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Frequent alterations of the tumor suppressor genes p53 and DCC in human pancreatic carcinoma
Frequent Alterations of the Tumor Suppressor Genes ~53 and DCC in Human Pancreatic Carcinoma BABETTE SIMON,* ROLF WEINEL,’ MARTIN HOHNE? GUNTHER KdRTNER,* and RUDOLF ARNOLD*
JUllA
*Department of Internal Medicine, Division of Gastroenterology and Metabolism, and slnstitute of Virology, University Giessen, Giessen, Germany
Background/Aims: The pathogenesis of pancreatic cancer is poorly understood. The multigenetic nature of carcinogenesis has been best documented in colon cancer. The relevance of this model was suggested for other epithelial tumors. Only advanced stages of pancreatic cancer are usually detected because of late diagnosis. Analysis of accumulated, diverse genetic changes could allow further understanding of putative mechanisms involved in tumor development. Activated c-Ki-ras oncogene has been shown to be a frequent event. However, additional alterations of tumor sup pressor genes are expected. Therefore, concomitant genetic changes of ~53 and deleted in colon carcinoma (EC) in pancreatic carcinoma cell lines and primary tumors were analyzed. Methods: ~53 protein and transcript expression were revealed by immunocytochemistry and immunohistochemistry, immunoassay, and Northern blot analysis. ~53 mutations were identified by sequence analysis. DCC expression was investigated by reverse-transcription polymerase chain reaction. Results: ~53 overexpression was observed in 9 of 12 cell lines. p53 point mutations were confirmed in seven cell lines overexpressing ~53. The majority of cell lines showed concomitant p53 and DCC alterations. Four of 6 primary tumors overexpressing p53 also showed loss of DCC expression. Conclusions: p53 and DCC genetic changes are associated with pancreatic cancer and the frequently activated c-Ki-ras oncogene. Therefore, the multihit model of carcinogenesis could prove relevant for pancreatic cancer.
WATZ,*
and ‘Department
lacking,
common
concept
of carcinogenesis
appears
to
be central to understanding neoplasia. The beststudied epithelial gastrointestinal tumor is colon cancer.’ Genetic changes include the activation of ras proto-oncogene and inactivation of tumor suppressor genes such as ~53 and deleted in colon carcinoma (DCC).‘.’ These genetic alterations occur according to a preferred sequence; however, the total accumulation of changes rather than the order appears to be important.’ Although proof is
could
model
neoplasms. cancer currently
also prove relevant
ranks as the fourth
cause of cancer death in humans,
the aggressive nancy.4
The
still poorly function
nature
of several
of this malig-
pathogenetic
mechanisms
are
Abnormalities
in structure
and
oncogenes
known to be associated
most
which reflects
and poor prognosis
underlying understood.
and
growth
with pancreatic
are
somatic
critical human
and early event occurring in most, if not all, pancreatic carcinomas.” However, it was sug-
gested
that
appears
mutational
factors
cancer.5 Notably,
c-K-ra.r
the neoplastic
to involve
tumor
activation
appears
to be a
pathway
in epithelial
cells
suppressor
genes rather
than
oncogenes,2 and mutations of ras oncogenes appear to have little effect on the neoplastic transformation of epithelial
cells in the absence
The demonstration cooperate
of other critical
that activated
with mutated
~53 in cellular
vitro ‘J and the observation
changes.’
ra.r oncogenes
can
transformation
in
that the incidence
of cancer,
including pancreatic carcinoma, is markedly increased in individuals with Li-Fraumeni syndrome who are heterozygous
for inherited
germline
mutations
of ~53’ sug-
gested ~53 as a further candidate to be altered in sporadic pancreatic cancer. This assumption could be confirmed by recent studies and the present work showing frequent ~53 alterations in this tumor.10-‘2 Furthermore, loss of the putative
vanced genetic
tumor
in pancreatic
Pancreatic he multistep
of Surgery, Philipps University, Marburg;
the colorectal
to pancreatic Pancreatic
observed
T
JORG SCHMIDT,*
cancers
suppressor
gene DCC was recently
carcinomas. are usually
’3 only diagnosed
at ad-
stages because of late diagnosis. Therefore, the changes associated with the development of the
tumor are difficult to assess, and only their accumulation can be analyzed. To provide further evidence that the multihit model might be relevant for pancreatic carcinoAbbreviations used in this paper: DCC, deleted in colon carcinoma; PCR, polymerase chain reaction. 0 1994 by the American Gastroenterological Association 001~5085/94/$3.00
1646
SIMON
genesis,
GASTROENTEROLOGY
ET AL.
we investigated
genetic
alterations
in this study
of the diverse tumor
~53 and DCC in a large number cell lines and some primary
phosphatase
suppressor
mL-’ pAB42 1 or pAB240 for 30 minutes
genes
washed with phosphate-buffered
carcinoma
with
tumors.
rabbit
Germany)
Cell Lines and Tumor Samples
staining.
1, MIAPaCa-2,
Pant-1,
carcinoma cell lines Capan-2, and BxPC-3
Type Culture
binding,
Collection
were obtained
(Rockville,
provided
by H. Kern (Marburg,
Cell lines were grown as adherent many) (PC-2,
PC-3,
Dulbecco’s
Pant-1,
and Dulbecco’s
Eagle medium
modified
10% fetal calf serum
and
and Capan-2).
samples
specimens
Tissue
of pancreatic
mors or normal histological
quantitate
Eagle
pancreatic
examination.
sections
using
Table1. Summary
the
of ~53
Alterations
in Pancreatic
HPAF 89888 8902 PC44 BXPC-3 MIAPaCa-2 Pant-1 PC-3 Patu II PC-2 AsPC-1 Capana Units expressed nuclear staining: b Ten micrograms
p53 protein
instructions
of pancreatic
and polyclonal
pAB240
pan-p53
to
carci-
The extracts
The ~53 mutant-specific
were was
were used
carci-
and CsF gradient (Hybond;
Amersham)
and
bromide staining.
conditions
were
pC53-SN3
Cell
kindly
described.20
provided
by random-priming
and washBlots
were
DNA fragment
by S. Friend (Boston,
DNA
triphosphate
(Stra-
of RNA was con-
Hybridization
as previously
in a
to a nylon
UV-irradiated
The integrity
The ~53 complementary
Germany)
separated
with the human ~53 complementary
with [32P]deoxycytidine
anti-alkaline
centrifugation,
gel, electrotransferred
firmed by ethidium
MA).”
were
Carcinoma
and Northern Blot Analyses
fragment
(Amersham,
technique.
was labeled Brauschweig,
Control hybridization
Lines
staining
~53 sequence
P53 assay’
p53 messenger RNAb
PAb 240
PAb 421
Codon
Exon
20 24 194 236 10 211 44 19 103 19 _
+ + ++ ++ + ++ + + ++ + _ _
++ +++ ++ +++ + +++ +++ +++ +++ _ _ _
++ +++ ++ +++ + +++ +++ +++ iii _ -
151 151 176 176 220 248 273 _ _ _ _
5 5 5 5 6 7 8
17
in extracts
Germany).
of plasmid
medium
~53
Cell line
Sci-
to the manufacturers’
tagene, Heidelberg, ing
sections
phosphate
selec-
1% agarose and formaldehyde
hybridized
alkaline
($53 mutant
mutant
isothiocyanate
and of Mutant p53
cells grown in complete
enzyme immunoassay
Total RNA was isolated from cell lines by guanidinium-
by standard
on glass slides and cryostat sections of primary pancreatic immunostained
and
Science
ELISA assay; Dianova GmbH/Oncogene
as described.”
membrane
Cells and tissue
by Dianova GmbH/Oncogene
RNA Isolation
of tu-
Total RNA was isolated as described
were fixed in acetone.
for counterpAB240
as tracer antibodies.
from resection
tissue were analyzed
carcinoma
used
antibodies
8988S,
below.
Pancreatic
was
monoclonal
used as a catcher antibody,
with
(8902,
Freeze-cut
lmmunocytochemistry lmmunohistochemistry Protein
Germany)
noma cell lines and the cells’ supernatant. prepared
and AsPC-l),
supplemented
were obtained
carcinomas.
hematoxylin
ence) was used according
Ger-
modified
MIAPaCa-2,
10% horse serum
Marburg,
Germany).‘”
A nonisotopic
with 10% (Patu II and HPAF) ot 15%
fetal calf serum (BxPC-3,
Meyers
tive quantitative
cells in RPM1 1640 supple-
and PC-44),
supplemented
Hamburg,
~53 Immunoassay
Germany).“-”
mented with 10% fetal calf serum (Gibco, Eggenstein, medium
incubated
(Dako,
MD); cell lines
and cell lines Patu II, 8902, 89888, and HPAF
Germany);14
saline, and further
complex (Behring,
Purified anti-p53
(Hamburg,
from the
with 2 /,tg
Fast red was used to localize the antibody
pAB42 1 were purchased
AsPC-
PC-2, PC-S, and PC-44 were a kind gift of M. Biilow (Maim, were gratefully
and
No. 6
at room temperature,
immunoglobulin
and APAAP
twice for 30 minutes.
American
noma
anti-mouse
Materials and Methods
Human pancreatic
were incubated
the concomitant
of pancreatic
pancreatic
samples
technique;”
Vol. 106,
analysis
Nucleotides CCC CCC TGC TGC TAT CGG CGT
TCC TCC AGC AGC - TGT - TGG - CAT
as nanogram per milligram total protein. Positive signals in ~53 immunocytochemistry were graded +, ~25%; ++, 25%-75%; and +++, 75%-1000/o. total messenger RNA. ~53 transcript was expressed as absent (-), weak (+), or strong (++),
-
Amino acids Pro - Se Pro - Se Cys - Se Cys - Se Tyr - Cys Arg - Trp His - Arg
for portion of cells with
June 1994
~53 AND DCC
of 1% ribosomal a ‘*P-labeled sequence
RNA
was performed
single-stranded
as described**
oligonucleotide
probe
the
Analysis of p53 Protein Expression in Pancreatic Carcinoma Cell Lines and Primary Pancreatic Carcinomas
’
5 ‘-ACGGTATCTGATCGTCTTCGAACC-3
Polymerase Chain Reaction Amplification and Sequencing of ~53 Genomic
DNA
from pancreatic
was isolated23 and subjected reaction
cell lines
to four separate polymerase
chain
for each cell line using
33mer
(PCR) amplifications
and 36mer oligonucleotide
carcinoma
primers
3’) and p53r (5’ ATGC-
GAATTCGGGATTCTGAGTGTAGACTGAAAAC amplification
of exons 5-9
tion was performed
with
of each deoxynucleoside
triphosphate,
2 mmol/L magnesium Elmer
Cetus,
Conditions
method
to
purified,
Sequencing
Version
(Perkin
elsewhere.“.‘*
PCR amplification
with EcoRI, and
sequencing
sequencing
by
Mutations
for exons primers
55°C
of PCR were
2.0; US Biochemicals,
was performed
sense and antisense
Products digested
of
per reaction.
II SK; one recombinant
double-stranded
(Sequenase
scribed
polymerase
and 72°C (3 minutes). into pBluescript
subjected
and 2.5 U of Amplitaq
CT) DNA
electrophoretically
subcloned
OH).
chloride,
Norwalk,
200 pm
a final concentration
5-9
clone was the
dideoxy
Cleveland, using
both
for each exon as de-
were confirmed
by a second
and resequencing.
and reverse transcribed
or NotI-
primers
scriptase DNA
(Pharmacia,
aliquots,
amplified
by the exon connection
complementary blot analyses desctibed.3
as described
DNA using
with pd(N)b Germany). strategy
Nine
Different
pancreatic
strong
tumors
nuclear
antibodies
as deprimers
and adjacent
patterns
emerged
in almost
1B). In HPAF
stained
nuclear
pattern.
with
primary
(Table
both
and 8902, about
25%
with both antibodies.
antibodies,
Capan-2,
pancreatic
staining. 1). PC-44,
and PC-3 showed
showing
No immunoreactivity
the cell lines PC-2, Four
both
pancreatic
nuclear
all cells with
of the cells showed nuclear staining BxPC-3
and six pri-
normal
Patu II, Pant-1,
staining
(Figure
antibodies,
12 cell lines
carcinomas
of 12 cell lines showed
staining
and AsPC-1
tumors
a weak
was observed
(722,
(Figure
228,
in 1A).
323,
and
BE) and one metastatic
tissue (from patient
BE) showed
positive
staining
the mutant-specific
monoclonal
antibody
pAB240
mal pancreatic normal
with
(Figure
lC), whereas surrounding
tissues (except for patient
tissue was available) carcinomas
pancreatic
were negative.
(929
expression
carcinoma
was further
Two primary negative
antibodies
(Table
quantitated
in the
cell lines by an immunoassay
the ~53 mutant-specific
monoclonal
nor-
BE, where no
and AS) showed
for pS.? with both monoclonal
2). p>3 protein
antibody
using
pAB240.
reverse tranwere PCR-
using DCC antisense
elsewhere.13
a DCC-specific
tissues.
monoclonal
were used to stain
Complementary
to 200 pg RNA,
fragments
Control amplifications
random
100 U MoMuLV
Freiburg,
corresponding
and sense primers
primers
using
was isolated
anti-p53
from human
staining
DCC Complementary DNA Amplification and Southern Blot Analysis scribed
derived
pancreatic
Total RNA from tissue specimens
different
and pAB421,
89SSS, MIAPaCa-2,
gene. Amplifica-
for PCR were 34 cycles of 94°C (1 minute),
(2 minutes), pooled,
of the humanp53
3’) for
100 pmol of each primer,
Two pAB240
mary pancreatic
p53f (5’ ATGCGAATTC-
TACTCCCCTGCCCTCAACAAGAT
1647
Results
using
with
IN PANCREATIC CANCER
Amplified
were analyzed
DCC
by Southern
57mer-oligonucleotide were performed
Table 2. ~53 Alterations and Loss of DCC Gene Expression in Primaty Pancreatic Carcinomas
as
with p-actin
as described.13
Figure 1. lmmunocytochemical staining of human ~53 with pAB240 in representative pancreatic carcinoma cell lines and primary pancreatic carcinoma tissue. (A) Absence of ~53 staining in Capan-2, (5) nuclear ~53 staining in MIAPaCa-2, and (C) ~53 staining in primary pancreatic carcinoma tissue (Original magnification X200).
Pancreatic carcinoma tumors 722 N T 927 N T 228 N T 323 N T AS T BE T M
Overexpression of p53
Loss of DCC expression
_ +
+ _
_ _
+ +
_
+ _
+ _ +
+ _
_
+a
+ +
_a _a
T, primary tumor; N, normal tissue; M, metastasis. a Obtained from previous work.13
1646
The highest in PC-44, Pant-I,
GASTROENTEROLOGY Vol. 106, No. 6
SIMON ET AL.
levels of mutated MIAPaCa-2,
~53 protein
and 8902,
were detected
less in Patu
the other pancreatic
carcinoma could
Table 1). Nop5.3 protein of the cultured
cell lines except AsPC- 1,
lines negative
be detected
was detected
(Figure
2 and
RNA
carcinoma
the transcript ~53 protein
transcript
3 and Table
correlated detected
2). The highest
bridization
8988S),
was analyzed
expression
in most
l), and the intensity
of
No transcript
of the p53 gene Hy-
amino
acid
carcinoma
mutation
the cell line Pant-1
at codon 220 in
cell line MIAPaCA-2
at codon
showed
273 in exon 8 (Figure
8902). One cell
mutation
248 in exon 7, and
a point
4 and Table
mutation
at codon
1).
Comparison of ~53 Alterations of DCC Gene Expression Loss of DCC expression mary pancreatic
that changes in the levels
were obtained
from our recent workI
region ofp53
after PCR amplification
encoding carcinoma of geno-
in four pri-
The DCC expression
carcinomas.
and pancreatic
2 and 3. In the primary
and Loss
was analyzed
tumors
of the ~53 gene in the pancreatic
cell lines by sequencing
176 (PC-44,
of two further
Sequence Analysis of the ~53 Gene in Pancreatic Carcinoma Cell Lines exons 5-9
were point
the deduced
RNA
ofp53 transcripts were not caused by RNA degradation and verified equal loading.
the genomic
All mutations
showed a point
exon 6. The pancreatic
Patu
and Capan-2.
of the same blot with the 18s ribosomal
We analyzed
Cell
did not show a mutation
and altered
and two at codon
showed a point
(Figure
in PC-44,
probe indicated
of the gene.
mutations
cell lines
protein.
of mutated
was observed
in the cell lines AsPC-1
oligonucleotide
in
blot anal-
was detectable the amount
forp53 expression
line (BxPC-3)
by the ~5.3 immunoassay
II, 8902, and MIAPaCa-2. was observed
with
region of the ~5.3
carcinoma
sequence. Four pancreatic carcinoma cell lines showed point mutations in exon 5, two at codon 151 (HPAF,
cell lines by Northern
ysis. A 2.8kilobasep53 cell lines (Figure
expression
in the conserved
immunohistochemicallyp53
in this domain missense
in the supernatants
cell lines (data not shown).
~53 messenger
Mutations
overexpressing
were found
Northern Blot Analyses of ~53 in Pancreatic Carcinoma Cell Lines 12 pancreatic
mic DNA.
gene were found in 7 of 9 pancreatic
in
and low levels of p53 expression
where no ~53 protein
II and
tumors
carcinoma
data
cell lines
and noted in Tables
of four patients
(722,
of one patient
(BE),
228, 323, and BE) and a metastasis
DCC expression was clearly reduced compared with adjacent normal tissues. In patients 927 and AS, expression of DCC was comparable ure 5A and Table expressing
with adjacent
2). Interestingly,
histochemically
abberant
normal tissue (Figpancreatic
tumors
~5.3 protein
also
showed loss of DCC gene expression, whereas in p53negative tumor tissues, no significant loss of DCC gene expression
was observed (Table 2). Except for one pancre-
atic carcinoma
cell line (MIAPaCa-2),
cell lines harboring
an altered ~53 protein,
reduced or lost DCC gene expres-
sion could be observed.
One cell line PC-2 had lost DCC
gene expression without immunohistochemical indication for an alteredp53 expression, although the immunoassay
2.8kb Figure 2. p53 protein expression of pancreatic carcinoma cell lines by a ~53 mutant selective quantitative enzyme-linked immunosorbent assay. Quantitation is achieved by the construction of a standard curve using known concentrations of mutant ~53 protein and comparing the absorbance. The amount of mutant ~53 protein is given as the mean of two assays. Values are given as nanogram per milligram of total protein concentration 2 SEM.
showed
low-level
~53
protein
expression
and
P53
Figure 3. Northern blot analysis of human ~53 transcript in human pancreatic carcinoma cell lines (top). Control hybridization with 18s oligonucleotide (bottom).
~53 AND DCC
June1994
@AC G
A
0 Wildtype T
A”;taG”’ T
72
927
N
T
N
IN PANCREATIC CANCER
228 T
1649
323 Ni
Ni
Mutant
B
1
2
345
678
41000
Pigure 4. Sequencing autoradiograms of ~53 gene in the conserved region for PCR-amplified DNA samples from pancreatic carcinoma. (A) exon 5, codon 151, C-T transition; (B) 8902, exon 5, codon 89883, 176, T-A transversion; (C) BxPC-3, exon 6, codon 220, A-G transition; (0) MIAPaCa-2, exon 7, codon 248, C-T transition; and (E) Panc-I, exon 8, codon 273, A-G transition. Sequences in A and 8 are shown in anti-sense direction, and C, D, and E are shown in sense direction. On the left is the wildtype sequence, and on the right is the mutated sequence. Arrows indicate mutated nucleotides.
Plgure 5. Expression of DCC in primary pancreatic carcinomas and normal adjacent tissue. (A) Southern blot analysis of PCR-amplified DCC complementary DNA. (8) pactin control amplification of tissue samples shown in A. T, pancreatic tumor tissue; N, normal adjacent tissue.
lines and primary pancreatic tumors and the association with loss of DCC gene expression in primary tumors. We
Northern (Table
blot
analysis
3 and Figures
showed
a weak ~53
transcript
2 and 3).
In this study, we analyzed alterations suppressor
Table
gene ~53 in human
3. Alterations
of c-Kis-ras,
Carcinoma
Cell Lines
Cell line HPAF 89888 8902 PC44 BxPC-3 MIAPaCa-2 Pant-1 PC-3 Patu II PC-2 AsPC-1 Capan-
c-KCras mutation codon 1213 + + + + ND ND ND wr -t W-T ND ND
NOTE. +, present; -, absent. ND, not determined; WT, wildtype.
observed
expression
immunologic
pancreatic
~53,
of the tumor carcinoma
cell
and DCC in Pancreatic
~53 mutation codon (exon)
Loss of DCC expression”
151(5) 151(5) 176 (5) 176 (5) 220 (6) 248 (7) 273 (8) _ _ _ _ _
_ _ ND + _ _ + +
evidence
in 9 of 12 pancreatic
suggesting a posttranslational the cellular environment,25
Discussion
bp
of abnormal
carcinoma
stabilizing complexing
~53
cell lines,
by changes in to other pro-
teins,26m29 or mutating of the p>3 protein coding sej0 We could show by DNA sequencing that in
quence.
seven pancreatic
carcinoma
cell lines, the abnormal
stain-
ing pattern arose in cells that had undergone ~33 mutations in the evolutionary conserved region of the gene. All ~53 mutations were missense mutations, and most of them were GC to AT transitions. However, the base changes showed no specific mutational spectrum that could indicate an association with an individual mutagen shown for other tumor types.24.31 Most of the mutations were detected in exon 5 in codons 15 1 and 176, which is consistent with sequencing data from Kalthoff et al.” in which 4 of 7 altered cell lines showed mutations in exon 5, notably, two of them in codon 176. Although Scarpa et aL3’ recently reported a preferential location of p53 mutations in primary pancreatic tumors in exons 5 and 7, no specific site appeared to be preferentially affected. The frequent mutational alterations in exon 5 are intriguing; however, codon 176 cannot be considered as a hotspot in pancreatic cancer based on the observations
1650
in these pancreatic tions
RNAs
carcinoma
may represent
mutated lines
GASTROENTEROLOGY Vol. 106, No. 6
SIMON ET AL.
p53 proteins
and
perhaps
cell lines. The ~53 muta-
the basis for increased in the pancreatic
also of certain
as suggested
carcinoma
mutated
by the remarkable
RNA overexpression
stability
of cell
messenger
~53 messenger
shown by some cell lines. However,
it can not be excluded
that other unidentified
the synthesis
or stability although
primary
pancreatic
tumors.
changes in pancreatic
immuno-
did not show
c-K-ras
mutations
duction
disrupt
the ~53 pathway
without
altering
the underlying
or genetic
complexing the protein
of the ~53 gene.
of other
and alter the level of expression
cause ofpS3
essarily be a mutational
alterations
with ~53 protein29834 could coding sequence. overexpression
alteration
Interaction
Therefore,
may not nec-
in the conserved
region
other proteins
could
with
in the tumorigenic pathway
were detected
vas proteins
tumorigenesis
involved
in cell adhesion
carcinoma
localizations
the concept
in pancreatic
of a multigenetic
cancer cell line in which the
noprecipitation were negative using the same mutantspecific monoclonal antibody pAB240. Recently, Ruggeri et al.” described some of the pancreatic carcinoma cell this study. Surprisingly, they reported ber of different mutational alterations
~53 alterations in lines analyzed in a very high numin individual cell
lines, which we could not confirm because we observed no more than one point mutation per cell line. This discrepancy is probably caused by random errors due to
in the pancreatic
of these diverse
cancer provides nature
support
of this tumor
gefor type.
mutations and ~53 alterations are the most common cancer-related alterations and are observed in a wide
rearrangement
enzyme-linked immunosorbent assay showedpS3 protein expression, whereas immunohistochemistry and immu-
of
ras
a genomic Capan-2’l
drigues et a1.35 in a colorectal
inhibition
these three genes show differand functions
cells. The accumulation
cancers.3.13z4’ Because
intriguing because nopS3 expression could be found by immunocytochemistry using the same monoclonal antibody pAB240. Th’is observation was also made by Ro-
The ~53 gene
processes that could be relevant
for metastases.40 Therefore,
lack of ~53 overexpression cannot predict a ~53 protein. Southern blot analyses indicated
pression using the sensitivep13 immunoassay and a weak pJ3 transcript found in Northern blot analysis. This is
trans-
apoptosis,‘” whereas the putative tumor suppressor gene DCC is expressed on the cell surface and appears to be
However, functional
gene in the cell line
the signal
across the membrane.36
gene expression
in thep53
to 3.13
appear to be involved
process by altering
in the conserved region of thepS3 gene in immunocytochemically negative pancreatic carcinoma cell lines.
that could explain the lack of a ~53 transcript and ~53 protein expression. Interestingly, the pancreatic carcinoma cell line PC-2 showed weak ~53 protein ex-
study
in Table
through
to
netic changes
No mutations
in an earlier
as summarized
contribute
ability to detect mutant ~53 protein in cells. Thus, the percentage of p53 alterations in pancreatic carcinomas analysis.
of these
cell lines. These
activator37,38 that might
ent cellular
try and sequence
accumulation
carcinoma
encodes a nuclear transcriptional
also mask antigenic epitopes on the ~53 protein and thus interfere with immunohistochemical or the immunoassay
may be even higher than shown by immunohistochemis-
frequent
cell lines have also been shown harbor
region
genes
p53 alterations
earlier13 showed
The membrane-bound
of the pS3 gene,
Comparing
genetic
point mutations in the conserved region of the ~53 gene. Intronic point mutations,33 mutations in the promoter cellular
alterations
genes ~53 and DCC in 4 of 6
suppressor
as shown in this study and loss of DCC gene expression
may have occurred in these cell lines. Interestfor ~53 protein,
In this study, we could observe concomitant of the tumor
was
a second PCR sample.
shown
transcripts
positive
by resequencing
molecular
affecting
ingly, the cell lines Patu II and PC-),
confirmed
because this group analyzed
In our study, each mutation
of the pS3
alterations
cytochemically
of Taq polymerase
infidelity
only one PCR reaction.
variety
of human
intestinal
tumors,
tumor
types. In contrast,
appears to be confined
loss of DCC
to certain gastro-
such as colonic, gastric, and pancreatic mutational
alterations
of the ras
and ~53 genes are also frequently observed in colorectal and gastric cancers, 1’42’43pancreatic cancer shows a common set of genetic changes with these tumors that might be relevant for tumor development and progression.
References 1. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-767. 2. Vogelstein B, Kinzler KW. The multistep nature of cancer. Trends Genet 1993;9:138-141. 3. Fearon ER, Cho KR, Nigro JM, Kern SE, Simons JW, Ruppert JM, Hamilton SR, Preisinger AC, Thomas G, Kinzler KW, Vogelstein B. Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 1990; 247:49-56. 4. Cello JP. Carcinoma of the pancreas. In: Sleisenger MH, Fordtran JS, eds. Gastrointestinal disease: pathology, diagnosis and management. Philadelphia: Saunders, 1989:1872-1884. 5. Lemoine NR, Hall PA. Growth factors and oncogenes in pancreatic cancer. Ballieres Clinical Gastroenterol 1990;4:815-832. 6. Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53:549-554. 7. Eliyahu D, Raz A, Gruss P. Givol D, Oren M. Participation of p53 cellular tumor antigen in transformation of normal embryonic cells. Nature 1984;312:646-649.
~53 AND DCC
June 1994
8.
9.
10.
Parada LF, Land H, Weinberg RA, Wolf D, Rotter W. Cooperation
Participation of p53 protein in the cellular response to DNA dam26.
Malkin D, Li FP, Strong LC, Fraumeni JF, Nelson CE, Kim DH, Kassel J, Gryka MA, Bischoff FZ, Tainsky MA, Friend SH. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 1990; 250:1233-1238.
27.
age. Cancer Res 1991;51:6304-6311. Sarnow P, Ho YS, Williams J, Levine AJ. Adenovirus Elb58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd protein in transformed cells. Cell 1982;28:387-394. Werness BA, Levine AJ, Howley PM. Association of human papillcmavirus types 16 and 18 E6 proteins with ~53. Science 1990; 248:76-79. Pinhashi-Kimhi 0. Michalovitz D, Ben-Zoov A, Oren M. Specific interaction between the p53 cellular tumor antigen and major heat shock proteins. Nature 1986;320:182-185. Momand J, Zambetti GP, Olson DC, George D, Leine Al. The mdm2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 1992;69:12371245. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancer. Science 1991;253:49-53. Chiba I, Takahashi T, Nau MM, Amico D, Curie1 D, Mitsudomi T, Buchhagen DL, Carbone D, Piantadosi S, Koga H, Reissman PT, Slamon DJ, Holmes EC, Minna JD. Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Oncogene 1990;5:1603-1610. Scarpa A, Capelli P, Mukai K, Zamboni G, Oda T, lacono C, Hirohashi S. Pancreatic adenocarcinomas frequently show p53 gene mutations. Am J Pathol 1993;142:1534-1543. Miller CW, lmai Y, Aslo A, Li L, Koeffler HP. Sublocalization of transcriptional activation domain of ~53. Proc Am Assoc Cancer Res 1992; 33:386. Vogelstein B. A deadly inheritance. Nature 1990;348:681-682. Rodrigues NR, Rowan A, Smith MEF, Kerr IB, Bodmer WF, Gannon JV, Lane DP. p53 mutations in colorectal cancer. Proc Natl Acad Sci USA 1990;87:7555-7559. Bos JL. ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682-4689. Fields S, Jang SK. Presence of a potent transcription activating sequence in the p53 protein. Science 1990;249:1046-1049. Farmer G, Bargonetti J, Zhu H, Friedmann P, Prywes R, Prives C. Wild-type p53 activates transcription in vitro. Nature 1992; 358:83-86. Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 1993; 3621847-849. Narayanan R, Kenneth GL, Schaapveld RQJ, Cho KR, Vogelstein B, Tran PB-V, Osborne MP, Telang NT. Antisense RNA to the putative tumor suppressor gene DCC transforms RAT-l fibroblasts. Oncogene 1992; 7:553-561. Uchino S, Tsuda H, Noguchi M, Yokota J, Terada M, Saito T, Kobayashi M, Sugimura T, Hirohashi S. Frequent loss of heterozygosity at the DCC locus in gastric cancer. Cancer Res 1992;52:3099-3102. Sugimura T, Hirohashi S. Genetic alterations in human gastric cancer. Cancer Cells 1991;3:49-52. lmazeki F, Omata M, Nose H, Ohto M, lsono K. p53 gene mutations in gastric and esophageal cancers. Gastroenterology 1992; 103:892-896.
Barton CM, Staddon SL, Hughes CM, Hall PA, Sullivan CO, Klop pel G, Theis B, Russel RCG, Neoptolemos J, Williamson RCN, Lane DP, Lemoine NR. Abnormalities of the p53 tumor suppressor gene in human pancreatic cancer. Br J Cancer 1991;64: 1076-1082.
28.
Ruggeri B, Zhang SY, Caamano J, Flynn SD, Klein-Szanto AJP. Human pancreatic carcinomas and cell lines reveal frequent and multiple alterations in the p53 and RB-1 tumor suppressor genes. Oncogene 1992;7:1503-1511.
29.
12.
Kalthoff H, Schmiegel W, Roeder C, Kasche D, Schmidt A, Lauer G, Thiele HG, Honold G, Pantel K, Riethmuller G, Scherer E, Maurer J, Maacke H, Deppert W.p53 and K-ras alterations in pancreatic epithelial lesions. Oncogene 1993;8:289-298. Hohne MW, Halatsch ME, Kahl GF, Weinel RJ. Frequent loss of expression of the potential tumor suppressor gene DCC in ductal pancreatic adenocarcinoma. Cancer Res 1992; 52:2616-2619. Bulow M, Scharfe T, Kloppel G, Kern HF. Establishment and characterization of continuous tumor cell lines from human pancreatic carcinoma in vitro. Digestion 1982; 25:17-18. Kim YW, Kern HF, Mullins TD, Koriwchak MJ, Metzgar RS. Characterization of clones of a human pancreatic adenocarcinoma cell line representing different stages of differentiation. Pancreas 1989;4:353-362. Metzgar RS, Gaillard MT, Levine SJ, Tuck FL, Bossen EH. Borowitz MJ. Antigens of human pancreatic adenocarcinoma cells defined by murine monoclonal antibodies. Cancer Res 1982;42:601608. Elslsser HP, Heidtmann HH, Lehr U, Agricola B, Kern HF. Structural analysis of a new highly metastatic cell line Patu 8902 from a primary human adenocarcinoma. Virchows Arch B Cell Pathol 1993;64:201-207.
30.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
1651
between gene encoding p53 tumor antigen and ras in cellular transformation. Nature 1984;312:649-651.
11.
13.
IN PANCREATIC CANCER
Cordell JL. Falini B, Erber WN, Ghosh AK, Abdulaziz Z, Macdonald S, Pulford KAF, Stein H, Mason DY. lmmunoenzyme labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 1984; 12:219-229. Gannon JV, Greaves R, lggo R, Lane DP. Activating mutations in p53 produce common conformational effects. A monoclonal antibody specific for the mutant form. EMBO J 1990;9:15951602. Simon B, Podolsky DK, Moldenhauer G, lsselbacher KJ, GattoniCelli S, Brand SJ. Epithelial glycoprotein is a member of a family of epithelial surface antigens homologous to nidogen, a matrix adhesion protein. Proc Natl Acad Sci USA 1990;87:2755-2759. Baker SJ, Markowitz S, Fearon ER, Willson JKV, Vogelstein B. Suppression of human colorectal carcinoma cell growth by wildtype ~53. Science 1990; 249:912-915. Szyf M, Milstone DS, Schimmer BP, Parker KL, Seidman JG. Cis modification of the steroid 21-hydroxylase gene prevents its expression in the Yl mouse adrenocortical tumor cell line. Mol Endocrinol 1990;4:1144-1152. Decker HJ. Gemmell RM, Neumann HP, Walter TA, Sandberg AA. Loss of heterozygosity on 3p in a renal cell carcinoma in von hippel lindau syndrome. Cancer Genet Cytogenet 1989;39:289293. Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC. Mutational hotspot in the p53 human hepatocellular carcinomas. Na ture 1992;350:427-428. Kastan MB, Onyekwere 0, Sidransky D, Vogelstein B, Craig RW.
31.
32.
33.
34. 35.
36. 37. 38.
39.
40.
41.
42. 43.
Received August 9,1993. Accepted January 25,1994. Address requests for reprints to: Babette Simon, M.D., Division of Gastroenterology, Department of Internal Medicine, Philipps University, BaldingerstraBe, 35033 Marburg, Germany. Fax: (49) 6421288922. Suppported by a Dornier research grant. The authors thank Dr. Eric Fearon for critically reviewing this manuscript.
JUllA
*Department of Internal Medicine, Division of Gastroenterology and Metabolism, and slnstitute of Virology, University Giessen, Giessen, Germany
Background/Aims: The pathogenesis of pancreatic cancer is poorly understood. The multigenetic nature of carcinogenesis has been best documented in colon cancer. The relevance of this model was suggested for other epithelial tumors. Only advanced stages of pancreatic cancer are usually detected because of late diagnosis. Analysis of accumulated, diverse genetic changes could allow further understanding of putative mechanisms involved in tumor development. Activated c-Ki-ras oncogene has been shown to be a frequent event. However, additional alterations of tumor sup pressor genes are expected. Therefore, concomitant genetic changes of ~53 and deleted in colon carcinoma (EC) in pancreatic carcinoma cell lines and primary tumors were analyzed. Methods: ~53 protein and transcript expression were revealed by immunocytochemistry and immunohistochemistry, immunoassay, and Northern blot analysis. ~53 mutations were identified by sequence analysis. DCC expression was investigated by reverse-transcription polymerase chain reaction. Results: ~53 overexpression was observed in 9 of 12 cell lines. p53 point mutations were confirmed in seven cell lines overexpressing ~53. The majority of cell lines showed concomitant p53 and DCC alterations. Four of 6 primary tumors overexpressing p53 also showed loss of DCC expression. Conclusions: p53 and DCC genetic changes are associated with pancreatic cancer and the frequently activated c-Ki-ras oncogene. Therefore, the multihit model of carcinogenesis could prove relevant for pancreatic cancer.
WATZ,*
and ‘Department
lacking,
common
concept
of carcinogenesis
appears
to
be central to understanding neoplasia. The beststudied epithelial gastrointestinal tumor is colon cancer.’ Genetic changes include the activation of ras proto-oncogene and inactivation of tumor suppressor genes such as ~53 and deleted in colon carcinoma (DCC).‘.’ These genetic alterations occur according to a preferred sequence; however, the total accumulation of changes rather than the order appears to be important.’ Although proof is
could
model
neoplasms. cancer currently
also prove relevant
ranks as the fourth
cause of cancer death in humans,
the aggressive nancy.4
The
still poorly function
nature
of several
of this malig-
pathogenetic
mechanisms
are
Abnormalities
in structure
and
oncogenes
known to be associated
most
which reflects
and poor prognosis
underlying understood.
and
growth
with pancreatic
are
somatic
critical human
and early event occurring in most, if not all, pancreatic carcinomas.” However, it was sug-
gested
that
appears
mutational
factors
cancer.5 Notably,
c-K-ra.r
the neoplastic
to involve
tumor
activation
appears
to be a
pathway
in epithelial
cells
suppressor
genes rather
than
oncogenes,2 and mutations of ras oncogenes appear to have little effect on the neoplastic transformation of epithelial
cells in the absence
The demonstration cooperate
of other critical
that activated
with mutated
~53 in cellular
vitro ‘J and the observation
changes.’
ra.r oncogenes
can
transformation
in
that the incidence
of cancer,
including pancreatic carcinoma, is markedly increased in individuals with Li-Fraumeni syndrome who are heterozygous
for inherited
germline
mutations
of ~53’ sug-
gested ~53 as a further candidate to be altered in sporadic pancreatic cancer. This assumption could be confirmed by recent studies and the present work showing frequent ~53 alterations in this tumor.10-‘2 Furthermore, loss of the putative
vanced genetic
tumor
in pancreatic
Pancreatic he multistep
of Surgery, Philipps University, Marburg;
the colorectal
to pancreatic Pancreatic
observed
T
JORG SCHMIDT,*
cancers
suppressor
gene DCC was recently
carcinomas. are usually
’3 only diagnosed
at ad-
stages because of late diagnosis. Therefore, the changes associated with the development of the
tumor are difficult to assess, and only their accumulation can be analyzed. To provide further evidence that the multihit model might be relevant for pancreatic carcinoAbbreviations used in this paper: DCC, deleted in colon carcinoma; PCR, polymerase chain reaction. 0 1994 by the American Gastroenterological Association 001~5085/94/$3.00
1646
SIMON
genesis,
GASTROENTEROLOGY
ET AL.
we investigated
genetic
alterations
in this study
of the diverse tumor
~53 and DCC in a large number cell lines and some primary
phosphatase
suppressor
mL-’ pAB42 1 or pAB240 for 30 minutes
genes
washed with phosphate-buffered
carcinoma
with
tumors.
rabbit
Germany)
Cell Lines and Tumor Samples
staining.
1, MIAPaCa-2,
Pant-1,
carcinoma cell lines Capan-2, and BxPC-3
Type Culture
binding,
Collection
were obtained
(Rockville,
provided
by H. Kern (Marburg,
Cell lines were grown as adherent many) (PC-2,
PC-3,
Dulbecco’s
Pant-1,
and Dulbecco’s
Eagle medium
modified
10% fetal calf serum
and
and Capan-2).
samples
specimens
Tissue
of pancreatic
mors or normal histological
quantitate
Eagle
pancreatic
examination.
sections
using
Table1. Summary
the
of ~53
Alterations
in Pancreatic
HPAF 89888 8902 PC44 BXPC-3 MIAPaCa-2 Pant-1 PC-3 Patu II PC-2 AsPC-1 Capana Units expressed nuclear staining: b Ten micrograms
p53 protein
instructions
of pancreatic
and polyclonal
pAB240
pan-p53
to
carci-
The extracts
The ~53 mutant-specific
were was
were used
carci-
and CsF gradient (Hybond;
Amersham)
and
bromide staining.
conditions
were
pC53-SN3
Cell
kindly
described.20
provided
by random-priming
and washBlots
were
DNA fragment
by S. Friend (Boston,
DNA
triphosphate
(Stra-
of RNA was con-
Hybridization
as previously
in a
to a nylon
UV-irradiated
The integrity
The ~53 complementary
Germany)
separated
with the human ~53 complementary
with [32P]deoxycytidine
anti-alkaline
centrifugation,
gel, electrotransferred
firmed by ethidium
MA).”
were
Carcinoma
and Northern Blot Analyses
fragment
(Amersham,
technique.
was labeled Brauschweig,
Control hybridization
Lines
staining
~53 sequence
P53 assay’
p53 messenger RNAb
PAb 240
PAb 421
Codon
Exon
20 24 194 236 10 211 44 19 103 19 _
+ + ++ ++ + ++ + + ++ + _ _
++ +++ ++ +++ + +++ +++ +++ +++ _ _ _
++ +++ ++ +++ + +++ +++ +++ iii _ -
151 151 176 176 220 248 273 _ _ _ _
5 5 5 5 6 7 8
17
in extracts
Germany).
of plasmid
medium
~53
Cell line
Sci-
to the manufacturers’
tagene, Heidelberg, ing
sections
phosphate
selec-
1% agarose and formaldehyde
hybridized
alkaline
($53 mutant
mutant
isothiocyanate
and of Mutant p53
cells grown in complete
enzyme immunoassay
Total RNA was isolated from cell lines by guanidinium-
by standard
on glass slides and cryostat sections of primary pancreatic immunostained
and
Science
ELISA assay; Dianova GmbH/Oncogene
as described.”
membrane
Cells and tissue
by Dianova GmbH/Oncogene
RNA Isolation
of tu-
Total RNA was isolated as described
were fixed in acetone.
for counterpAB240
as tracer antibodies.
from resection
tissue were analyzed
carcinoma
used
antibodies
8988S,
below.
Pancreatic
was
monoclonal
used as a catcher antibody,
with
(8902,
Freeze-cut
lmmunocytochemistry lmmunohistochemistry Protein
Germany)
noma cell lines and the cells’ supernatant. prepared
and AsPC-l),
supplemented
were obtained
carcinomas.
hematoxylin
ence) was used according
Ger-
modified
MIAPaCa-2,
10% horse serum
Marburg,
Germany).‘”
A nonisotopic
with 10% (Patu II and HPAF) ot 15%
fetal calf serum (BxPC-3,
Meyers
tive quantitative
cells in RPM1 1640 supple-
and PC-44),
supplemented
Hamburg,
~53 Immunoassay
Germany).“-”
mented with 10% fetal calf serum (Gibco, Eggenstein, medium
incubated
(Dako,
MD); cell lines
and cell lines Patu II, 8902, 89888, and HPAF
Germany);14
saline, and further
complex (Behring,
Purified anti-p53
(Hamburg,
from the
with 2 /,tg
Fast red was used to localize the antibody
pAB42 1 were purchased
AsPC-
PC-2, PC-S, and PC-44 were a kind gift of M. Biilow (Maim, were gratefully
and
No. 6
at room temperature,
immunoglobulin
and APAAP
twice for 30 minutes.
American
noma
anti-mouse
Materials and Methods
Human pancreatic
were incubated
the concomitant
of pancreatic
pancreatic
samples
technique;”
Vol. 106,
analysis
Nucleotides CCC CCC TGC TGC TAT CGG CGT
TCC TCC AGC AGC - TGT - TGG - CAT
as nanogram per milligram total protein. Positive signals in ~53 immunocytochemistry were graded +, ~25%; ++, 25%-75%; and +++, 75%-1000/o. total messenger RNA. ~53 transcript was expressed as absent (-), weak (+), or strong (++),
-
Amino acids Pro - Se Pro - Se Cys - Se Cys - Se Tyr - Cys Arg - Trp His - Arg
for portion of cells with
June 1994
~53 AND DCC
of 1% ribosomal a ‘*P-labeled sequence
RNA
was performed
single-stranded
as described**
oligonucleotide
probe
the
Analysis of p53 Protein Expression in Pancreatic Carcinoma Cell Lines and Primary Pancreatic Carcinomas
’
5 ‘-ACGGTATCTGATCGTCTTCGAACC-3
Polymerase Chain Reaction Amplification and Sequencing of ~53 Genomic
DNA
from pancreatic
was isolated23 and subjected reaction
cell lines
to four separate polymerase
chain
for each cell line using
33mer
(PCR) amplifications
and 36mer oligonucleotide
carcinoma
primers
3’) and p53r (5’ ATGC-
GAATTCGGGATTCTGAGTGTAGACTGAAAAC amplification
of exons 5-9
tion was performed
with
of each deoxynucleoside
triphosphate,
2 mmol/L magnesium Elmer
Cetus,
Conditions
method
to
purified,
Sequencing
Version
(Perkin
elsewhere.“.‘*
PCR amplification
with EcoRI, and
sequencing
sequencing
by
Mutations
for exons primers
55°C
of PCR were
2.0; US Biochemicals,
was performed
sense and antisense
Products digested
of
per reaction.
II SK; one recombinant
double-stranded
(Sequenase
scribed
polymerase
and 72°C (3 minutes). into pBluescript
subjected
and 2.5 U of Amplitaq
CT) DNA
electrophoretically
subcloned
OH).
chloride,
Norwalk,
200 pm
a final concentration
5-9
clone was the
dideoxy
Cleveland, using
both
for each exon as de-
were confirmed
by a second
and resequencing.
and reverse transcribed
or NotI-
primers
scriptase DNA
(Pharmacia,
aliquots,
amplified
by the exon connection
complementary blot analyses desctibed.3
as described
DNA using
with pd(N)b Germany). strategy
Nine
Different
pancreatic
strong
tumors
nuclear
antibodies
as deprimers
and adjacent
patterns
emerged
in almost
1B). In HPAF
stained
nuclear
pattern.
with
primary
(Table
both
and 8902, about
25%
with both antibodies.
antibodies,
Capan-2,
pancreatic
staining. 1). PC-44,
and PC-3 showed
showing
No immunoreactivity
the cell lines PC-2, Four
both
pancreatic
nuclear
all cells with
of the cells showed nuclear staining BxPC-3
and six pri-
normal
Patu II, Pant-1,
staining
(Figure
antibodies,
12 cell lines
carcinomas
of 12 cell lines showed
staining
and AsPC-1
tumors
a weak
was observed
(722,
(Figure
228,
in 1A).
323,
and
BE) and one metastatic
tissue (from patient
BE) showed
positive
staining
the mutant-specific
monoclonal
antibody
pAB240
mal pancreatic normal
with
(Figure
lC), whereas surrounding
tissues (except for patient
tissue was available) carcinomas
pancreatic
were negative.
(929
expression
carcinoma
was further
Two primary negative
antibodies
(Table
quantitated
in the
cell lines by an immunoassay
the ~53 mutant-specific
monoclonal
nor-
BE, where no
and AS) showed
for pS.? with both monoclonal
2). p>3 protein
antibody
using
pAB240.
reverse tranwere PCR-
using DCC antisense
elsewhere.13
a DCC-specific
tissues.
monoclonal
were used to stain
Complementary
to 200 pg RNA,
fragments
Control amplifications
random
100 U MoMuLV
Freiburg,
corresponding
and sense primers
primers
using
was isolated
anti-p53
from human
staining
DCC Complementary DNA Amplification and Southern Blot Analysis scribed
derived
pancreatic
Total RNA from tissue specimens
different
and pAB421,
89SSS, MIAPaCa-2,
gene. Amplifica-
for PCR were 34 cycles of 94°C (1 minute),
(2 minutes), pooled,
of the humanp53
3’) for
100 pmol of each primer,
Two pAB240
mary pancreatic
p53f (5’ ATGCGAATTC-
TACTCCCCTGCCCTCAACAAGAT
1647
Results
using
with
IN PANCREATIC CANCER
Amplified
were analyzed
DCC
by Southern
57mer-oligonucleotide were performed
Table 2. ~53 Alterations and Loss of DCC Gene Expression in Primaty Pancreatic Carcinomas
as
with p-actin
as described.13
Figure 1. lmmunocytochemical staining of human ~53 with pAB240 in representative pancreatic carcinoma cell lines and primary pancreatic carcinoma tissue. (A) Absence of ~53 staining in Capan-2, (5) nuclear ~53 staining in MIAPaCa-2, and (C) ~53 staining in primary pancreatic carcinoma tissue (Original magnification X200).
Pancreatic carcinoma tumors 722 N T 927 N T 228 N T 323 N T AS T BE T M
Overexpression of p53
Loss of DCC expression
_ +
+ _
_ _
+ +
_
+ _
+ _ +
+ _
_
+a
+ +
_a _a
T, primary tumor; N, normal tissue; M, metastasis. a Obtained from previous work.13
1646
The highest in PC-44, Pant-I,
GASTROENTEROLOGY Vol. 106, No. 6
SIMON ET AL.
levels of mutated MIAPaCa-2,
~53 protein
and 8902,
were detected
less in Patu
the other pancreatic
carcinoma could
Table 1). Nop5.3 protein of the cultured
cell lines except AsPC- 1,
lines negative
be detected
was detected
(Figure
2 and
RNA
carcinoma
the transcript ~53 protein
transcript
3 and Table
correlated detected
2). The highest
bridization
8988S),
was analyzed
expression
in most
l), and the intensity
of
No transcript
of the p53 gene Hy-
amino
acid
carcinoma
mutation
the cell line Pant-1
at codon 220 in
cell line MIAPaCA-2
at codon
showed
273 in exon 8 (Figure
8902). One cell
mutation
248 in exon 7, and
a point
4 and Table
mutation
at codon
1).
Comparison of ~53 Alterations of DCC Gene Expression Loss of DCC expression mary pancreatic
that changes in the levels
were obtained
from our recent workI
region ofp53
after PCR amplification
encoding carcinoma of geno-
in four pri-
The DCC expression
carcinomas.
and pancreatic
2 and 3. In the primary
and Loss
was analyzed
tumors
of the ~53 gene in the pancreatic
cell lines by sequencing
176 (PC-44,
of two further
Sequence Analysis of the ~53 Gene in Pancreatic Carcinoma Cell Lines exons 5-9
were point
the deduced
RNA
ofp53 transcripts were not caused by RNA degradation and verified equal loading.
the genomic
All mutations
showed a point
exon 6. The pancreatic
Patu
and Capan-2.
of the same blot with the 18s ribosomal
We analyzed
Cell
did not show a mutation
and altered
and two at codon
showed a point
(Figure
in PC-44,
probe indicated
of the gene.
mutations
cell lines
protein.
of mutated
was observed
in the cell lines AsPC-1
oligonucleotide
in
blot anal-
was detectable the amount
forp53 expression
line (BxPC-3)
by the ~5.3 immunoassay
II, 8902, and MIAPaCa-2. was observed
with
region of the ~5.3
carcinoma
sequence. Four pancreatic carcinoma cell lines showed point mutations in exon 5, two at codon 151 (HPAF,
cell lines by Northern
ysis. A 2.8kilobasep53 cell lines (Figure
expression
in the conserved
immunohistochemicallyp53
in this domain missense
in the supernatants
cell lines (data not shown).
~53 messenger
Mutations
overexpressing
were found
Northern Blot Analyses of ~53 in Pancreatic Carcinoma Cell Lines 12 pancreatic
mic DNA.
gene were found in 7 of 9 pancreatic
in
and low levels of p53 expression
where no ~53 protein
II and
tumors
carcinoma
data
cell lines
and noted in Tables
of four patients
(722,
of one patient
(BE),
228, 323, and BE) and a metastasis
DCC expression was clearly reduced compared with adjacent normal tissues. In patients 927 and AS, expression of DCC was comparable ure 5A and Table expressing
with adjacent
2). Interestingly,
histochemically
abberant
normal tissue (Figpancreatic
tumors
~5.3 protein
also
showed loss of DCC gene expression, whereas in p53negative tumor tissues, no significant loss of DCC gene expression
was observed (Table 2). Except for one pancre-
atic carcinoma
cell line (MIAPaCa-2),
cell lines harboring
an altered ~53 protein,
reduced or lost DCC gene expres-
sion could be observed.
One cell line PC-2 had lost DCC
gene expression without immunohistochemical indication for an alteredp53 expression, although the immunoassay
2.8kb Figure 2. p53 protein expression of pancreatic carcinoma cell lines by a ~53 mutant selective quantitative enzyme-linked immunosorbent assay. Quantitation is achieved by the construction of a standard curve using known concentrations of mutant ~53 protein and comparing the absorbance. The amount of mutant ~53 protein is given as the mean of two assays. Values are given as nanogram per milligram of total protein concentration 2 SEM.
showed
low-level
~53
protein
expression
and
P53
Figure 3. Northern blot analysis of human ~53 transcript in human pancreatic carcinoma cell lines (top). Control hybridization with 18s oligonucleotide (bottom).
~53 AND DCC
June1994
@AC G
A
0 Wildtype T
A”;taG”’ T
72
927
N
T
N
IN PANCREATIC CANCER
228 T
1649
323 Ni
Ni
Mutant
B
1
2
345
678
41000
Pigure 4. Sequencing autoradiograms of ~53 gene in the conserved region for PCR-amplified DNA samples from pancreatic carcinoma. (A) exon 5, codon 151, C-T transition; (B) 8902, exon 5, codon 89883, 176, T-A transversion; (C) BxPC-3, exon 6, codon 220, A-G transition; (0) MIAPaCa-2, exon 7, codon 248, C-T transition; and (E) Panc-I, exon 8, codon 273, A-G transition. Sequences in A and 8 are shown in anti-sense direction, and C, D, and E are shown in sense direction. On the left is the wildtype sequence, and on the right is the mutated sequence. Arrows indicate mutated nucleotides.
Plgure 5. Expression of DCC in primary pancreatic carcinomas and normal adjacent tissue. (A) Southern blot analysis of PCR-amplified DCC complementary DNA. (8) pactin control amplification of tissue samples shown in A. T, pancreatic tumor tissue; N, normal adjacent tissue.
lines and primary pancreatic tumors and the association with loss of DCC gene expression in primary tumors. We
Northern (Table
blot
analysis
3 and Figures
showed
a weak ~53
transcript
2 and 3).
In this study, we analyzed alterations suppressor
Table
gene ~53 in human
3. Alterations
of c-Kis-ras,
Carcinoma
Cell Lines
Cell line HPAF 89888 8902 PC44 BxPC-3 MIAPaCa-2 Pant-1 PC-3 Patu II PC-2 AsPC-1 Capan-
c-KCras mutation codon 1213 + + + + ND ND ND wr -t W-T ND ND
NOTE. +, present; -, absent. ND, not determined; WT, wildtype.
observed
expression
immunologic
pancreatic
~53,
of the tumor carcinoma
cell
and DCC in Pancreatic
~53 mutation codon (exon)
Loss of DCC expression”
151(5) 151(5) 176 (5) 176 (5) 220 (6) 248 (7) 273 (8) _ _ _ _ _
_ _ ND + _ _ + +
evidence
in 9 of 12 pancreatic
suggesting a posttranslational the cellular environment,25
Discussion
bp
of abnormal
carcinoma
stabilizing complexing
~53
cell lines,
by changes in to other pro-
teins,26m29 or mutating of the p>3 protein coding sej0 We could show by DNA sequencing that in
quence.
seven pancreatic
carcinoma
cell lines, the abnormal
stain-
ing pattern arose in cells that had undergone ~33 mutations in the evolutionary conserved region of the gene. All ~53 mutations were missense mutations, and most of them were GC to AT transitions. However, the base changes showed no specific mutational spectrum that could indicate an association with an individual mutagen shown for other tumor types.24.31 Most of the mutations were detected in exon 5 in codons 15 1 and 176, which is consistent with sequencing data from Kalthoff et al.” in which 4 of 7 altered cell lines showed mutations in exon 5, notably, two of them in codon 176. Although Scarpa et aL3’ recently reported a preferential location of p53 mutations in primary pancreatic tumors in exons 5 and 7, no specific site appeared to be preferentially affected. The frequent mutational alterations in exon 5 are intriguing; however, codon 176 cannot be considered as a hotspot in pancreatic cancer based on the observations
1650
in these pancreatic tions
RNAs
carcinoma
may represent
mutated lines
GASTROENTEROLOGY Vol. 106, No. 6
SIMON ET AL.
p53 proteins
and
perhaps
cell lines. The ~53 muta-
the basis for increased in the pancreatic
also of certain
as suggested
carcinoma
mutated
by the remarkable
RNA overexpression
stability
of cell
messenger
~53 messenger
shown by some cell lines. However,
it can not be excluded
that other unidentified
the synthesis
or stability although
primary
pancreatic
tumors.
changes in pancreatic
immuno-
did not show
c-K-ras
mutations
duction
disrupt
the ~53 pathway
without
altering
the underlying
or genetic
complexing the protein
of the ~53 gene.
of other
and alter the level of expression
cause ofpS3
essarily be a mutational
alterations
with ~53 protein29834 could coding sequence. overexpression
alteration
Interaction
Therefore,
may not nec-
in the conserved
region
other proteins
could
with
in the tumorigenic pathway
were detected
vas proteins
tumorigenesis
involved
in cell adhesion
carcinoma
localizations
the concept
in pancreatic
of a multigenetic
cancer cell line in which the
noprecipitation were negative using the same mutantspecific monoclonal antibody pAB240. Recently, Ruggeri et al.” described some of the pancreatic carcinoma cell this study. Surprisingly, they reported ber of different mutational alterations
~53 alterations in lines analyzed in a very high numin individual cell
lines, which we could not confirm because we observed no more than one point mutation per cell line. This discrepancy is probably caused by random errors due to
in the pancreatic
of these diverse
cancer provides nature
support
of this tumor
gefor type.
mutations and ~53 alterations are the most common cancer-related alterations and are observed in a wide
rearrangement
enzyme-linked immunosorbent assay showedpS3 protein expression, whereas immunohistochemistry and immu-
of
ras
a genomic Capan-2’l
drigues et a1.35 in a colorectal
inhibition
these three genes show differand functions
cells. The accumulation
cancers.3.13z4’ Because
intriguing because nopS3 expression could be found by immunocytochemistry using the same monoclonal antibody pAB240. Th’is observation was also made by Ro-
The ~53 gene
processes that could be relevant
for metastases.40 Therefore,
lack of ~53 overexpression cannot predict a ~53 protein. Southern blot analyses indicated
pression using the sensitivep13 immunoassay and a weak pJ3 transcript found in Northern blot analysis. This is
trans-
apoptosis,‘” whereas the putative tumor suppressor gene DCC is expressed on the cell surface and appears to be
However, functional
gene in the cell line
the signal
across the membrane.36
gene expression
in thep53
to 3.13
appear to be involved
process by altering
in the conserved region of thepS3 gene in immunocytochemically negative pancreatic carcinoma cell lines.
that could explain the lack of a ~53 transcript and ~53 protein expression. Interestingly, the pancreatic carcinoma cell line PC-2 showed weak ~53 protein ex-
study
in Table
through
to
netic changes
No mutations
in an earlier
as summarized
contribute
ability to detect mutant ~53 protein in cells. Thus, the percentage of p53 alterations in pancreatic carcinomas analysis.
of these
cell lines. These
activator37,38 that might
ent cellular
try and sequence
accumulation
carcinoma
encodes a nuclear transcriptional
also mask antigenic epitopes on the ~53 protein and thus interfere with immunohistochemical or the immunoassay
may be even higher than shown by immunohistochemis-
frequent
cell lines have also been shown harbor
region
genes
p53 alterations
earlier13 showed
The membrane-bound
of the pS3 gene,
Comparing
genetic
point mutations in the conserved region of the ~53 gene. Intronic point mutations,33 mutations in the promoter cellular
alterations
genes ~53 and DCC in 4 of 6
suppressor
as shown in this study and loss of DCC gene expression
may have occurred in these cell lines. Interestfor ~53 protein,
In this study, we could observe concomitant of the tumor
was
a second PCR sample.
shown
transcripts
positive
by resequencing
molecular
affecting
ingly, the cell lines Patu II and PC-),
confirmed
because this group analyzed
In our study, each mutation
of the pS3
alterations
cytochemically
of Taq polymerase
infidelity
only one PCR reaction.
variety
of human
intestinal
tumors,
tumor
types. In contrast,
appears to be confined
loss of DCC
to certain gastro-
such as colonic, gastric, and pancreatic mutational
alterations
of the ras
and ~53 genes are also frequently observed in colorectal and gastric cancers, 1’42’43pancreatic cancer shows a common set of genetic changes with these tumors that might be relevant for tumor development and progression.
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Received August 9,1993. Accepted January 25,1994. Address requests for reprints to: Babette Simon, M.D., Division of Gastroenterology, Department of Internal Medicine, Philipps University, BaldingerstraBe, 35033 Marburg, Germany. Fax: (49) 6421288922. Suppported by a Dornier research grant. The authors thank Dr. Eric Fearon for critically reviewing this manuscript.