and H-ras (esophagus)
mutations (17, 18). It thus appears that in some tissues these
transformation-associated
genes cooperate in malignant trans
formation (19). The simultaneous occurrence of p53 and ras
mutations has also been observed in human colorectal cancer
(20, 21) but does not seem to apply to all tissues. In studies on
human esophageal carcinomas from high-incidence areas, p53
base substitutions were found in 44% of neoplasms, while ras
mutations were completely absent (22).
Regarding the location ofp53 mutations, the results obtained
were unexpected in two respects. Rather than being randomly
Table 1 p53 point mutations in rat tumors induced by N-nitroso compounds
Mutations in codon 204 (GAG -»AAG) and codon 213 (CGC —¿Â»
CAG) cause amino acid substitutions (Glu —¿>
Lys and Arg —¿â€¢
Gin, respectively. Point mutations
other than in exon 6 are as described in the table footnotes.
CarcinogenKidneyCarcinoma
Tumor
Tumors with
or mor
p53mutations7/8
5\"1*0001'0AAT;
NDMAMesenchymal
NDMANephroblastoma
tumor
NEUNephroblastoma
SpontaneousEsophagusCarcinoma/papilloma
(88%)6/8
(75%)8/8(100%)8/12(75%)7/9
NMUGlandular
(78%)1/12(8%)0/7
Exon 6
2131&
(88%)4
(13%)3
(13%)2
(50%)8(100%)9 (38%)8(100%)4 (25%)8(100%)4
(75%)6
(33%)5
(56%)00Codons204
(56%)00Exon
(67%)00Codon2131
stomachCarcinoma
NMUBrainOligodendroglioma
NEU°
Thr.*
Codon 140, ACA -»ACG; Thr -»
Tyr.'
Codon 135, TGC -. TAC; Cys -.
Ile.'
Codon 274, GTT —¿
ATT; Val -.
Cys.'
Codon 238, TGC —¿
TGT; Cys —¿
Tyr.f
Codon 238, TGC —¿
TAC; Cys —¿
codon'Codon
Codon 243, TGC -»TAC; Cys -•Tyr; and
144, CAG —¿
CAA; Glu —¿
Glu.one
(33%)5
80 7
Exon
00
Ie1"
01'
0y
o0
00
(0%)239,
0
»
Asn.Codon2047
AAC —¿cExonAsn —¿
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pS3 MUTATIONS
ACGT
ACGT
ACGT
IN NITROSAMINE-INDUCED
TUMORS
suggests that O 6-alkyldeoxyguanosines
are responsible
for
these mutations.
There is increasing evidence that chemical carcinogens or
their ultimate reactive metabolites cause specific base substitu
tions in oncogenes and tumor suppressor genes (2, 3, 29-31).
Our observation of the same mutational hotspots in tumors of
two different tissues (kidney and esophagus) and in three hiscodon
G
tologically different types of kidney tumors (carcinomas, mes
213
°
enchymal tumors, and nephroblastomas) suggests a selective
targeting of codons 204 and 213 in neoplasms induced by
alkylating /V-nitroso compounds in rats. Identification of such
selective mutations in human neoplasms would allow an esti
mation of the actual contribution of carcinogenic /V-nitroso
compounds to the overall human tumor load. However, this is
the first report on p53 mutations in rats, and it cannot be
excluded that mutations at these sites predominate because they
lead to a species-specific growth advantage of initiated cells,
irrespective of the chemical nature of the inducing agent. Also,
site-specific DNA repair deficiency may contribute to the selec
codon
tive mutations of codons 204 and 213 (28). To resolve this
204
G A
question, analyses of a wide range of chemically induced animal
tumors will be necessary. However, ¡tmay be worthwhile to
analyse exon 6 of p53 in WBC (32) and in second primary
malignancies of patients who underwent chemotherapy with
methylating (procarbazine, dacarbazine, streptozotocin) or
Nephroblastoma
chloroethylating[l,3-bis(2-chloroethyl)-l-nitrosoureaand
l-(2Normal kidney
Renal mesenchymal
chloroethyl)-3-cyclohexyl-1 -nitrosourea] agents.
tumor
It is noteworthy that spontaneous nephroblastomas in WAB/
Fig. 1. DNA sequencing autoradiographs of the rat p53 gene (exon 6) in the
region of codons 202 to 217. The renal mesenchymal tumors induced by NDMA
Not rats showed p53 point mutations similar to, but less fre
contain a transition mutation (GAG —¿Â»
AAG) in codon 204, the normal alÃ-ele
quently than, neophroblastomas induced by NEU (Table 1).
being lost. The nephroblastoma transplacentally induced by NEU shows the same
type of mutation in codons 204 (GAG -> AAG) and 213 (CGG —¿
CAG), but the
The pathogenesis of spontaneous nephroblastomas has not been
normal alÃ-elesare still detectable.
fully elucidated (10). Since their incidence is very low (0.5% of
breeders surviving up to 2 years), it is unlikely that these
neoplasms are due to oncogenic viruses or genetic predisposi
distributed over exons 5 to 8, mutations were heavily (89%)
tion. Our findings suggest rather that nephroblastomas in
clustered in exon 6, and within this exon they selectively af
WAB/Not rats are not truly spontaneous but may result from
fected codons 204 and 213, leading to amino acid substitutions
low-level dietary exposure to environmental /V-nitroso com
Glu —¿Â»
Lys and Arg —¿>
Gin, respectively (Table 1). Point
pounds or related carcinogens.
mutations in codon 204 have not previously been reported in
It has been suggested that the biological properties of mutant
any human or animal neoplasm. Codon 213, on the other hand,
p53 proteins depend on the site of point mutations. Base
is a fairly frequent site for p53 substitution mutations in a substitutions at codons 151, 247, and 273 exert a dominant
variety of human neoplasms (1, 23). Furthermore, the third
negative effect on the phenotype of cotranslated wild-type hu
base of codon 213 shows an approximately 2-11% background
man p53, driving the latter into the mutant phenotype form,
level of CGA -»CGG (Arg -»Arg) polymorphism (24-26). In
whereas the p53 protein encoded by a mutated codon 248, i.e.,
the rat, this mutation creates a typical intron-exon splicing
the germ-line mutation underlying the Li-Fraumeni syndrome
acceptor sequence. Another unexpected finding was the simul
(33, 34), does not show this effect (35). Studies are under way
taneous presence in renal (42%) and esophageal tumors (56%) to determine the effects of point mutations at codons 204 and
of mutations in both hotspot codons. This was particularly
213 of the ratp53 gene and their possible relationship to organevident in nephroblastomas transplacentally induced in rats by specific malignant transformation.
NEU (9). All eight tumors analyzed contained mutations in
Human tumors frequently exhibit either a loss of both alÃ-eles
both codons 204 and 213, in addition to a G —¿Â»
A transition
of the p53 gene, the loss of one p53 alÃ-elewith an associated
mutation in codon 12 of the K-ras gene (17). It remains to be point mutation, insertion or deletion of the remaining alÃ-ele,or
clarified whether these double mutations are present in the an inactivation of thep53 gene in one alÃ-elebut a normal (wildsame tumor cell population or whether there are different
type) sequence in the other. In the present study, sequencing
neoplastic cell types containing point mutations at either codon
gels showed consistently weaker signals for the mutated base
204 or 213. In human neoplasms, double mutations appear to when compared to the corresponding normal (wild-type) se
be a very rare event (26, 27).
quence (Fig. 1). This may be due to the fact that only a fraction
It has been established that 0-alkylated bases are the major
of tumor cells carry the mutation. A loss of the normal alÃ-ele
promutagenic DNA lesions produced by simple alkylating N- was observed in only one renal mesenchymal tumor (Fig. 1),
nitroso compounds. The pathway for production of point mu
but Southern blot analyses with highly polymorphic markers
tations by O 6-methyI- and O "-ethyldeoxyguanosine is mispairmay reveal a higher incidence of allelic loss.
ing during DNA replication with deoxythymidine (28). Our
In conclusion, this study shows that renal and esophageal
tumors induced by carcinogenic /V-nitroso compounds in rats
finding that most base substitutions are G —¿Â»
A transitions
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p53 MUTATIONS
IN NITROSAMINE-INDUCED
contain an unusually high incidence of p53 mutations which
are clustered in codons 204 and 213. Unlike human neoplasms,
a high proportion of tumors contain double mutations in both
hotspot codons.
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Selective Mutation of Codons 204 and 213 of the p53 Gene in
Rat Tumors Induced by Alkylating N-Nitroso Compounds
Hiroko Ohgaki, Gordon C. Hard, Norio Hirota, et al.
Cancer Res 1992;52:2995-2998.
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