(CANCER RESEARCH 51. 3.ÃŽ56-.W1.July I. 1991]
Detection of Aberrations of the p53 AlÃ-elesand the Gene Transcript in Human
Tumor Cell Lines by Single-Strand Conformation Polymorphism Analysis'
Yoshinori Murakami,2 Kenshi Hayashi, and Takao Sekiya
Oncogene Division, \ational Cancer Center Research Institute, I-I, Tsukiji. 5-chome, Chuo-ku. Tokyo 104, Japan
ABSTRACT
MATERIALS
Aberrations of the p53 gene and its transcript in ten human tumorderived cell lines were investigated by single-strand conformation poly
morphism analysis of polymerase chain reaction products. This method
can detect loss of one of the two alÃ-elesand a point mutation in the
remaining alÃ-elesimultaneously. Aberrations were newly found in four
tumor cell lines. Three cell lines had lost one of the alÃ-elesand had a
point mutation in the other. These point mutations, resulting in amino
acid substitutions, were in the region of the p53 gene, which is highly
conserved in different species. All the mutated genes »ereexpressed and
produced mRNAs with point mutations. One cell line did not contain any
detectable transcript of the p53 gene, although no structural aberration
of the gene was detected in the regions analyzed. Most of the cell lines
found to carry aberrations of the p53 gene in this work have previously
been reported to have mutations in oncogenes or in a tumor suppressor
gene other than the p53 gene. These results provide additional examples
of multiple genetic changes in the genesis of human tumors.
Cell Lines. Two large cell lung cancer cell lines (Lu65 and Lu99) and
a pancreatic cancer cell line (PSN1) were established at the National
Cancer Research Institute, Tokyo. Japan (8, 9). A promvelocytic leu
kemia cell line (HL60), a hepatoccllular carcinoma cell line (PLC/
PRF/5), and a tcsticular tumor cell line (ITO-II) were obtained from
the Japanese Cancer Research Resources Bank. A relinoblastoma cell
line (Y79) and two colon cancer cell lines derived from the same tumor
(COL0320 HSR and COLO320 DM) were obtained from the Ameri
can Type Culture Collection. A Wilms' tumor cell line (\V2) was kindly
INTRODUCTION
Many studies have demonstrated frequent loss of heterozygosity at loci on chromosome 17p, where the tumor suppressor
p53 gene is located, in a wide variety of human tumors (1-3).
Recently, by restriction fragment length polymorphism analysis
and nucleotide sequence analysis of polymerase chain reaction
products of the gene. Baker et al. (4) and Nigro et al. (5) clearly
demonstrated that, in human colon, lung, and breast cancers
and in brain tumors, both alÃ-elesof the p53 gene are mutated,
mostly by deletion of one alÃ-eleand a nucleotide substitution
or small deletion in the remaining alÃ-ele.These results indicated
the direct involvement of aberrations of the p53 gene in genesis
of these tumors.
We recently developed a rapid, sensitive method for detection
of single nucleotide substitutions, namely, SSCP' analysis of
PCR products (6, 7). In this work, by PCR-SSCP analyses of
genomic DNAs and the reverse transcription products of
mRNAs, we detected aberrations of the p53 gene and tran
scripts of these abnormal genes in several human tumor-derived
cell lines. The loss of one of the alÃ-elesand a point mutation in
the remaining alÃ-eleof the p53 gene in some of the cell lines
could be clearly demonstrated simultaneously. Moreover, the
presence of only mutated transcripts in these cells was also
demonstrated by this method. Our finding of aberrations of the
p53 gene in several human tumor cell lines provides additional
evidence for the involvement of p53 gene aberration in a reces
sive manner in human tumor cells.
Received 2/6/91; accepted 4/15/91.
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 I'.S.C. Section 1734 solely to indicate this fact.
' This work was supported by a Grant-in-Aid from the Ministry of Health and
Welfare for a Comprehensive 10-Year Strategy for Cancer Control. Japan: a
Grant-in-Aid for Cancer Research from the Ministry of Education. Science, and
Culture; and a grant from the Special Coordination Fund of the Science and
Technology Agency of Japan.
2To whom requests for reprints should be addressed.
"The abbreviations used are: SSCP. single-strand conformation polymor
phism; I'C'R. polymerase chain reaction: SSC. standard saline citrate. SDS.
AND METHODS
provided by Dr. M. Sekiguchi. Institute of Medical Science. University
of Tokyo. Japan. A colon cancer cell line (Cl) was derived from a
metastatic focus in the lung (10).
Southern Blot Analysis. Genomic DNA was prepared by the proteinase K-phenol-chloroform extraction method (II). Genomic DNA (5
A/g)was digested with the restriction endonuclcasc llind\\\. The prod
ucts were separated by electrophoresis in 0.7rr agarose gel and trans
ferred to a nylon membrane. Hybridi/ation was performed at 42°Cfor
12 h in 6x SSC (Ix SSC is 0.15 M NaCI/0.015 M sodium citrate)-10
irmi EDTA-5X Denhardt's solution-0.5'V SDS-100 ¿ig/mlof denatured
salmon testis DNA-10% dextran sulfate-50% formamide. The mem
brane was then washed with 2x SSC once and 0.2x SSC" 3 times at
50°Cfor 30 min and exposed to Kodak XAR-5 film for 24 h at -80°C
with an intensifying screen (Lightning Plus; Dupont Cronex).
A cDNA probe of 172 base pairs corresponding to positions 178 to
350 of p53 was prepared by the PCR from the reverse transcription
product of total cellular RNA isolated from normal human peripheral
lymphocytes using unlabeled primers. Nucleotide sequences of the
primers used are indicated in Table 1 (cDNA fragment. Dl and D2).
Recombinant plasmid p2R3.8, containing a 3.8-kilobase pair EcoRI
fragment corresponding to the 3' region of human RB cDNA, was
kindly provided by Dr. T. P. Dryja, Harvard Medical School. The p53
cDNA probe and 3.8-kilobase pair EcoR\ fragment were labeled as
described under Northern blot analysis.
Northern Blot Analysis. Total cellular RNA was obtained by the acid
guanidine thiocyanate-phenol-chloroform
extraction method (12).
Samples of about 4 jug of total cellular RNA, separated by electropho
resis in \% agarose gel containing 2.1 M formamide, were transferred
to nylon membranes. Hybridi/ation and autoradiography were per
formed in the same conditions as those for the Southern blot analysis
described above, except that the membranes were washed with 2x SSC
at 50°Cfor 30 min 3 times after hybridization.
The recombinant plasmid pR/iAc3'ut, containing a 1.3-kilobase pair
Hpa\\-EcoR\ fragment corresponding to the 3' region of the rat /i-actin
gene, was used to detect the human /i-actin gene transcript (13). The
probes for the p53 and /1-actin genes were labeled with [l:P|phosphate
using a multiprime-labeling kit (Multiprime rapid hybridization system;
Amersham) with |n-l:P|d(TP as a radioactive substrate.
PCR-SSCP Analysis of Gene Transcripts and Genomic DNA. Total
cellular RNA (1 /jg) was annealed with a synthetic DNA of 20 nucleotides (Table 1, cDNA fragment. Primer F2) complementary to the 3'
noncoding region of the p53 mRNA. Then it was transcribed with
Molony murine leukemia virus reverse transcriptase (Bethesda Re
search Laboratories) in the presence of the RNase inhibitor RNasin in
the reaction mixture (14 p\) described previously (14). A portion of the
reaction mixture (0.5 /ul)was subjected directly to the polymerase chain
reaction to amplify the regions of p53 cDNA (Fig. 3.4) in the mixture
(5 /jl) as described (15), using a pair ol appropriate oligonucleotides as
primers. For analysis of genomic DNA by the SSCP technique, 50 ng
sodium dodecyl sulfate; cDNA. complementary DNA: RB. retinoblastoma: PBL.
of DNA were similarly subjected to the PCR to amplify the regions of
peripheral blood lymphocyte.
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ABERRATIONS OF- THE pS.l GENE IN HUMAN TIMOR
Table 1 Primers used for amplification of the p53 cDSA and genomic DNA
CELL LINES
the gene transcript ¡nHL 60 cells were reported previously
(19), and our results confirmed the observation.
The p53 gene transcripts in the tumor cell lines were then
analyzed by Northern blot hybridization. As shown in Fig. 2,
in addition to HL60 cells (Lane 9), a large cell lung cancer cell
line Lu65 (Lane 2) and a hepatocellular carcinoma cell line
PLC/PRF/5 (Lane 5) did not give any detectable transcript,
although the signal for 0-actin mRNA (Fig. IB. Lanes 2, 5, and
9) as an internal control could be detected in these cell lines.
The greatly reduced amounts of the p53 transcript in PLC/
PRF/5 cells have been reported by Bressac et al. (20). The other
seven cell lines contained significant amounts of p53 transcripts
of 2.8-kilobase pairs.
Primers.Amplified
fragmentcDNA
fragmentAKC1)EFGenomic
fragmentABC1)EFGNameAlA2BlB:ClC2DlD2ElE2FIF2AlA2BlB2ClC2niD2ElE2FIF2ClG2Séquence5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5'5
DNA
12345678
Direct DNA Sequencing. Nucleotide sequences of DNA fragments
were determined by the dideoxy chain-termination method on asym
metric PCR products (17). Fifty cycles of the asymmetric PCR were
performed as described (17) with an uneven molar ratio of the two
primers (5 to 50:1). The amplification reaction mixture was diluted and
deionized in a Centricon 30 microconcentrator (Amicon), and the
single-strand DNAs obtained were annealed to a 5'-labeled primer.
kb.
-23.1
-9.4
-6.6
-4.3
--2.3
-2.0
B
the p53 gene (Fig. 3B). Primers used for amplification of cDNA and
genomic fragments are shown in Table 1. The 5' ends of these primers
were labeled with [>-3:P]ATP by the polynucleotide kinase reaction as
described previously (7, 16).
The product of the PCR was diluted with 100 to 500 volumes of
loading solution containing 90% formamide, 20 mM EDTA, and 0.05%
xylene cyanol and bromphenol blue; denatured at 80°C.and applied (1
/jl/lane) to 6% polyacrylamide gel containing 90 HIMTris-borate (pH
8.3) and 4 mivi EDTA, with or without 10% glycerol as specified.
Electrophoresis was performed at 30 W for 2 to 6 h under cooling with
a fan. The gel was dried on filter paper and exposed to X-ray film at
—80"Cfor 0.5 to 12 h with an intensifying screen.
9101112
.
_23.1
- 9.4
- 6.6
~ w -
-4.3
-2.3
-2.0
Fig. 1. Southern blot analysis of the p53 gene in ten human tumor-derived cell
lines. Hybridization was performed with a p53 cDNA probe for the 7.5- and 2.5kilobase pair DNA fragments of the p53 gene (A) (19) and a p2R3.8 probe for
DNA fragments of the RB gene of 2.0- to 9.4-kilobasc pairs (B) (27). Lanes I to
lìcontain samples from V79. Lu65. Lu99. CI. PLC/PRF/5. VV2. Itoli. PSNI.
HL60. COLO320 homogeneously staining regions, and COLO320 double-minute
cells and from peripheral blood lymphocytes of an individual, respectively, kb.
kilobases.
Chain elongation and termination were performed with a Sequenase
TM kit (United States Biochemical Corporation), and products were
analyzed in 6% polyacrylamide gel containing 7 M urea.
1 23456789
1011 12
kb.
RESULTS
-2.8
Southern Blot Analysis and Northern Blot Analysis. Before
PCR-SSCP analysis, we analyzed the p53 gene and its tran
script in ten human tumor cell lines by conventional methods.
The cell lines include large cell lung cancer, pancreatic cancer,
hepatocellular carcinoma, testicular tumor, retinoblastoma, co
lon cancer, Wilms' tumor, and promyelocytic leukemia. DNAs
isolated from the tumor cell lines were digested with restriction
enzyme Hind\\\ and subjected to Southern blot analysis using
the p53 cDNA probe. As shown in Fig. IA, all the cell lines,
except the promyelocytic leukemia cell line HL60 (Fig. \A,
Lane 9), gave the same signals as that of DNA from normal
PBL (Fig. \A, Lane 12) (the bands of 7.5-kilobase pairs and
2.5-kilobase pairs corresponding to exons 2 to 11 of the p53
gene: cf. Réf.18). Complete loss of the p53 gene and lack of
B
-2.0
Fig. 2. Northern blot analysis of the p53 gene transcript in ten human tumorderived cell lines. Hybridization was performed with a p53 cDNA probe for the
2.8-kilobase (ÄA)pair p53 transcript (A) (19) and a fi-actin probe for the 2.0kilobase pair .i-actin transcript (B) (13). Lanes I to 12 contain samples from the
same cells as described in the legend to Fig. 1.
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ABERRATIONS OF THE p53 GENE IN HUMAN TUMOR CELI. LINES
PCR-SSCP Analysis of the p53 Gene and Its Transcript. For
analysis of p53 mRNA by the PCR-SSCP method, total cellular
RNA from the tumor cell lines was transcribed into cDNA
using reverse transcriptase and oligonucleotide primers with
nucleotide sequences complementary to the 3' noncoding re
gion of the p53 mRNA. The single-stranded cDNA fragments
thus obtained were then amplified by the PCR to doublestranded DNA fragments using sets of two 12P-labeled oligo
nucleotide primers. The six regions of the p53 cDNA amplified
are indicated in Fig. 3A, the fragments with nucleotide lengths
of 254 to 331 base pairs obtained being designated as A to F.
These six fragments, which overlapped each other and covered
the whole coding sequences of the p53 cDNA of about 1.2kilobase pairs, were subjected to SSCP analysis.
Results on the cDNA Fragment D carrying the nucleotide
sequence of exons 5 to 7 are shown in Fig. 4A. Two comple
mentary strands were obtained from 8 of 10 tumor cell lines.
As expected from the results of Northern blot analysis (Fig.
2A), no PCR product from the p53 cDNA was detected in
HL60 cells and Lu65 cells. On the other hand, a significant
amount of the PCR product was detected in PLC/PRF/5 cells
(Fig. 4A, Lane 5), although the p53 gene transcript was scarcely
detectable by Northern blot analysis (Fig. 2A, Lane 5). In this
cell line, the separated strands showed different mobilities from
those of the fragment from normal peripheral lymphocytes, the
mobility shifts indicating a structural change in Fragment D.
Fragments showing abnormal mobilities were also observed
in COLO320 cells (Fig. 4A, Lanes 10 and II). These cells did
not give the slower moving band obtained with normal lympho
cytes, suggesting the absence of the normal transcript. In these
cases, only one of the complementary strands was informative,
and the faster moving strand carrying a structural change did
ATG
my
II
1
234
254bp
A
5
ft
6
.ftfttt
lili
7
TGA
I
8 9 10
J
,,
11
"
257bp
C
331bp
B
280bp
D
126-186 187-22J
302DP
F
t!
225-261
139 bp
D
F
PLC/PRF/5
and COLO320 cells, no fragment other than
Fragment D showed a mobility shift, suggesting the presence
of a structural change in only the sequence of Fragment D (data
not shown).
For analysis of genomic DNA, regions containing coding
exons of the p53 gene were amplified into 7 fragments with
nucleotide lengths of 139 to 408 base pairs, designated as
Fragment A to Fragment G (Fig. 3Ä).SSCP analysis of these
fragments confirmed the aberrations of the gene corresponding
to the abnormalities observed in Figs. 2 and 4A. Results for
genomic DNA Fragment D carrying exon 7 of the p53 gene are
shown in Fig. 4B. The loss of both p53 alÃ-elesin HL60 cells
was again confirmed (Fig. 4B, Lane 9). The result on Fragment
D from Lu65 DNA, which did not contain the p53 gene
transcript (Fig. 2A\ Fig. 4A, Lane 2), indicated the presence of
the p53 gene with a normal sequence of at least exon 7 (Fig.
4B, Lane 2). The mobility shifts of fragments shown in Fig.
4B, lanes 5, 10, and //, indicated the presence of a nucleotide
sequence change in exon 7 of the p53 gene in the DNAs from
PLC/PRF/5 and COLO 320 cell lines, respectively, resulting
in production of an abnormal cDNA Fragment D (Fig. 4A). No
mobility shift of genomic fragments other than Fragment D
was detected in PLC/PRF/5 and COLO 320 cell lines (data
not shown). It should be noted that the results shown in Fig.
4B. Lanes 5, 10. and //. clearly demonstrated loss of a normal
p53 alÃ-elein these cell lines. From these results, we concluded
that, in PLC/PRF/5 and COLO320 cell lines, one of the two
p53 alÃ-eleswas lost and that the remaining alÃ-elecarrying a
nucleotide sequence change in exon 7 was transcribed as an
abnormal mRNA species. The transcript in PLC/PRF/5 cells
may be unstable and so could be detected by PCR-SSCP analy
sis, but not by Northern blot hybridization.
Similar analyses of the transcripts and genomic DNAs in the
region corresponding to exon 5 of the p53 gene were performed,
and the results obtained are shown in Fig. 5, A and B. respec
tively. Mobility shifts of Fragment C of the p53 cDNA (Fig.
5A, Lane 8) and Fragment C amplified from genomic DNA
(Fig. 5B, Lane 8) together with the absence of a fragment with
the normal mobility in both the transcript and genomic DNA
clearly demonstrated that PSN1 cells contained only the p53
alÃ-elewith a structural aberration within exon 5 and the tran
script of the mutated alÃ-ele.
We analyzed all the fragments from transcripts and genomic
DNAs shown in Fig. 3 of ten tumor cell lines. However, we
detected no aberrations in them other than those described
above.
Nucleotide Sequence Analysis of Fragments with Mobility
Shift. To elucidate the structural alterations that caused mobil
ity shifts in fragments detected by the PCR-SSCP analysis
described above, we determined the nucleotide sequences of
these fragments. Fragment D carrying exon 7 of the p53 gene
was amplified from DNAs of PLC/PRF/5 and COLO320 cells,
and Fragment C carrying exon 5 of the gene was amplified
from DNAs of PSN1 cells. These fragments were separated
from other unrelated PCR products and primers by the SSCP
method, eluted from polyacrylamide gel, and subjected to direct
sequencing by the asymmetric PCR method.
¿t»
t
262-306 307-331
330 bp
E
10
139 bp
not show a mobility shift. Such false negative results are some
times obtained on PCR-SSCP analysis. However, all of the
known nucleotide substitutions including point mutations and
DNA polymorphisms so far analyzed could be detected by
mobility shift of either one of the complementary strands.4 In
202 bp
G
Fig. 3. Regions of p53 cDNA and the p53 gene amplified and subjected to
SSCP analysis. In A, the coding regions of p53 cDNA were divided into six
fragments (A to F). Their positions are indicated by bars with arrows at both ends,
and their nucleotide lengths are indicated under the bars. ATG and TUA indicate
the initiation and termination codons, respectively, of the open reading frame of
the p53 cDNA. In B, the seven regions of the p53 gene (Fragments A to G)
corresponding to exons 2 to 11 are indicated by bars with arrows at both ends.
Their nucleolidc lengths are indicated under the bars. The coding regions of the
exons are indicated by solid boxes with amino acid positions, while noncoding
regions are indicated by open boxes. In both .-1 and B. the locations of point
mutations reported by Nigro et al. ( 12) are indicated by vertical arrows.
4 Unpublished results.
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Aßi-'KK
\no\s
or Tin:
CENEIN WM\N n AIORCKI.I UNES
B
A
1 2 3 4 5 6 7 8 9101112
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 4. Analyses of 1)N.\ fragments carry
ing the nuclcotidc sequence of e\on 7 of the
p53
gene. /)SSCP
analysis
of the Fragment
p53 cDNA/)
l-'ragmenl
(Ai and
the genomic
of the p53 gene (fl). Fleclrophoresis was per
formed in d'i polyucrylamidc gel containing
10'V glyccrol at 30 W ¡it25°Cfor 5 h (A) or 3
h (H). Lanci I to 12 contain p53 cDNA Fragment lìobtained from Y79. Lu65. Lu99. C'l
PLC ,'1'KI75. \\2.
Itoli. PSM.
UI.60
("OI.O320 homogeneously staining regions
and COI.O320 double-minute cells and Iron
peripheral lymphocytes, respectively. C. nu
cleolide sequence analysis of the genomic
Fragment /) of the p53 gene from PU'/PRF/
PLC/PRF/5
T
Colo320HSR( -)
3'
T
C
G A
normal lymphocyte
3' T
C G A
C G A
TG248Arg249SerC
5. COI.O320 homogeneously staining regions,
and COI.O320 douhle-minute cells and from
normal hlootl lymphocytes.
Colo320DM(3'
248
GG
C
G
CA
TGCT
T!
G
TA
TG248Arg249ArgC
•
•»
GG
C
G
CA
TGCGG•«ttP•
AGÕ/
•
normal
lymphocyte
PSN1
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 5. Analysis of DNA fragments carry
ing the nueleotide sequence of exon 5 of the
p53 gene. SSCP analysis of the p53 cDNA
Fragment ( (AÌ
and genomic Fragment C of
the p53 gene (/?). Flectrophoresis \vas per
formed in 6'< polyacrylamidc gel with (Ai or
without (B) IO<Vglyccrol at 30 \\ for 5 h at
25°C.Art/ic'.v/ to 12 contained samples from
1 2 3 4
B
5 6 7 8 9 10 11 12
the same cells as described in the legend to
Fig. 4. C. nueleotide sequence analysis of gen
omic Fragment ( of the p53 gene from PSN I
cells and normal lymphocytes.
Cl cells (Fig. 6A, Lane 4) and B strand from PLC/PRF/5 cells
The sequencing ladders of Fragment D shown in Fig. 4C
(Fig. 6A. Lane 5) revealed that a nueleotide substitution at
revealed that, in PLC/PRF/5 cells, the arginine codon (AGG)
codon 72 resulted in two different mobilities of Strands.-l (CGC.
at position 249 of the p53 gene is mutated to a serine codon
(ACT) by a G to T transversion at the third letter. In COLO
arginine codon) and B (CCC. proline codon) (Fig. 6(T). Using
this polymorphism, in Y79 cells, two p53 alÃ-elescould be
320 cells (HSR and DM lines), a C to T transition at the first
distinguished (Fig. 6A. Lane /). whereas in Lu65, Lu99, Cl,
letter of the 248th codon resulted in replacement of arginine
\V2, and Itoli cell lines and PBL from an individual, the alÃ-eles
(CGC) by tryptophan (TGG) (Fig. 4C). The results on Frag
ment C shown in Fig. 5C indicated that, in PSN1 cells, the
were found to be homozygous. In accordance with loss of one
132nd codon for lysine (AAG) of the p53 gene was changed to of the p53 alÃ-elesshown in Figs. 4 and 5. only one alÃ-elewas
a glutamine codon (CAG) by an A to C transversion.
detected in PLC/PRF/5 (Fig. 6.4. Lane 5). PSN I (Lane 8). and
Nucleotide Sequence Polymorphisms Detected by PCR-SSCP.
COLO320 cell lines (Lanes JO and //).
Buchman et al. ( 18) have detected two p53 proteins with distinct
SSCP analysis of Fragment B amplified from cDNA by the
electrophoretic mobilities on SDS-polyacrylamide gel electroPCR (Fig. 3.4) could also detect this nueleotide sequence poly
phoresis due to polymorphism and suggested a nueleotide se
morphism in all the tumor cell lines except Lu65 and HL60
quence polymorphism within a coding exon of the p53 gene.
cells, which lacked the p53 transcript (Fig. 6#. Lanes 2 and 9).
By PCR-SSCP analysis, we could detect this polymorphism in In Y79 cells, both Strands A and B were observed (Fig. 6ß.
Fragment A carrying exon 4 of the p53 gene (Fig. 6.4) amplified
Lane /). suggesting that both the p53 alÃ-eleswere transcribed.
from genomic DNAs of the tumor cell lines. As seen in Fig. As the p53 gene was not present in these cells, the minor
6/Õ,the slower moving strand of the genomic Fragment B fragments observed in HL60 cells (Fig. 6ß,Lane 9) might be a
showed two distinct mobilities, .4 and B. These results suggested
PCR product from other than the p53 transcript. The same
the presence of a nueleotide sequence polymorphism within
fragment was also observed in other cell lines, although the
Fragment B. Nucleotide sequence analysis of the .4 strand from
amount of the fragment was less than that in HL60 cells.
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ABERRATIONS OF THE p53 GENE IN HUMAN TIMOR CELL LINES
B
PLC/PRF/5
(B)
123456789101112
Fig. 6. Nucleotide sequence polymorphism
within c\on 4 of the p53 gene. SSCP analysis
of the genomic Fragment B of the p53 gene
(A ) and cDNA Fragment B of the p53 gene
(B). Electrophoresis was performed in 6%
polyaerylamide gel with 10rr glycerol at 30 \V
for 1.5 h at 25°C.Lanes I to 12 eontained
B
1 23456789101112
C1
(A)
TCGATCGA
B
samples from the same cells as described in
the legend to Fig. 4. C nucleotide sequence
analysis of genomic Fragment B of the p53
gene from PLC/PRF/5 and CI cells.
DISCUSSION
In HL60 and Lu65 cells, no appreciable levels of p53 tran
scripts were detected, even by PCR-SSCP analysis. In HL60
In most cases examined so far, inactivation of the p53 gene cells, in addition to a point-mutated N-ras gene and an ampli
has been found to be due to loss of one of the two p53 alÃ-eles fied c-myc gene (25), loss of both of the p53 alÃ-elesand the
and a point mutation or a small deletion in the other alÃ-ele(4, absence of the gene products have been reported (19). We
5). The simple, sensitive PCR-SSCP method can detect both confirmed these aberrations by the PCR-SSCP method together
loss of a gene and subtle genetic change in the remaining alÃ-ele with conventional Southern and Northern blot hybridization.
at the same time. Therefore this method is suitable for analyzing
The homozygous or hemizygous p53 gene observed in Lu65
aberrations of tumor suppressor genes (21). Transcripts from cells and the absence of transcripts of the gene suggest loss of
the tumor suppressor gene can also be analyzed by this method.
one of the p53 alÃ-elesin this cell line. As we could not detect
In this work, we detected aberrations of the p53 gene and its any aberration in the fragments amplified from genomic DNA,
transcript for the first time in four human tumor-derived cell
the p53 gene in Lu65 cells might have a mutation in a region
lines. In a pancreatic cancer cell line (PSN1), a colon cancer
other than the coding sequence. This cell line has also been
cell line (COLO320), and a hepatocellular carcinoma cell line reported to have amplifications of the point-mutated K.\-ms2
(PLC/PRF/5), loss of one of the two p53 alÃ-elesand a point
gene and the c-myc gene, loss of one of the two RB alÃ-eles,and
mutation in the remaining alÃ-elewere observed. The finding of a point mutation introducing a stop codon in the remaining RB
the p53 gene aberration in PSN1 cells is the first evidence for alÃ-ele(21, 26). Therefore, these two cell lines are additional
the possible involvement of the gene in pancreatic cancers,
examples of cells carrying multiple genetic changes.
although a mutated p53 gene has been reported in a pancreatic
cancer as one of the tumors in a rare cancer syndrome (22).
REFERENCES
Mutation of the ras gene has often been detected in pancreatic
cancers (23), and PSN1 cells also have a Ki-ras2 gene with a
1. Borgslorm. G. H., V'uopio. P.. and de la Chapella. A. Abnormalities of
chromosome No. 17 in myeloproliferative disorders. Cancer Genet. Cytogepoint mutation in the 12th codon together with an amplified
net.. 5: 123-125. 1982.
c-niyc gene (9). Therefore, this cell line is a good example of a
2. Mackay, J.. Steel. C. M.. Elder. P. A., and Forrest. A. P. M. AlÃ-eleloss on
cancer cell having multiple genetic changes including ihose of
short arm of chromosome 17 in breast cancers. Lancet. 2: 1384-1385. 1988.
3. James. C. D.. Carlbom. M.. Nordenskjold. M.. Collins. V. I*., and Ca\ance.
two oncogenes (Ki-ras and c-myc) and a tumor suppressor gene
W. K. Mitotic recombination of chromsome 17 in astrocytomas. Proc. Nail.
(p53).
Acad. Sci. USA. 86: 2858-2862. 1989.
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3361
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Detection of Aberrations of the p53 Alleles and the Gene
Transcript in Human Tumor Cell Lines by Single-Strand
Conformation Polymorphism Analysis
Yoshinori Murakami, Kenshi Hayashi and Takao Sekiya
Cancer Res 1991;51:3356-3361.
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