Download Harvey ras (H-ras) Point Mutations Are Induced by 4

[CANCER RESEARCH54, 5310—5317,
October 15, 19941
Harvey ras (H-ras) Point Mutations Are Induced by 4-Nitroquinoline-1-oxide in
Murine Oral Squamous Epithelia, while Squamous Cell Carcinomas and
Loss of Heterozygosity Occur without Additional Exposure1
Bo Yuan, Briana W. Heniford, Douglas M. Ackermann, Brian L. Hawkins, and Fred J. Hendier@
Departments of Biochemistry fB. Y., F. J. H.), Surgery [B. W. H., B. L H., F. J. H.], Pathology (D. M. A.], and Medicine (F. J. H.], the Henry Vog: Research Institute of the
James Graham Brown Cancer Center (B. 1'., B. W. H., B. L H., F. J. H.], University of Louisville, Louisville VA Medical Center (F. J. H.], and Alliant Hospitals (D. M. A.],
Louisville, Kentucky 40292
ABSTRACT
tumors are typically well-differentiated tumors (3, 9). Lesions develop
Tumorigenesis is a multistep genetic process requiring several somatic
mutations for neoplastic transformation.
These mutations appear to be
sequential, random, and independent events. However, we find linked,
nonrandom
ma mutations
duced tumorigenesis
occurring
during
4-thtroqulnoline-1-oxide-in
months after exposure to the carcinogen
had ceased.
Thecarcinogenhadbeentopicallyappliedto the oralcavityof CBAmice
for 4 to 16 weeks. Dysplasia developed
after 24 weeks, and carcinoma
in
situ and squamous cell carcinoma developed after 28 weeks. H-ms muta
lions were detected In 13 of 25 tissue specimens (10 of 14 invasive card
nomas and 2 of 4 carcinoma in situ, 1 of S dysplastic tissue, and 0 of 2
normal tissues). Approximately
one-half of the tumors had C to A point
mutations at codon 12 of the cellular H-ms proto-oncogene
on mouse
chromosome
7. None had codon
11, 13, or 61 mutations.
Loss of heterozy
gosity occurred in 5 of 14 invasive cancers. Larger invasive squamous cell
carcinomas consistently lost the wild-type allele, whereas preneoplastic
lesions and small tumors were heterozygous for ma. This suggests a causal
relationship between carcinogen treatment, H-ms activation, and initia
tion of tumorigenesis.The wild-typoallelein mousechromosome7 is lost
with the progression
of tumorigenesls
long after
exposure
to the carcino
gen. Thus, loss of heterozygosity of the ras gene appears to occur without
multiple carcinogen-induced mutations, i.e., as a result of a cascade of
events
induced
by an earlier
ras mutation.
SCCs3 in the aerodigestive tract are typically chemical carcinogen
induced human malignancies closely associated with tobacco expo
sure and alcohol consumption (1, 2). To define the molecular events
involved in oral squamous mucosa neoplastic transformation, we have
established a murine model which mimics human oropharyngeal
SCCs using the chemical carcinogen, 4NQO (3). 4NQO is a complete
chemical carcinogen which acts as an alkylating agent causing G to A
transversion (4, 5). Topical application to the oral cavity without
subsequent exposure to a tumor promoter resulted in preneoplastic
and oral cavity 5CC in rodents (6, 7, 8). In similar studies, tn-weekly
application of 4NQO to the oral cavity for up to 16 weeks produced
5CC in 100% of CBA mice after 50 weeks (3). Morphologically, the
that develop
resemble
human head and neck SCC. The pro
gression from preneoplastic to invasive carcinoma is orderly. The
Received 2/9/94; accepted 8/18/94.
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.
I Supported
in part
by
the
Department
of
Veterans
Affairs,
the
Alliant
Community
Trust Foundation, the James Graham Brown Cancer Center Foundation, and the Norwich
Eaton Resident Award.
2 To whom
requests
for reprints
should
H-ras mutations have been implicated in human and munine squa
mous carcinogenesis (10, 11). ras oncogene activation occurred dun
ing the early stages of skin carcinogenesis induced by the carcinogen,
DMBA, and the promoter, TPA (12, 13). Continuous exposure of
squamous cells to DMBA and TPA induced H-ras mutations on
chromosome 7 in greater than 90% of mice (14). H-ras appeared to be
activated by specific mutations which can be affected by the initiating
carcinogen (15). Since tumors do not develop immediately, the acti
vated ras oncogene may be detected only when neoplastic develop
ment and clonal expansion has occurred. The wild-type ras allele is
frequently lost with continued exposure to carcinogen and/or tumor
promoter (16, 17, 18). This LOH has been associated with tumor
progression. An increase in the ratio of mutant H-ras to normal H-ras
correlated with both progressive chromosomal changes and morpho
logical evidence of neoplastic transformation (16, 17, 18, 19).
In vitro chemical modification of critical oncogenes by carcinogens
INTRODUCTION
lesions
in areas of dysplasia surrounded by apparently “normal―
tissues.
Transformation appears clonal as demonstrated by EGF receptor
overexpression (9). Neoplastic transformation occurs in the absence of
any inflammation at least 3 months after the cessation of exposure to
the carcinogen.
be addressed,
at Division
of Medical
Oncology!
such as ras with subsequent DNA transfection has been used to
determine the carcinogenic potential of various compounds (20, 21).
Chemical modification of the plasmid proto-ras oncogene in vitro
with 4NQO led to codon 12 mutations. Subsequent DNA transfection
resulted in activated H-ras (22). This demonstrates a direct interaction
between the initiating carcinogen and the critical ras DNA sequences,
implying that ras oncogene activation is involved in the initiation of
neoplasia.
DMBA-induced skin (12, 19) and oral cavity SCCS are frequently
associated with H-ras mutations (23), whereas 4NQO-induced ras
oral cavity lesions have not been reported previously. ras mutations in
human
head and neck cancer
are detected
in approximately
10% of
tumors in Western civilizations (24, 25). However, H-ras mutations at
codons
12 and 61 occur in 35% of oropharyngeal
5CC in India (26).
Since the 4NQO-induced lesions closely resembled human head and
neck cancer, the incidence of H-ras mutations was investigated in
4NQO-induced oral cavity tumors using highly sensitive molecular
techniques. H-ras point mutations were detected in approximately
60% of tumor tissues. Most remarkably, long after exposure to 4NQO
has ceased, most larger invasive tumors with ras mutations lose the
normal ras allele and only the mutant ras remains, i.e., LOH occurred.
This observation suggests that mutant ras alleles confer a selective
growth advantage in tumor progression, and LOH at codon 12 occurs
without exposure to tumor promoters or carcinogens.
Hematology, J. Graham Brown Cancer Center, 529 S. Jackson Street, Louisville, KY
40292.
3 The
abbreviations
used
are:
SCC,
squamous
cell
carcinoma;
4NQO,
MATERIALS AND METHODS
4-nitroquinoline
1-oxide; DMBA, 7,12-dimethylbenz(a)anthracene;
TPA, 12-O-tetradecanoylphorbol-13acetate; LOH, loss of heterozygosity; CIS, carcinoma in situ; PCR, polymerase chain
reaction; dNTP, 3'-deoxynucleoside-5'-triphosphate; RFLP, restriction fragment length
polymorphism; MAMA, mismatch amplification
conformational polymorphism.
mutation assay; SSCP, single-strand
4NQO Treatment
Seventy
female
CBA mice (Charles
River,
Boston,
MA) approximately
9
weeks of age and 23 to 27 g were treated three times per week from 4 to 16
5310
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1994 American Association for Cancer Research.
4NQO-INDUCED H-ms MUTATIONS
weeks with 5 mg/ml 4NQO (Sigma Chemical Co., St. Louis, MO) in propylene
glycol and topically applied to the posterior oropharynx (3). Untreated, pro
pylene
glycol,
and carcinogen-treated
mice were
observed
Identification
for a total of 49
Gross lesions, when present, were identified, and the tissues were immediately
frozen in liquid nitrogen and stored at —70°C.
The lesions produced were
identified and graded as normal, mild dysplasia, moderate dys
plasia, severe dysplasia,
Abnormal
CIS, and invasive
Mutations
of H-ins
Genomic
DNA.
Genomic
DNA
cancer.
Cell Isolation
andSi (Fig.2) at 94,55, and72°C
for 1 mmandfor 60 cycles.EachPCRwas
carried out in a SOjil solution containing 10 m@iTris-HCI (pH 8.3), 50 mM
KC1,1.5 mMMgCl2, 1 ,LMof each oligonucleotide primer,200 @M
of each
dNTP (Pharmacia, Piscataway, NJ), and 0.1 unit4d of Taq DNA polymerase
(Boehnnger Mannheim, Indianapolis IN). Additional Taq DNA polymerase
(0.025 unit/pA)was added at 30 cycles. A nested PCR was used to obtain
Cryopreserved tissues were sectioned (6—8 @&m)
and fixed in buffered
formalin. To reduce the contamination of tissue specimens with nonpreneo
plastic or nonmalignant cells, sections were stained with polychrome (3.6 g of
specific
toluidine blue 0 and 1.36 g of basic fuchsin in 500 ml of 30% ethanol; Sigma;
normal tissues and on two sections from dysplastic tissues.
Fig. 1). Abnormal cells were identified, physically isolated using a scalpel
and transferred
to a 0.5-ml
in 5 p1 of the
mixture from the isolated lesions was amplified by PCR using the primers Al
weeks. Groups of mice were sacrificed at 16, 20, 24, 28, 33, 38, and 49 weeks.
histologically
Amplification
of H-ms
fication
microscope,
tube. A 50-@l
solution containing
0.5% Nonidet P-40, 10 m@i Tris-HC1 (pH 8.0), 10 m@i
1 @.dof the Si
+ Al
PCR product with
was carried out on at least three different
sections
from tumor and
RFLP Analysl& RFLP analysis was used to determine the allelic status of
H-ms
under a dissecting
H-ras DNA by amplifying
internalprimersA2 and 52 (Fig. 2) for an additional30 cycles.Each ampli
point mutations.
cation were digested
PCR products
obtained
with the restriction
NaCl, 3 mMMgCl2, and 0.5% sodium dodecyl sulfate was added to the tissue
to the recommendations
and incubated at room temperature for 2 h.
cuts wild-type
from the nested
enzymes,
PCR amplifi
MnlI and PvuII, according
of the supplier (USB, Cleveland, OH). PvuII digestion
and mutant H-ras
similarly
(data not shown).
MnII cuts the
Fig. 1. Physical isolation of tumor cells. Cryo
preserved tissues were sectioned (6—8pm), stained
with polychrome, and washed with phosphate-buff
ered saline buffer. Abnormal areas were identified
and physically isolated using a scalpel under
a X 2.5 dissecting microscope. Genomic DNA was
extracted and used in subsequent PCRS.A-C, poly
chrome-stained tissue sections prior to dissection
(X 100); D-F, the same sections viewed under the
dissecting microscope (X 2.5); G-1, isolated dis
sected tissue viewed under the dissecting micro
scope (X 2.5); J-L, the isolated tissue (X 100). A,
D, G, and J, dysplastic tissue (tissue no. 1; Table
1); B, E, H, and I, CIS (tissue no. 2; Table 1); 5CC
(tissue no. 4; Table 1). The circles in A-F and
arrows in G-l indicate the area of the tissue which
was dissected.
5311
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1994 American Association for Cancer Research.
4NQO-INDUCED H-ras MUTATIONS
@
@
MnlI
Fig. 2. Murine H-ms exon I. Location of restric
tion sites, oligonucleotide primer sequences, and
PCR products. The genomic sequences of murine
exon I were adapted from Brown et a!. (39). The
Al
mismatchedpnmer(PAA)
CAT-3' (14); Al, 5'-CACCFCFGGCACCFAG
GCAGAGC-3' (14); S2, 5'-TGGCAGGTGG
codon12
and A2, 5'-GAGCTCACCTC
TATAGTGGGATC-3' were developed for this
study. The mismatched
MnlI
I
oligonucleotide primer sequences used in standard
PCR were: Si, 5'-Cfl'GGCFAAGTGTGmCT
GGCAGGAGC-3';
Pvull
mismatched PCR
PCR primer, PAA, (5'-
lO2bp
cTFGTGGTGGTGGGCGCFAA-3'), was devel
@
oped by Cha el al. (27). MnlI and Pvull indicate the
location of these restriction sites in exon I. The
fragments, 206, 167, and 102 base pairs, indicate
the length of the PCR-amplified products with the
indicated oligonucleotide primers.
wild-type
H-ras
segment,
resulting
in three fragments
nested __________________________I @7
PCR
_________________________________________________
of 19, 73, and 75 base
(1 ,.@g!ml) in 0.SX ThE (1 X ThE = 90 m@iTris-Borate,
pairs, whereas digestion of H-ras mutant at co-Jon 12 results in two fragments
of 19 and 148 base pairs. Following digestion, fragments were separated by
electrophoresis at 100 V on 3% agarose gels containing ethidium bromide
A
2.0 mM EDTA) buffer
(Fig. 3).
MAMA. Since in preneoplastic lesions or small tumors only a few cells
might contain the mutation, the more sensitive MAMA was used to detect
infrequent
@
@
206 bp
first PCR
H-ras point mutations
(27, 28). The point mutation detected
in the
tissues studied was a G to A transversion at the second base of codon 12. Since
,@ @4SSSIol@IOuS
Hs4@
Homozyg@*
‘I.wv
$1.A1
52.52
hb@I i*@
tSi.*i
32.5.2
thJ
the DNA
sequences
in this region of the H-ras gene are identical
between
mouse and rat, the optimal nucleotide sequence for the mismatched primer
was selected from the 11 primers described by Cha et a!. (27) for detecting
a 0 to A transversion at codon 12 of rat H-ras (Fig. 2). The primer PAA
is mismatched
20
@
73.
for the wild-type
sequence
3'-terminus
and is mismatched
transversion
at only the terminal 3'-base.
in the last two bases at the
for the codon
12 mutant
with
a 0 to A
PCR cycle number, temperature,
duration of primer extension, and magnesium and glycerol concentration
were all tested (27). Briefly, DNA isolated from tissue sections was
amplified by nested PCR as described as above and diluted 1:100. Mis
matched PCR was carried out using PAA and A2 primer at 94°Cfor 1 mm
and 55°Cfor 1 mm for 35 cycles with 0.35 pmol4tl each primers, 0.1 mM
dNTP, 0.015 unit/pi of Taq polymerase, and 10% glycerol. PCR products
were then separated by electrophoresis at iOO V on 3% agarose gels
containing ethidium bromide (1 @Wml)
in 0.5X TBE buffer.
—@
B
SSCP Analysis.
SSCP was used to physically separate and isolate H-ras
DNA segments for direct sequencing (29). The PCR was performed for 40
cycles in 50 @l
containing 5 pi of genomic DNA, 0.5 p@lof [a-P32]dATP
(3,000 Ci/mmol; Amersham, Arlington Heights, IL), 0.2 m@tdNTPs, and 1
@LM
of Si and Al primers
with melting
at 94°C for 1 mm, annealing
at 65°C
for 1 mm, and elongation at 72°Cfor 1 mm. The 206-base pair PCR product
was removed
C
from low melting
agarose
gel by elution
with 0.1% sodium
dodecylsulfate, 0.5 Mammoniumacetate, and 10 mMmagnesiumacetate
at 37°Cfor 12 hours by shaking and purified through a Chroma-Spin 100
(Clontech, Palo Alto, CA). The isolated PCR fragment was diluted 1:10 in
@:
mt
loading
wt
Fig. 3. Identification of H-ras mutations by RFLP analysis. A, nested PCR. Tumor
DNA was isolated (see “Materials
and Methods―).
PCR amplification of H-ms using Si
solution
(96%
formamide,
20
mM
EDTA,
0.05%
xylene
cyanol,
and
bromophenol blue), denatured at 95°Cfor 3 mm, and applied (5 id/lane) to
a 5% polyacrylamide (acrylamide:bis, 50:1) per 0.5X ThE gel with 5%
(v/v) glycerol. Electrophoresis was carried out at 16°Cand 40 W for 3 h in
a 40 x 20 x 0.035
cm gel (Hoefer
Scientific,
San Francisco,
CA). The gel
and Al primers was performed (Lanes Sl+AI). The synthesized nucleotide segments
were subsequently amplified with primers S2 and A2 (167 base pairs; Lanes S2+A2).
was dried on filter paper and exposed to X-ray film (Kodak XRP-1;
The second segment was digested with MnII and subjected to electrophoresis in 0.5X
TBE, 1 @tg/mlethidium bromide at room temperature, and 150 V for 2 h using a 4%
agarose gel. Wild-type H-ras was digested at both MnIl sites yielding three fragments
Eastman Kodak, Rochester, NY) for 2 h.
Sequence Analysis. Abnormal fragments detected by SSCP analysis were
eluted and amplified by PCR using nested 52 and A2 primers. The PCR
of 75, 73, and 19 base pairs, which are unresolved on this gel, whereas mutant ras was
digested yielding only two fragments of 148 and 19 base pairs. Normal ras. tissue no.
5; Heterozygous Ha-ras, tissue no. 3; Homozygous Ha-ras, tissue no. 4. Ha-ras, H-ras.
Arrows, the location of the 73-, 75-, 148-, 167-, and 206-base pair fragments. B and
C, allelic analysis of H-ras point mutations at codon 12. Nested PCR with subsequent
RFLP was applied to all 25 tissue specimens (Table 1). Standard, DNA amplified
from H-ras segments inserted into plasmids. Lane A, RFLP of Mnll-digested PCR
products amplified from normal H-ras (tissue no. 5); Lane B, 1:1 mixture of mutant
and wild-type plasmid DNA from tissues nos. 4 and 5; Lane C, homozygous mutant
H-ras (tissue no. 4); Lane D. undigested PCR product (tissue no. 4). Lanes 1—15and
C, Lanes 16—25,
MnIl digests of tissues nos. 1—25
(Table 1). Molecular weights (MW)
are a HaeIII digest of PBR 322 (Sigma). mt, mutation type; wt, wild type.
product was ligated into a pCR II vector and cloned according to the
recommended procedures of the provider (In vitrogen, San Diego, CA).
Colonies were selected, minipreps of DNA were made, and the isolated
DNA was alkaline denatured. DNA sequencing was performed using the
2-pg
denatured
plasmid
DNA and 0.5 pmol
of M13 reversed
primer
(—40
base pairs), 5 p@Ci[a-S35]dATP and Sequenase Kit reagents as recom
mended by the provider (United States Biochemicals). Wild-type mouse
genomic DNA was similarly cloned from PCR products synthesized with
A2 and 52 primers and sequenced. Wild-type and mutant H-ras cloned
5312
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1994 American Association for Cancer Research.
4N00-INDUCEDH-ms MUTATIONS
@
DNA segments were used as internal controls in the RFLP, MAMA, and
SSCP reactions.
no glycerol
A
5%glycerol
1O%glycerol
1f@
11
RESULTS
To determine if H-ras mutations occurred during malignant trans
formation, 25 morphologically characterized tissues with a range of
histologically identified pathological lesions were selected from
4NQO-treated mice. Normal tissues were obtained from either un
treated or vehicle-treated (propylene glycol) mice. Cryosections were
stained with polychrome; the presence or absence of a morphological
lesion was confirmed microscopically (Fig. 1). DNA was isolated, and
INr—@
mt 0 0.02 0.2 2 0 0.02 0.2 2
MW
I 2
3 4 5
6
7
8
0 0.02 0.2 2 (p9)
9
10 11 12
the H-ras gene in the region of the first and second exons was
amplified by PCR (Fig. 2). PCR amplification of cryosection DNA
with primers specific for H-ras exon 1, Al, and 51, resulted in
multiple bands with a predominant segment of 206 base pairs. The
subsequent amplification with internal oligonucleotide primers, A2
and S2, resulted
in a single fragment
of 167 base pairs (Figs. 2 and 3).
PCR amplification in the absence of genomic DNA or with human
genomic DNA resulted in no detectable product (data not shown).
Isolation of 4NQO-affected Tissues. Initially, whole tissue sec
tions were used for analysis. However, even in tissue specimens
which contained invasive SCC, sections were contaminated with
tissue other than squamous epithelium and/or morphologically normal
squamous epithelium. Thus, DNA was isolated from a mixture of
normal and abnormal cells. DNA from nontumor cells, with wild-type
C MWI 2 3 4 5 6 7 8 910111213141516MW
H-ras, decreases the detection of mutant H-ras. To reduce dilution by
unaffected tissue, the lesions were morphologically identified on
stained cryosections and dissected (Fig. 1). Genomic DNA was iso
lated from the lesion and amplified by nested PCR.
RFLP Analysis. 4NQO acting as a DNA alkylating agent should
result in 0 to A transversion (4,5). Of the codons previously impli
cated in H-ras activation, codon 12 has two available G residues. The
restriction enzyme, Mn!!, cuts the wild-type DNA when both Os are
present at codon 12 but not when a mutation had occurred in H-ras
DNA at codon 12 (Figs. 2 and 3). The MnlI digests yield the following
DNA segments: (a) wild-type H-ras: 75, 73, and 19 base pairs; (b)
mutant codon 12 H-ras: 148 and 19 base pairs; and (c) heterozygous
mutant H-ras: 148, 75, 73, and 19 base pairs (Figs. 2 and 3). Eleven
of 25 lesions contained putative codon 12 mutations (Fig. 3, B and C).
In 5 of 11 invasive 5CC, the wild-type ras allele was absent; LOH had
occurred. All five with apparent LOH were relatively large carcino
mas. Only one of the CIS and none ofthe early, dysplastic lesions had
H-ras mutations as determined by RFLP analysis. Nested PCR and
Mn!! digestion had been carried out with cloned wild-type (Fig. 3B,
Lane A) and mutant H-ras serving as positive controls (Fig. 3B, Lane
C); a 1:1 mixture of mutant and wild-type H-ras was used as control
for heterozygous H-ras (Fig. 3B, Lane B). PCR amplificationin the
absence of genomic DNA yielded no detectable products. The RFLP
studies were performed at least three times on different sections from
each tumor and control tissue specimen and twice from the dysplastic
lesions. When H-ras mutations were detected by RFLP, they were
present in all amplifications of that tissue specimen. In lesions het
erozygous for H-ras, there was no significant variation in the ratio of
mutant to wild-type H-ras observed by electrophoresis. No H-ras
codon 61 mutations were detected using RFLP analysis of tissues
sections amplified with primers specific for exon II (14) and digested
with Xba I (data not shown).
Missense Amplification Mutational Analysis. The presence of
mutant H-ras oncogenes in preneoplastic and neoplastic lesions asso
ciates H-ras with neoplastic transformation. Yet, these observations
do not define the timing of ras oncogene activation in the multistep
process of carcinogenesis. In an attempt to ascertain the point at which
Fig. 4. Detection of H-ms mutations with MAMA. A, effect of glycerol on the
selectivity of mismatched PCR. MAMA was performed on cloned H-ms DNA obtained
from plasmids using PAA, a sense primer mismatched at the 3'-terminus (Fig. 2) which
amplifies only mutant H-ms (27) and the Al primer at 94°Cfor 1 mm and 55°Cfor 1 mm
for 35 cycles. PCR was performed in the presence of 20 pg of wild-type plasmid H-ms
DNAwithvaryingamounts(0—2
pg)of mutantplasmidH-ras(clonedfromtissueno.4),
0.35 pmol/pJ each of primers, 0.1 mat dNTP, 0.015 unit/pJ of Taq polymerase, and 10%
glycerol. Electrophoresis was carried out on 4% agarose gel (Fig. 3). The amplified
segment is 102 base pairs. B, MAMA detection of H-ms point mutations in tissue DNA.
MAMA was performed as in (A). However, DNA samples obtained from tissues were first
amplified by nested PCR to obtain specific H-ms fragments. Subsequently, the PCR
products were diluted 1:100 and amplified with the PAA and A2 primers as in (A).
Control DNA was obtained from wild-type H-ms plasmid (Lane 1), codon 12 mutant
H-ras plasmid (Lane 2), or normal mouse liver (Lanes 3—4).Tissue DNA was obtained
either from dissected tissue as in Fig. 1 (Lanes 5—8)or from the entire tissue sections
which contained both tumor and normal cells (Lanes 9—12).Lane 1, 20 pg wild-type
H-ras plasmid DNA only; Lane 2, mixture of 0.2 pg mutant H-mas plasmid DNA and 20
pg wild-type H-ras plasmid DNA Lanes 3 and 4, 0.5 gsg normal mouse liver DNA; Lane
5, dissected tissue no. 4; Lane 6, dissected tissue no. 10; Lane 7, dissected tissue no. 15;
Lane 8, dissected tissue no. 9; Lane 9, tissue section no. 4; Lane 10, tissue section no. 10;
Lane 11, tissue section no. 15; Lane 12, tissue section no. 9. The tissues are described in
Table 1. C, detection of H-ras point mutations in early lesions. Control DNA and DNA
isolated from dissected tissues were treated as in (B). Lanes 1—3
(ms plasmid controls).
MAMA performed in the presence of2O pgofwild type plasmid H-ras DNA(Lanes 1-3).
Lane 1, contains only 20 pg wild type H-ma; Lanes 2 and 3, contain 0.2 and 2 pg of mutant
H-ras plasmid DNA, respectively. Lanes 4—7(tissues nos. 21, 22, 23, and 24) are all
invasive SCC, which demonstrate that the results with MAMA are consistent with RFLP
analysis in Fig. 3. In Lanes 8—16(tissues nos. 1, 2, 6, 7, 13, 16—18,and 20), MAMA was
carried out on CIS and dysplastic tissues. MAMA detected H-ras mutations in tissue nos.
16 and 20, which were not detected by RFLP. Arrows, locations of 102-base pair segment.
H-ras oncogene activation occurs, we have searched for H-ras muta
tions in normal, premalignant, and malignant tissue. To increase the
sensitivity of our ability to detect H-ras mutations, these assays were
repeated using MAMA (Fig. 4; Ref. 27). As described below, 4NQO
induced a G to A transversion at the second base of codon 12. This is
identical to the point mutation on rat H-ras detected by MAMA by
Cha et a!. (27). The assay is very sensitive since only mutant se
quences are amplified under appropriate conditions. Thus, it is pos
5313
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1994 American Association for Cancer Research.
@
:@
@
4NQO-INDUCED
A1
2
3
4
@
@—@-
H-ms MUTATIONS
5
B GA@
C
GATC
::::@
@GAA
Fig. 5. Verification of H-ms mutations. A. detection of putative mouse H-mas mutations by SSCP. DNA amplified from whole tissue sections was amplified with 51 and Al primers
in the presence of [a-P@2JdATP. The product was electrophoresed, and the band was eluted and electrophoresed (see “Materialsand Methods―).The autoradiograph was the result of
a 16 h exposure. Arrows indicate the migration of mutant H-ras. Lanes 1—5,
tissues nos. 1—5
(Table 1); Lanes 1,2, and 5, normal H-ras; Lane 3, heterozygous mutant H-ras; Lane 4.
homozygous mutant H-ras. B, DNA sequence analysis. The DNA shown in (A) was isolated, amplified by nested PCR, and cloned into a pCR II plasmid. DNA sequencing was
performed using 2 j.tg of double-stranded DNA as template and M13 (—40base pairs) oligonucleotide primer in the [a-S35]dATP. The sequencing gel was exposed for 48 h. Arrow
indicates the G to A transversion at the codon 12 of H-ras. Mutant, the DNA isolated from Lane 4 (tissue no. 4); Wild type is from Lane I (tissue no. 5) of the gel shown in (A).
sible to detect H-ras mutation even when only a few mutant cells are
present among many unaffected cells. Since the rat and mouse H-ras
sequences are identical in this region of gene, their primers were
essentially used as described. Optimal conditions for the assay were
developed using the mismatched primer PAA shown to have the
greatest selectivity and specificity (Fig. 2). Cloned mutant and wild
type H-ras-containing plasmids were used as positive and negative
controls. To obtain purified ras template for MAMA, DNA isolated
from tissue sections was amplified using external and nested primers.
When the amplified
DNA was diluted
1:100 prior to MAMA,
only the
mismatched product was detected with ethidium bromide staining
(Fig. 4). Glycerol concentration was critical in defining the specificity
ofthe reaction. Melting the H-ras plasmid DNA at 94°Cfor 1 mm and
annealing at 55°Cfor 1 mm in the presence of 10% glycerol for 35
cycles yielded maximal synthesis of mutant sequence without signif
icant synthesis of the wild-type H-ras. Mutant H-ras was detected
even when wild-type
H-ras was in 1000-fold
excess (Fig. 4A). If only
oralTissue4NQO
Table
1H-ras point mulations are highly associated with 4
cavity SCCsNQO-induced
size―no.Pathology(wk)
(wk)(mm)H-ras1Dysplasia4
exposure
mumine
ObservationTumor
49—I—2CIS16
24—!—3SCC12
491.5i!—4SCC12
494.5W+5SCC8
491.5—!—6Normalsolvent
49—!—7Dysplasia4
49—I—8Normalsolvent
49—I—9SCC12
492.0+!—10SCC16
383.0+R11SCC12
491.5—!—125CC4
491.5+!—13Dysplasia12
49—!--14SCC8
491.5—I—15SCC16
283.0+!+16Dysplasia12
49+!@“17Dysplasia16
38—I—18CIS8
49—1—19CIS8
49+!—20CIS8
these
point
mutations
detected
to controls
(data
not shown).
The
Similar SSCP experiments were performed to search for codon 61
mutations within the H-ras exon II. Varying the temperature, power,
and glycerol concentration of the electrophoresis, no significant band
shifts were observed in the five H-ras-negative tumors tested (data not
shown). Presumably, there were no H-ras codon 61 point mutations in
4NQO-induced tumors.
To prove that 4NQO induced point mutations at codon 12, the
wild-type
ras
detected;
+!—,
wild-type
and
mutant
ras
by MAMA
but negative
in RFLP
and SSCP
SSCP-isolated
DNA was amplified
and cloned.
Using denatured
dou
ble-stnanded DNA as template, DNA sequencing showed that the
mutation was a G to A transversion at codon 12, which alters the
present; +!+, LOH, i.e., no significant wild-type ras detected.
b H-ras
compared
H-ras Homozygous H-ras).
333.0+!+
—!—, only
DNA fragments
DNA was digested with PvuII to show that the amplified comple
mentary DNA was intact (data not shown) and Mn1I to demonstrate
that the mutation had occurred at codon 12 (Fig. 3A, Heterozygous
aTumorsizewasmeasuredto thenearest0.5mm.+,mutantH-rasalleleatcodon12;
allele;
to 20% mutant DNA might be present, mutant and normal H-ras DNA
can be distinguished by SSCP (29). Following PCR amplification
with [a32P]dATP, the DNA strands were isolated from SSCP gels,
diluted, denatured, and resolved by polyacrylamide gel electro
phoresis (Fig. 5A). A mutated sequence can be detected as a
change of mobility caused by its altered secondary or tertiary
structures. DNA was obtained from tumors apparently homozy
gous at codon 12 for mutant H-ras, from tumors heterozygous for
mutant H-ras, and normal tissues by RFLP. Tissue no. 4 was
apparently homozygous at codon 12 for mutant H-ras, and tumor
no. 3 heterozygous for mutant H-ras and MAMA (Figs. 3 and 4).
By SSCP Tissues no. 3 and no. 4 contain DNA with different
mobility (Fig. 5A); presumably, these tissues have mutations in the
to secondary PCR using the A2 and S2 primers and [a-P32]dATP.
Secondary SSCP was performed to confirm the altered mobility of
491.5+!—22SCC8
491.5i!—23SCC4
491.5—1—24SCC16
332.0+!+25SCC16
ms
analysis.
Demonstration of Codon 12 Mutations. Although as little as 10
H-ras sequence. The DNA was eluted from the gel and subjected
49@,_b21SCC12
—, wild-type
0.1% of cells in a tissue specimen contain mutant H-ras, MAMA
should detect the mutation. Therefore, this assay is at least 100-fold
more sensitive than SSCP and RFLP analysis.
The mutant H-ras DNA was initially found in 13 of 25 tissue
specimens by SSCP and RFLP analysis (10 of 14 invasive carci
nomas; 1 of 4 CIS; 0 of 5 dysplasia; and 0 of 2 normal tissues;
Table 1). All specimens which had H-ras mutations at codon 12 by
RFLP had the same mutations identified with MAMA. In addition,
MAMA identified two additional mutations in a CIS and a dys
plastic lesion that were not detected by either SSCP or RFLP
analysis.
5314
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1994 American Association for Cancer Research.
4NQO-INDUCED H-mm MUTATIONS
amino acid from Gly to Asp. No codon 11, 13, or 61 mutations were
found in H-ras DNA. No H-ras mutations were detected in liver from
4NQO-treated mice.
premalignant
DISCUSSION
tissue section
termine the molecular events involved in neoplastic transformation.
We have observed that microdissection significantly reduces the con
tamination of uninvolved tissue and has increased our ability to detect
molecular events (Fig. 4B). To obtain adequate material for the
studies described, extensive PCR amplification and nested PCR are
required (Fig. 3A). The present report demonstrates our ability to
amplify genomic DNA from tissue sections without contamination
and to obtain internally consistent results. Each amplification shown
is the amplified PCR product isolated from a tissue or dissected
section. Where multiple reactions are shown from the same tissue,
each gel represents the product obtained from a single cryosection
from that same lesion. At least three sections from each tumor and two
lesions from each dysplastic lesion were amplified. No significant
variation was observed in assays of the amplified DNA.
H-ras Mutations as a Primary Event in Neoplastic Transfor
mation. The incidence of H-ras lesions reportedherein is 10 of 14
invasive SCC, 2 of 4 CIS, and 1 of 5 dysplastic lesions. The rate of ras
mutation is significantly greater than the 5% observed in Western
human head and neck cancers (24, 25), and is even higher than the
35% H-ras point mutations observed in tobacco-chewing patients
from India (26). The frequency of 4NQO-induced mutations is similar
but not identical to that observed by Balmain and Conti's laboratories
studying DMBA-induced murine skin squamous lesions (12, 13, 14)
and hamster buccal mucosa (23). They observed codon 61 H-ras
mutations in approximately 90% of papillomas and carcinomas. In
DMBATTPA-treated F1 mice, LOH was also detected, predominately
in spindle cell carcinomas (16, 17, 18, 19). H-ras mutations appear to
be somewhat less frequently detected in 4NQO-treated mice. The
does increase
in the invasive
SCC,
and the present
study
detected LOH at H-ras only in invasive 5CC. These observed differ
ences in the models may relate to the short exposure to 4N00,
tissue may result either because
H-ras
codon
12 muta
tions are relatively rare in dysplastic lesions or the assays were still
not sensitive enough to detect these mutations. These explanations are
not mutually exclusive. Amplification of DNA using MAMA to detect
codon 12 mutations has been reported to detect 1 in 10,000 sequences
(27). However, using identical conditions, we could detect only 1 in
1000 sequences (Fig. 4A). The amount of material dissected from the
Molecular Analysis of 4NQO-induced Lesions. 4NQO induces
an orderly progression of lesions in the oral cavity that eventually
leads to neoplastic transformation and tumorigenesis (3). These le
sions begin to be detected in the absence of inflammation 8 weeks
after cessation of exposure to the carcinogen. The largest tumors were
5 mm; most are 1.5 to 3 mm. Dysplastic lesions are microscopic. This
has necessitated the development of micromolecular analysis to de
incidence
mutations. However, when the abnormal tissues were isolated by
dissection and the more sensitive MAMA was used, H-ras mutations
were observed in two of nine tissues which were H-ras negative in
previous SSCP and RFLP analyses. The few mutations detected in
the
absence of tumor promoters, the more differentiated invasive 5CC in
the 4NQO model, or the different sensitivity to the carcinogens in the
oral cavity or difference in the mouse strains.
An alternative explanation might be differences in assay sensitivity
to identify H-ras mutations. None of the other laboratories dissected
the lesions from surrounding uninvolved tissue and subsequently used
PCR amplification, RFLP analysis, SSCP, and MAMA to identify
H-ras mutations in premalignant and malignant tissues. Therefore, it
is unlikely that the differences in incidence of ras mutations and the
fewer homozygous lesions is due to the sensitivity of our analysis. We
presume that the differences observed are due to either the carcinogen,
the susceptibility of the exposed tissue, and/or to differences in mouse
strain susceptibility.
The frequency of 4NQO-induced H-ras mutations detected in dys
plastic lesions is low considering the high incidence in tumors. If
H-ras mutations were the primary event occurring as a result of
exposure to the carcinogen, 4NQO, then these mutations should be
found in dysplastic tissue. In our initial screening of lesions using
which comprises
a dysplastic
lesion is minimal
(Fig. 1).
The tissue was amplified by primary and secondary PCR requiring
approximately 100 cycles to detect the amplified DNA. Under these
conditions, H-ras mutations were detected in some of the premalig
nant tissues with tissue purification and more sensitive assays. There
fore, H-ras mutations present in the lesion, but not in the tissue
section, could have been missed either by not being present or by not
being amplified. H-ras mutations might not be detected if they com
prised less than 1/1000 of the H-ras DNA. Since the dysplastic lesions
assayed
were contaminated
with nondysplastic
tissue,
it is impossible
to determine what proportion of dysplastic cells within a lesion
contain H-ras mutations. The frequency of mutant H-ras containing
cells is very low in the dysplastic tissue. It appears that these muta
tions were rarely associated with dysplasia. Therefore, H-ras activa
tion does not cause dysplasia.
Only some but not all dysplastic lesions eventually undergo malig
nant transformation. However, the apparent lack of H-ras mutations in
the dysplastic lesions implies that H-ras activation does not, in itself,
cause dysplasia. H-ras activation should confer a significant growth
advantage on cells within dysplastic lesions which have codon 12
mutations. Presumably, they would be more likely to transform,
eventually progressing to CIS and invasive 5CC. However, cells with
a single H-ras or p53 mutation are not transformed; additional muta
tions in the same gene or other genes are required for malignant
transformation (30, 31). Therefore, H-ras mutations or other muta
tions should be present in dysplastic cells which eventually transform.
Sequence analysis confirmed
that 4NQO-induced
tumors in CBA
mice with H-ras have G to A mutations at codon 12. CBA mice have
a rather high incidence of spontaneous H-ras mutations in the liver
(32). Greenhalgh et a!. (33) have reported spontaneous codon 61
mutations in cultured BALB/c keratinocytes. However, analysis of
liver tissue from 50 4NQO-treated mice has demonstrated no spon
taneous H-ras mutations (data not shown). Only codon 12 H-ras
mutations were detected in 4NQO-treated oropharyngeal tissue; no
mutations involving codons 11, 13, or 61 were observed. Since only
G to A transversionswere observed,this is presumptiveevidence that
the mutations were not spontaneous but were caused by exposure to
4NQO.
The reason for the observed high frequency in the mutational
activation of the H-ras gene in this CBA strain is not known. Studies
of liver carcinogenesis in CBA mice have demonstrated spontaneous
and carcinogen-induced H-ras mutations with higher frequency than
observed in most other strains (32). With respect to chemically in
duced tumorigenesis, 4NQO is a direct-acting, highly mutagenic,
DNA alkylating agent that preferentially reacts with guanosine resi
dues at the N7 position (4, 5). The predicted consequence of the DNA
adduct formation is to change the coding sequence from G to A, which
is the observed
mutation.
Presumably,
the activating
mutations
in
H-ras genes were the consequence of a direct interaction between
4NQO and the ras sequences. An alternative explanation, which is
unlikely because of the specificity of codon 12 mutations, is that the
RFLP analysis, none of the dysplastic lesions had evidence of H-ras
G to A mutationin H-ras preexists in certaincells in the epithelium
5315
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1994 American Association for Cancer Research.
4NQO-INDUCED
H-mac MUTATIONS
and that 4NQO treatment only manifest and facilitates the clonal
expansion of such cells.
H-ras Mutations Affecting Tumor Progression. The data show
that H-ras mutations are associated with the initiation of tumorigen
esis and affect progression by conferring a more aggressive and
invasive tumor type. Since a single point mutation in H-ras rarely is
associated with invasive 5CC, our data is consistent with Finney and
Bishop's (34) in vitro analysis that a single H-ras mutation is not
dominant and in itself not sufficient for the completion of tumorigen
involving H-ras, leads to malignant transformation. Consistent with
this observation, LOH, i.e., loss of the normal H-ras allele, has been
found in many human tumors, including human oral squamous cell
carcinomas (35, 36), and frequently occurs during murine skin squa
mous cell transformation (16, 17). This observation suggests that the
has been associated with human tumors (35, 36, 38), but presumably
these occur in the setting of continued exposure to carcinogens.
The mechanism by which the second normal H-ras allele is lost is
unclear. Is it induced by the presence of a H-ras mutation or merely
selected for by the growth advantages associated with an H-ras
mutation? Bremner and Balmain (17) have demonstrated that tnisomy
occurs in DMBA-induced skin lesions. They have speculated that this
is an intermediate event with a loss of the chromosome containing the
normal H-ras allele resulting in LOH (17). However, Bianchi et al.
(16) suggested that mitotic recombination followed by gene conver
sion might be the mechanism for LOH at H-ras. These studies (16, 17)
have used F1 mice, while the present study used inbred CBA mice.
Hence, the mechanism(s) by which H-ras LOH occurs may differ in
inbred strains and F1 mice, and the mechanism by which the normal
ras allele is lost in inbred mice remains obscure.
absence of the normal gene product may facilitate transformation.
LOll at Codon 12. By dissecting tumors and amplifying DNA
ACKNOWLEDGMENTS
esis. At least one additional
event,
often a gross chromosomal
event
from these cells, we have been able to perform allelic analysis of
H-ras mutations. Even though the cellular DNA present in the PCR
varied as a result of the number of cells isolated from the tissue
sections, the first PCR for 60 cycles generated saturated yields of the
targeted DNA fragments. The second, nested PCR yielded H-ras
specific DNA products (Fig. 3). Loss of wild-type ras was demon
strated in 5 of 14 invasive SCC; these tumors were moderately to well
differentiated morphologically and larger than 1.5 mm. The sequence
analysis
of these mutations
demonstrated
G to A transversion
We thank Dr. R. Barker for synthesizing many of the oligonucleotide
primers and Michael Eisenbach for tissue preparation. We also thank Des.
N. Martin, B. Ozanne, and S. Peiper for critically reading this manuscript.
REFERENCES
1. Mattson, M. E., and Winn, D. M. Smokeless tobacco: association with increased
cancer risk. Nail. Cancer Inst. Monogr., 8: 13—16,
1989.
2. Binnie, W. H., Rankin, K. V., and Mackenzie, I. C. Etiology of oral squamous cell
carcinoma. J. Oral Pathol., 12: 11—29,
1983.
at codon
3. Hawkins, B. L, Heniford, B. W., Ackermann, D. M., Leonberger, M., Martinez,
12. All of the other tumors were heterozygous at codon 12. If some
lesions
were significantly
contaminated
with normal
tissue,
S. A., and Hendler, F. J. 4NQO carcinogenesis: a mouse model of oral cavity
squamous cell carcinoma, Head Neck, 16: 424-432, 1994.
4. Thomas, D. C., Husain, I., Chancy, S. 0., Panigrahi, G. B., and Walker, I. G.
Sequence effect on incision by (A)BC exonuclease of 4N00 adducts and UV
the assay
would not necessarily discriminate homozygous from heterozygous
mutations. Thus, the incidence of LOH in the smaller benign lesions
may be underestimated, but the incidence of ras mutations and LOH
in the larger lesions is accurate.
Once tumonigenesis was initiated, neoplastic cells undergo succes
sive genetic
changes,
resulting
in the loss of wild-type
H-ras.
photoproducts. Nucleic Acids Res., 19: 365—370,1991.
5. Panigrahi, G. B., and Walker, I. G. The N2-guanineadduct but not the C@-guanineor
N6-adenine adducts formed by 4-nitroquinoline 1-oxide blocks the 3'-S' exonuclease
action of T4 DNA polymerase. Biochemistry, 29: 2122—2126,1990.
6. Steidler, N. E., and Reid, P. C. Experimental induction of oral squamous cell
carcinomas in mice with 4-nitroquinoline-l-oxide.
Oral Surg., 57: 424—531,
1984.
7. Steidler, N. E., and Reade, P. C. Initiation and promotion of experimental oral
As
Finney and Bishop (34) have demonstrated that only homozygosity of
H-ras can transform cells, it is possible that H-ras mutations are
responsible for the transformation in 4NQO-induced SCC. The data
does not support this speculation. LOH at H-ras was observed fre
quently in lesions that were invasive 5CC but not in CIS and dys
plastic tissues. Although the in situ lesions were contaminated with
nonmutant H-ras, it is unlikely that the contamination would approach
50% of the sample necessary to achieve the observed distribution of
H-ras alleles (Fig. 1). The proportion of mutant and normal H-ras
DNA detected in the heterozygous 5CC and in situ carcinomas
mucosal carcinogenesis in mice. J. Oral Pathol., 15: 43—47,1986.
8. Prime, S. J., Malamos, D., Rosser, T., and Scully, C. Oral epitheial atypia and
acantholytic dyskeratosis in rats painted with 4-nitroquinoline N-oxide. J. Oral
PathoL, 15: 280—283,1986.
9. Heniford, B. W., Shum-Siu, A., Leonberger, M., and Hendler, F. J. Variation in
cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase
polymerase chain reaction. Nucleic Acids Res., 21: 3159—3166,1993.
10. Balmain, A., and Brown, K. Oncogene activation in chemical carcinogenesis@Adv.
Cancer Res., 51: 147—182,
1988.
11. Barbacid, M. ras genes. Annu. Rev. Biochem., 56: 779—827,1986.
12. Quintanilla,
suggests that these tissues are heterozygous for H-ras. Apparently, the
13. Klein-Szanto, A. J. P., Larcher, F., Bonfil, R. D., and Conti, C. J. Multistage chemical
loss of the second normal H-ras allele is a late event in 4NQO
tumorigenesis,
after neoplastic
transformation
has occurred.
carcinogenesis protocols produce spindle cell carcinomas of the mouse skin. Carci
nogenesis
(Land.),
10:2169—2172,
1989.
The ccl
14. Brown, K., Buchmann, A., and Balmain, A. Carcinogen-induced mutations in the
mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression.
lular population may be heterogenous for H-ras at early stages of
tumorigenesis, but invasive tumors have selected a dominant clone
which has LOH at H-ras. Based on in situ studies performed with
probes for epidermal growth factor receptor mRNA and DNA, mor
phologically identical cells in these tumors exhibit focal overexpres
sion of epidermal growth factor receptor RNA (4). It is likely that
similar selective pressures are leading to H-ras homozygosity in
association with tumorigenesis.
H-ras LOH, reported herein, developed in the absence of exposure
to 4NQO in mice that had not received 4NQO for at least 4 months
and typically for more than 6 months before tumors developed. LOH
and gross chromosomal changes have been shown to develop in the
presence of either carcinogen and/or tumor promoter (16, 17, 37).
However, we report LOH at a mutant allele without the continued
pressure of either a carcinogen or a tumor promoter. H-ras instability
M., Brown, K., Ramsden, M., and Balmain, A. Carcinogen-specific
mutation and amplification of H-ms during mouse skin carcinogenesis. Nature
(Land.), 322: 78—80,1986.
Proc. Natl. Acad. Sci. USA, 87: 538-542,
1990.
15. Sukumar, S. An experimental analysis of cancer: role of masoncogenes in multistep
carcinogenesis. Cancer Cells (Cold Spring Harbor), 2: 199—204,1990.
16. Bianchi, A .B., Navone, N. M., Aldaz, M. C., and Conti, C. J. Overlapping loss of
heterozygosity by mitotic recombination on mouse chromosome 7F1-ter in skin
carcinogenesis. Proc. Nail. Acad. Sci. USA, 88: 7590—7594, 1991.
17. Bremner, R., and Balmain, A. Genetic changes in skin tumor progression: correlation
between presence of a mutated ms gene and loss of heterozygosity on mouse chr 7.
Cell, 61: 407—417,1990.
18. Buchmann, A., Rugged, B., Klein-Szanto, A. J. P., and Balmain, A. Progression of
squamous carcinoma cells to spindle carcinomas of mouse skin is associated with an
imbalance of H-ms alleles on chromosome 7. Cancer Res., 51: 4097-4101, 1991.
19. Bianchi, A. B., Aldaz, M. C., and Conti, C. J. Nonrandom duplication of the
chromosome bearing a mutated H-ras-1 allele in mouse skin tumors. Proc. NatI.
Acad. Sci. USA, 87: 6902-6906, 1990.
20. Marshall, C. J., Vousden, K. H., and Phillips, D. H. Activation of c-Ha-mas-l
proto-oncogene by in vitro modification with a chemical carcinogen. Nature (Lond.),
310: 586—589,1984.
21. Yuan, B., and Wong, J. L. Inactivity of acrylonitrile epoxide to modify a H-ras DNA
5316
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1994 American Association for Cancer Research.
4NQO-INDUCED H-ras MUTATIONS
in a non-focus
transfection-transformation
assay.
Carcinogenesis
(Land.),
C.C. Harris,S. Hirohashi,N. Ito (eds.), MultistageCarcinogenesis,pp.97—108.
12:
787—791,1991.
22. Hashimoto,Y., Kawachi, E., Shudo, K., Sekiya, T., and Sugimura, 1. Transforming
activity of human c-H-mas-l proto-oncogene generated by the binding of 2-amino-6methyl-dipyrido[l,2-a:3',2'-djimidazole
and 4-nitroquinoline N-oxide: direct cvi
Tokyo: JapanScientific Societies Press, 1990.
31. Fearon, E. R, and Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell,
61: 759—767,1990.
32. Buchmann, A., Bauer-Hofmann, R., Mahr, 1., Drinkwater, N. R., Lox, A., and
Schwarz, M. Mutational activation of the c-H-ms gene in liver tumors of different
rodent strains: correlation with susceptibility to hepatocarcinogenesis. Proc. NatI.
Acad. Sci. USA, 88: 911—915,1991.
dence of cellular transformation by chemically modified DNA. Jpn. 1. Cancer Res.,
78: 211—215, 1987.
23. Gimenez-Conti, I. B., Bianchi, A. B., Stockman, S. L., Conti, C. J., and Slaga, T. J.
Activating mutation of the Ha-ras gene in chemically induced tumors of the hamster
cheek pouch. Mol. Carcinog., 5: 259—263,1992.
24. Rumsby, G., Carter, R. L, and Gusterson, B. A. Low incidence of ras oncogene
33. Greenhalgh, D. A., Welty, D. J., Strickland, J. E., and Yuspa, S. H. Spontaneous
Ha-ms gene activation in cultured primary murine keratinocytes: consequences of
Ha-ras gene activation in malignant conversion and malignant progression. Mol.
Carcinog., 2: 199—207,1989.
activation in human squamous cell carcinomas. Br. J. Cancer, 61: 365—368,1990.
25. Warnakulasuriya, K. A., Chang, S. E., and Johnson, N. W. Point mutations in the
H-ras oncogene are detectable in formalin-fixed tissues of oral squamous cell carci
nomas, but are infrequent in British cases. J. Oral Pathol. Med., 21: 225—229,
1992.
26. Saranath, D., Chang, H., Bhoite, L, Panchal, R., Mehta, A. R., Johnson, N., and Deo,
M. 0. High frequency mutations in codons 12 and 61 of H-ras oncogene in tobacco
related human oral carcinomas. Br. J. Cancer, 63: 573—578,1991.
27. Cha, R. S., Zarbl, H., Keohavong, P., and Thilly, W. 0. Mismatch amplification
mutation assay (MAMA): application to the c-H-ms gene. PCR Methods and Appl.,
34. Finney, R. E., and Bishop, M. J. Predispositionto neoplastic transformation caused by
gene replacement of H-masl. Science (Washington DC), 260: 1524—1527,1993.
35. Saranath, D., Bhoite, L T., Mehta, A. R., Sanghavi, V., and Deo, M. 0. Loss of allelic
heterozygosity at the harvey ras locus in human oral carcinomas. 1. Cancer Res. Gin.
Oncol., 117: 484—488,1991.
36. Howell, R. E., Wong, F. S., and Fenwick, R. G. Loss of Harvey ras heterozygosity in
oral squamous carcinoma. J. Oral Pathol. Med., 18: 79—83, 1989.
37. Furstenberger, G., Schurich, B., Kaina, B., Petrusevska, R. T., Fusenig, N. E., and
Marks, F. Tumor induction in initiated mouse skin by phorbol esters and methyl
2: 14—20,1992.
28. Nelson, M. A., Futscher, B. W., Kinsella, T., Wymer, J., and Bowden, G. T. Detection
methanesulfonate:
of mutant Ha-ms genes in chemically initiated mouse skin epidermis before the
development of benign tumors. Proc. Natl. Acad. Set. USA, 89: 6398—6402,1992.
29. Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. A rapid and sensitive detection of
point mutations and genetic polymorphisms using polymerase chain reaction. Genom
ics, 5: 874—879,1989.
30. Balmain, A., Kemp, C. J., Burns, P. A., Stoler, A. B., Fowls, D. J., and Akhurst, R. J.
Functional loss of tumor suppressor genes in multistage chemical carcinogenesis. In:
correlation between chromosomal damage and conversion (“stage
I of tumor promotion―)
in vivo. Carcinogenesis (Land.), 10: 749—752,1989.
38. Fearon, E. R., Feinberg, A. P., Stanley, H. H., and Vogelstein, B. Loss of genes on
the short arms of chromosome 11 in the bladder cancer. Nature (Land.), 318:
377—380,1985.
39. Brown, K., Bailleul, B., Ramsden, M., Fee, F., Krumlanf, R., and Balmain, A.
Isolation and characterization of the 5' flanking region of the mouse c-Harvey-ms
gene. Mol. Carcinog., 1: 161—170,
1988.
5317
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1994 American Association for Cancer Research.
Harvey ras (H-ras) Point Mutations Are Induced by
4-Nitroquinoline-1-oxide in Murine Oral Squamous Epithelia,
while Squamous Cell Carcinomas and Loss of Heterozygosity
Occur without Additional Exposure
Bo Yuan, Briana W. Heniford, Douglas M. Ackermann, et al.
Cancer Res 1994;54:5310-5317.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/54/20/5310
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1994 American Association for Cancer Research.