Download Screening of a Specific Point Mutation in Tumor Suppressor p53

Mol. Cells, Vol. 1, pp. 491-4%
Screening of a Specific Point Mutation in Tumor Suppressor p53
Gene of Korean Hepatocellular Carcinoma Tissues
Bok-Soo Lee, Chang-Duk Jun, Young-Whan Chun, Sang-Gi Paik l , Baik-Whan Cho2,
Chan ChoP, Kwon-Mook Chae4 and Hun-Taeg Chung*
Department of Microbiology and Immunology, 3Department of Pathology and 4Department of
Surgery, School of Medicine, Wonkwang University, Iri 570-749, Korea; IDepartment of Biology,
Chungnam National University, Tafjon 305-764, Korea; 2Department of Surgery, School of
Medicine, Chonbuk National University, Chunju 560- 756, Korea
(Received on November 30, 1991)
The point mutation at a specific site (the third base of codon 249 of exon 7) in the
p53 gene was not found in the 8 hepatocellular carcinoma samples from Korean patients.
This result is quite different from the report on Chinese and South African patients that
showed the point mutations at the same site with the frequency of 50% in hepatocellular
carcinoma sample. Even though this particular point mutation was not found in Korean
samples, there might be mutations at other sites of p53 gene, because expression of mutated
p53 gene was detected in the nuelei of hepatocellular carcinoma samples by using monoelonal antibodies which are specific for muta nt-type p53 proteins. Also the change of DNA
content was found in the hepatocellular carcinoma samples.
Originally p53 had been considered to be an oncogene, but several researchers have indicated that the
wild type gene product actually functions as a tumour
suppressor gene (Finlay et al., 1989; Sturzbecher et al.,
1988; Wolf and Rotter, 1985). In human tumours, the
short ann of chromosome 17 is often deleted or the
remaining (not-deleted) p53 alleles were found to contain mutations and several reports suggest that p53
mutations play a role in the development of many
common human malignancies (Baker et aI., 1989; Eliyahu et aI., 1989). Especially, hepatocellular carcinoma
(HCC) is a prevalent cancer in Africa and Eastern
Asia, abnormalities in the structure and expression
of the p53 are frequent in HCC cell lines and allelic
loses from chromosome 17p have beeen found in
HCCs from China and Japan (Bressac et al., 1991).
Mutations occur at specific sites (at the third base
of codon 249), and this specificity could reflect exposure to a liver-specific carcinogen, being aflatoxin B1.
The human p53 gene of 11 exons is 20 kilobases (kb)
long, and the first and second exons are separated
by an intron of 10 kb (Peter and Lionel, 1986).
Baker et ai., (1990) reported that chromosome loss
occurs near the mutation sites of suppressor gene in
many cases. Loss of a series of chromosomes appears
to occur early in the development of many cancers,
with other gene changes usually coming in later (Baker et al., 1990; Marx, 1991). Mutations in the p53
gene are known to be the most common genetic alterations in diverse human cancers. Recently, two
groups of researchers reported that primary hepatocel* To whom correspondence should be addressed
lular carcinomas (HCC) from endemic hepatitis areas
and abundant aflatoxin B had showed high frequency
of point mutation at codon 249 of exon 7 in the p53
gene (Bressac et al., 1991; Hsu et aI., 1991). Korea is
known as an endemic area of hepatitis and many
Korean foods are also known to contain much aflatoxin.
In this study, the authors tried to see wheter or not
there is a specific point mutation at codon 249 of
exon 7 in the p53 gene. Small fragment (110 base
pairs) of the gene which contains codon 249 of exon
7 of p53 gene from the DNA of normal HCC tissue
was amplified by using polymerase chain reaction.
The amplified fragments from the wild type p53 gene
could be digested by the restriction enzyme HaeIII.
The authors screened the point mutation at the third
base of codon 249 of exon 7 in the p53 gene by the
electrophoresis of digested DNA segments of amplified
DNA fragments from HCC tissue DNA.
In addition, the nuclear phosphoprotein p53 was
originally discovered in extracts of transformed cells,
reacting with antiserum from animals with tumours
induced by simian virus 40 (SV40). Because large T
antigen is needed to maintain the transformed phenotype, it was suggested that this interaction is important
for transformation (Werness et al., 1990). Wild-type p53
seems to negatively regulate cell growth and division,
but overproduction or mutated p53 protein inhibits
wild-type p53, either by complexing with normal p53
The abbreviations used are: HCC, hepatocellular carcinoma,
PBS, phosphate-buffered saline solution; PCR, polymerase
chain reaction.
© 1991 The Korean Society of Molecular Biology
492
Mutations of p53 Gene in Hepatoma
protein or by competitive inhibition (Reich and Levine, 1984). These recent studies suggest that mutated
p53 not only relieves the tumor suppressor effect of
wild-type p53 but also exerts a direct oncogenic effect
(Marshall, 1991). In tumour cells, there was an increase in GI phase and then a steady elevated or slightly increasing level throughout Sand G2M phases.
Also, aneuploid populations showed a uniform high
p53 content (Morke and Didrik, 1991). Although it
is .not a specific property of malignant cells, a high
level is strongly suggestive of malignancy, and combined with DNA measurement could increase the possibility of discriminating between normal and malignant
cells in tumor tissue.
The authors also measured the DNA content and
p53 gene product by using flow cytometer after staining the nuclei from HCC tissue by propidium iodide
and fluorescein isothiocyanate-labeled monoclonal antibodies which is specific for p53 gene product.
Materials and Methods
DNA Preparation
Paraffin-embedded tissue HCC samples were obtained from Chonbuk University. The samples were extracted with histoclear or xylene to remove paraffin
and were rehydrated in a sequence of decreasing ethanol concentrations. The tissue was submerged in distilled water overnight. After discarding distilled water,
3 ml of 0.5% pepsin solution was added and incubated
for I h at 37 °C, minced to tiny pieces with surgical
blades. The resulting solution was then filtered through a 40 IJIIl nylon mesh, centrifuged, and washed
in phosphate-buffered saline solution (PBS). The samples were dried under vacuum and added with digestion buffer (50 mM Tris-HC1, pH 8.5, 1 mM EDTA,
plus 0.5% Tween 20) containing 200 fll/ ml proteinase
K The samples were incubated for 3 h at 55 °C and
then at 95 °c for 8 min to inactivate the protease.
Any residual tissue pellet was removed by centrifuging
f~r about 30 sec and collected supernatant was used
for PCR.
Polymerase chain reaction
Amplification took place in 25-fll reaction mixtures
containing I fll (100 ng) of prepared supernatant in
50 mM Tris-HCl (PH 8.3), 3 mM MgCh, 20 mM KCl,
250 f.1g borine serum albumin, each primer (P3 and
R3) at 0.25 M, each dNTP at 200 /lM, and 2 units
of Taq polymerase (KIST). The oligonucleotide primers were purchased from KIST. PCR reactions were
carried out for 31 cycles which consisted of 94 °C (1
sec), 55 °C (1 sec), and 72 °c (5 sec). After the last
cycle, the sample was left at 72 °c for an _additional
10 sec to ensure that the PCR products are fully double stranded.
Analysis of mutation site
Amplified PCR products were extracted with phenol, chloroform and precipitated with ethanol. After
Mol. Cells
the sample was dried under vacuum and resuspended
in distilled water, it was digested with HaeIII for 1
h at 37 °C. The digested products were loaded onto
2% Seakem agarose (FMC) gel contating 5 f.1g/ml ethidium bromide in 1X TBE buffer. q>X174/RF HaeIII
fragments (NEB) were used as size markers.
Analysis of DNA ploidity
Thin sections (5 f.1m) were deparaffinized in histoclear and rehydrated through a sequence of ethanol
changes. The deparaffinized tissues were washed with
distilled water twice. After discarding distilled water,
3 ml of 0.5% pepsin solution was added and incubated
for 1 h at 37 "c, minced to tiny pieces with surgical
blades. The resulting solution was then filtered through a 4O-1JIIl nylon mesh, centrifuged, and washed
in PBS. After washing in PBS, 100 fll RNase I (I
mg/ml, Sigma) was added and the nuclei were resuspended in staining solution (propidium iodide (PI)
25 f.1g/ml in PBS, 1 X 106 cells). The samples were
analysed on an FACstar (Beckton Dickinson). The
laser excitation wavelength was 488 nm at 200 mW.
Green (FITC) and red (PI) fluorescence were separated by a 560 nm dichroic mirror. In addition, the
green and red photomultiplier tubes (PMT) were guarded by a 530 nm bandpass and a 630 nm longpass
filter, respectively. Spectral overlap between green and
red signals was compensated by using the subtraction
unit of the flow cytometer. At least I X 105 cells were
counted for each sample.
Analysis of p53 oncoprotein produced
Prior to immunostaining, nuclear suspensions were
prepared from paraffin-embedded tissues. The primary
monoclonal antibodies (diluted to a final concentration of 2.5 f.1g antibody/20 fll) PAbl801 I, II, and III
were added separately in the cell suspension (approximately 5 X 105 nuclei) in PBS with 0.5% BSA. The
suspension was incubated for 30 min in a refrigerator.
Following incubation, the cells were washed once in
PBS with 0.1% Triton X-lOO, resuspended in 50 fll
FITC-conjugated goat anti-human IgG, dilute 1:20, incubated for 30 min at room temperature. After incubation, they were again washed with PBS/Triton and
resuspended in PBS. The samples were analysed on
flow cytometer by the same method as the one in
analysis of DNA ploidity.
Results
DNA content analysis of HCC samples
Aneuploidy is almost always found in many human
cancers, the most common cancer-related genetic change known as point mutations, allelic loss, rearrangements, deletion, and insertions. These aberrations, together with alterations of oncogenes and other tumour
suppressor genes, make up the mutational network
leading to malignancy. Figure I shows representative
DNA histograms demonstrating a typical normal
DNA diploidy (panel A), a tumour with a DNA
Vol. 1 (1991)
Bok-Soo Lee et af.
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Figure 1. DNA content of nonnal and cancer cells. Nuclei were prepared from paraffin-embedded tumor tissues and stained
with propidiun iodide. DNA content of each cell was measured by flow cytometer at the wavelength of 488 nm. A, diploid
from nonnal tissues; B, C, and D, aneuploid from cancer tissues.
aneuploidy containing hypoploidy (panel B), a abnormal DNA aneuploidy containing hyperploidy (panel
C), and a tumour with DNA aneuploidy containing
tetraploidy (panel D). About 60% of total HCC patients showed abnormal pattern in DNA analysis. In
the case of panel C, the number of S-phase cells increased. Abscissa represents fluorescence intensity
(DNA content) and ordinate shows the number of
cells. The results of this analysis represent that changes of DNA content have influenced on malignancy
of HCC samples.
Analysis of mutant p53 protein in HCC tissues
Some of the tumours, there was an increase in p53
in G 1 phase and then a steady elevated or slightly
increasing level throughout Sand G 2M phases. Also,
aneuploidy populations showed a high mutant p53
protein content in Figure 2. Panel A shows negative
control, and panel B shows nonspecific binding to
nuclear protein when only secondary antibody was
used. Panel C shows positive expression of specific
mutated p53 nuclear phosphoprotein. The monoclonal
antibody PAb 1801 used in this study reacts only with
human p53, recognizes a more stable epitope on the
p53 protein and might also detect product of mutated
p53 gene. The normal p53 protein is assumed to play
an important role in the regulation of cell cycle, and
the protein itself is strictly regulated with a very short
half-life. It is also known that quiescent and dividing
lymphocytes synthesize different forms of p53. The
change between these two forms occors at the GO-G 1
transition. Possi~ily, this reflects a structural or posttranslational change in the protein. In many transformed cell lines and other cancers, the steady state levels
of p53 are very high, and the tumorigenic efficiency
seems to be related to the extent of p53 overexpression. In the present study, almost all HCC samples
were p53 positive, and thus these results correlate with
other findings of p53 being associated to high-grade
malignancy.
Screening of point mutation on specific site in p53
gene
The p53 gene has a constant source of fascination
494
Mutations of p53 Gene in Hepatoma
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FLUORESCENCE INTENSITY
Figure 2. Typical pattern of nuclear protein P53 expression in Korean HCCs. The nuclei prepared from paraffin-embedded
tissue were stained with monoclonal antibody PAb 1801 , which recognizes human p53 mutant. This figure shows that tumor
cells produce the p53 mutant. A, negative control; B, only secondary antibody used; C, PAb-II + secondary antibody
used.
since its discovery nearly a decade ago. Most tumours
with allelic deletions contain p53 point mutations resulting in amino-acid substitutions. Such p53 gene
mutations are clustered in four hot-spots which exactly coincide with the four most highly conserved regions of the gene, reflecting its functional importance
and selection in the p53 protein in the development
of many common human malignancies. These domains and exons 5-8 are frequently the sites of mutation. · Recently, in an area of high incidence of hepatitis B virus, and aflatoxin B which is both a mutagen
(inducing G to T substitution) and a liver-specific carcinogen, the high mutation frequency (50%) was observed at a specific site in exon 7 in hepatocellular carcinoma patients. This indicates that this position is
a hot spot associated with HCC development in this
geographic region. For this reason, p53 gene was amplified in the exon 7 region including codon 249
known as specific mutation site, using polymerase
chain reaction. Amplified products were of 110 base
pairs and digested with restnctlOn enzyme HaeIII
which specifically recognizes the wild type codon 249
sequence. Because the mutated codon 249 is not recognized by this enzyme, two different types of this region could be detected.
Figure 3 shows results from Korean HCC samples.
All amplified products had 11O-bp fragments and were
digested with HaeIII yielding 75-bp and 35-bp fragments. Lanes A and E represent size markers pBR322
/BstNI and X174RF/HaeIII. Lane B shows results from
normal tissues. Lanes C, D and E show results from
HCC tissues. Of these lanes, the left line is the amplified product and the right one is the digested products. These results indicate that HCC patients in Korea have no specific point mutations on codon 249
of exon 7 in p53 gene.
Relation to p53 mutations and DNA content in Korean
hepatocellular carcinomas
Table 1 shows the summarized data of mutations
Vol. 1 (1991)
Bok-Soo Lee et al.
A B
c
Discussion
D
E
bp
1,857
1,060
383
121
Figure 3. Restriction enzyme analysis of nonnal tissue, hepatocellular carcinoma tissue and tumor cell line DNAs demonstrates that codon 249 mutations did not occur. DNAs were
amplified by PCR using synthetic oligonucleotide primers,
which can specifically recognize DNA segment of exon 7
of p53 gene. The amplified products of 110 bp were digested
with HaeIII and then separated on agarose gels. All HaeIIIdigested tumor and non-tumor DNAs yield 75- and 35-bp
fragments. A, pBR322/BstNl size markers; B, nonnal tissue;
C, HCC tissue; D, HepG2; E, <l>X174RF/HaeIII size markers.
Table 1. p53 mutations and DNA contents in Korean hepatocellular carcinomas
Patient
Age
Sex
p53 mutation
DNA ploidy
I
2
3
4
63
49
35
53
WT"
WT
WT
WT
S
SI
M
M
M
M
M
M
M
M
Aneuploid
Aneuploid
Aneuploid
Aneuploid
Aneuploid
Aneuploid
Aneuploid
Aneuploid
Aneuploid
48
6
7
54
8
58
Cell line (HepG2)
WT
WT
WT
WT
495
aWT indicates the wild-type.
on specific site in p53 gene and DNA content of Korean hepatocellular carcinomas. All samples have wild
type at the third base of codon 247 on exon 7 in
p53 gene, and the nuclei represent the chromosomal
abnormality and the expression of mutant p53 protein.
Although dietary exposure to hepatitis B virus and
aflatoxin B is an epidemiologically defined risk factor
for HCC in Southern Africa and China, this hot spot
mutation was not found among HCC patients in Korea.
The current understanding of p53 in cancer is that
overproduction of a mutated p53 protein inhibits wildtype p53, either by cOIl1plexing with normal p53 protein or by competitive inhibition, but its precise function is unknown (Banks et aI., 1986; Eliyahu et al.,
1985). Mutant forms of the murine p53 clones can
immortalize primary rat embryo fibroblast cells and
cooperate with an activated ras oncogene to transform
cells. They have transforming activity and are transdominant over wild-type p53. The trans-dominant
phenotype of the mutated p53 protein may be explained by its ability to oligomerize with wild-type p53,
drawing it into this complex and effectively inactivating it (Halevy et ai., 1990; Herskowitz, 1987). The SV
40 large T antigen and adenovirus ElB 55-kDa proteins form complexes with p53 protein resulting in
increased half-life, presumably also inactivating its normal function \" as a negative regulator of cellular growth. This change in conformation is associated with
enhanced protein stability (Levine et aI., 1991; Gannon
et al., 1990).
There is evidence for both dominant loss-of-function
mutations in transformed cells and gain-of-function
mutations in tumourigenesis assays. The two are not
mutually exclusive. Overexpression of the wild-type
protein with mutant p53 protein or other oncogene
products suppresses transformation, cell growth, and
the tumourigenic potential of the cells. Thus, the ratio
of mutant to wild-type p53 in a cell could be critical
in regulating cell division. p53 would negatively regulate entry into S phase. The p53 protein also associates
with the viral DNA replication complexes in cells infected with herpesvirus. Perhaps p53 acts in the late
G 1 phase of the cell cycle to promote or prevent the
assenmbly of a DNA replication-initiation complex
(Diller et aI., 1990; Raycroft et al., 1990).
Alternatively, p53 could act as a transactivator of
gene transcription, either promoting or repressing messenger RNA synthesis. Some mutations that activate
transformation affect gene transcription. Transformation-activating mutations alter the DNA-binding abilities of p53. It now needs to be determined whether
the p53 DNA-binding region recognizes a specific
DNA sequence. Thus p53 could have a role in regulating gene transcription, perhaps of a set of genes that
effect the passage from late G 1 to S phase of the cycle
(Finlay et aI., 1989; Marshall, 1991). Sequencing of the
p53 gene in mammals, amphibians, birds, and fish
has revealed five highly consezved dimains, four of
which fall within exons 5 through 8. Domains 3, 4,
and 5 are included in the two binding regions for
SV40 large T antigen (Werness et al., 1990). The numerous amino acids that presumably alter the biological function of the p53 protein when substituted are
dispersed among four conserved domains as well as
among intervening sequences, suggesting that no single domain is responsible for maintaining p53 tumour suppressor function. Integration of hepatitis B
496
Mutations of p53 Gene in Hepatoma
virus DNA is a frequent event associated with HCC.
And the mutagen aflatoxin B1, which is the main
aflatoxin species found in foods in Africa and China,
binds preferentially to G residues in G+C-rich regions and induces G to T substitutions almost exclusively (Bressac et ai., 1991 ; Hsu et ai., 1991). Thus, aflatoxin B1 is a potent hepatocarcinogen in different species and dietary exposure to it is an epidemiologically
defined risk factor for HCC, so it is possible that p53
mutations caused by aflatoxins or other environmental
carcinogens might contribute to the high incidence
of HCC in these areas. p53 gene mutations in HCCs
were G to T transversions in seven of the 16 tumours
in individuals from the region of Qidong, China, all
at the third base pair position of codon 249. The mutation was not present in non-malignant cells of these
individuals. Four out of 10 HCCs in individuals from
a different population at high risk of liver cancer
(Southern Africa) also contained G to T transversions,
three of which were at codon 249. Other than the
clusters of transitions at rare CpG dinucleotide sites,
p53 mutations in most human cancers are dispersed
over the midregion of the coding sequence (Finlay
et al., 1989; Nigro et al., 1989; Hollstein et al., 1991).
They found that 12 of 26 HCCs examined from two
high-risk groups contained a mutation at the same
codon, and other mutations as well in some cases.
And so far, several oncogenes and tumour suppressor
genes have been identified in a mutant form in human cancers (Nigro et ai., 1989; Hunter, 1991). The
number of such genes will continue to grow, and it
is also possible that genes will be identified to alter
cell growth when perturbed by completely different
mechanisms. Determining how these combinations disturb cellular growth control should enable us to understand the vast diversity of cancers.
In this study, a point mutation at the specific site
(the third base of codon 249 of exon 7) was not found
in the p53 gene. These results are quite different from
those which showed point mutations at the specific
site at the frequency of 50 percents from Chinese and
South African patients, but coincide with the results
in 22 HCCs from Japanese patients. Even though the
point mutation was not found, there might be mutations at other sites of p53 gene because expression
of mutated p53 protein was detected by using monoclonal antibodies which are specific for mutant-type
p53 protein and because DNA content was changed
in the hepatocellular carcinoma samples. So, other
mutations will be searched by sequencing of various
amplified PCR products.
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