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Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
Int. J. Cancer: 82, 187–190 (1999)
r 1999 Wiley-Liss, Inc.
DIFFERENT FREQUENCIES OF p53 CODON-249 HOT-SPOT MUTATIONS
IN HEPATOCELLULAR CARCINOMAS IN JIANG-SU PROVINCE OF CHINA
Yasuhito SHIMIZU,1 Ji-Jiang ZHU,1 Fang HAN,2 Takatoshi ISHIKAWA1 and Hideaki ODA1*
1Department of Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
2Department of Pathology, Nanton Cancer Hospital, Nanton, People’s Republic of China
Environmental carcinogens often induce specific mutations
in the p53 gene, apparent in tumors. The relation between
aflatoxin B1(AFB1)-related hepatocellular carcinomas (HCCs)
and hot spot at codon 249 of the p53 gene has received a great
deal of attention, but its significance is still controversial. To
clarify this problem, we analyzed the p53-mutational status of
HCCs in Jiang-su province in China, where AFB1 contamination of the staple food significantly differs between the
northern and southern parts (prominent only in the latter),
while other conditions are quite similar. Background liver
status and mutations in exons 5 to 8 of p53 in a total of 31
cases were divided approximately equally between the 2
areas. In all, 15 tumors exhibited a total of 17 mutations in the
p53 gene; 9 cases from the southern part of the province had
the hot-spot mutation at codon 249 (9/16, 56%), but only one
case from the northern part (1/15, 8%). These results suggest
that AFB1 contamination may correlate with codon-249
mutations in HCC. Int. J. Cancer 82:187–190, 1999.
r 1999 Wiley-Liss, Inc.
Mutations of the p53 tumor-suppressor gene have been reported
in about 50% of human cancers (Hollstein et al., 1991; Hussain and
Harris, 1998). Although they are observed in the highly conserved
region of p53, the pattern and exact positions vary with the type of
cancer. It is well known that environmental carcinogens can induce
specific mutations in this gene. For example, skin cancers related to
ultraviolet-light exposure demonstrate transition mutation at dipyrimidine sites (Brash et al., 1991; Tornaletti et al., 1993). Lung
cancers in cigarette-smoking patients are closely correlated to
G-to-T transversions (Takeshima et al., 1993). In hepatocellular
carcinomas, p53 mutations vary in different geographic regions,
probably due to differences in etiological factors. However, a hot
spot at codon 249 has been reported in HCCs from Qidong in China
(Hsu et al., 1991), South Africa (Bressac et al., 1991) and Senegal
(Coursaget et al., 1993). This hot spot has been considered to be
associated with dietary contamination by aflatoxin B1 (AFB1) in
these regions. HCCs from other areas such as Taiwan (Sheu et al.,
1992), Thailand (Hollstein et al., 1993) and Mexico (Soini et al.,
1996) show a mutation at codon 249 at low frequency while in
HCC in Europe, Japan and other areas without AFB1 contamination this type of mutation is rare (Kress et al., 1992; Murakami et
al., 1991; Challen et al., 1992). Furthermore, Aguilar et al. (1993)
reported that human hepatocytes exposed to AFB1 in vitro showed
the same G-to-T mutation at codon 249 of the p53 gene as already
observed in HCCs.
However, there are some inconsistent data. None of the HCCs
induced by AFB1 in non-human primates showed p53 mutations at
codon 249 (Fujimoto et al., 1992) and hyperplastic nodules caused
by this carcinogen in the rat liver exhibited no specific p53
mutations (Hulla et al., 1993). Furthermore, Hsieh and Atkinson
(1995) reported that the level of AFB1-DNA adducts does not
correlate with p53 mutations at codon 249. Thus, the relation
between the codon 249 hot spot in HCC and AFB1 contamination
in the diet remains controversial.
From the molecular-epidemiological point of view, it is interesting to compare the p53-mutational status of HCCs in a restricted
area with the same climate, same ethnicity and similar lifestyle
except for AFB1 contamination. In Jiang-su province of China
(Fig. 1), people living in Qidong, Haimen and Nanton, in the
southern part, consume corn as their staple food, while those in the
northern part have eaten rice for a long time. AFB1 contamination
has been observed only in corn (Armstrong, 1980; Yu, 1995). We
therefore focused attention on the 2 areas of Jiang-su province, and
analyzed the p53-mutational status of HCCs developing in this
inhabitants. Comparison of the northern and southern parts provided evidence that hot-spot codon-249 mutations may correlate
with AFB1 contamination.
MATERIAL AND METHODS
Patients
A total of 31 HCC cases was analyzed. Patient details are
summarized in Table I. According to the patient’s addresses, 15
cases (1 to 15) were from the northern part of Jiang-su province and
the other 16 (16 to 31) from the southern part. Ages of the patients
ranged from 35 to 67, with an average of 45. Although information
on virus infection was limited, 5 out of the 15 northern patients and
12 of the 16 southern patients were HBs-antigen-positive. Almost
all the patients evidenced features of chronic hepatitis or cirrhosis
as a background liver status.
DNA preparation, PCR and sequencing
All HCC materials were resected surgically in Nanton Cancer
Hospital. DNA was extracted from formalin-fixed paraffinembedded tissues as described by Oda et al. (1995). Briefly, 10
serial sections (10 µm thick) from paraffin blocks were attached to
glass slides. The first and last sections were stained with hematoxyline-eosin for making sure of tumor parts and non-tumorous liver
tissues under a microscope. From the remaining 8 sections, we
separated each part and put into tubes with xylene. After elimination of paraffin in this way, xylene was replaced by ethanol, and the
samples were dried followed by digestion in lysis buffer with
proteinase K (50 mM Tris-HCl, pH 8.0, 0.5 mg/ml of proteinase K)
at 55°C for 24 hr. Genomic DNA was precipitated with ethanol
after phenol-chloroform extraction and dissolved into TE buffer.
All possible precautions were taken to avoid contamination. Tumor
tissue and adjacent non-tumorous liver tissues were examined in all
cases except case 1, for which normal materials were not available.
Using the genomic DNA, exons 5 to 8 of the p53 gene were
amplified independently by the polymerase chain reaction (PCR).
The primers used have been described (Oda et al., 1995). Genomic
DNA (0.5 µg) was dissolved in a total volume of 50 µl of solution
containing 1 ⫻ PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl,
1.5 mM MgCl2, 0.001% gelatin (w/v)), 0.2 mM of dNTP, 1 µM of
each primer and 2.5 of Taq polymerase. After 40 cycles of PCR, the
products were sub-cloned into the EcoRV site of pBluescript
(Pharmacia, Uppsala, Sweden).
Sequencing was performed for at least 50 sub-clones of mixed
recombinant colonies, by the dideoxy ribonucleotide chain termina-
Grant sponsors: Ministry of Education, Science, Sports and Culture,
Japan (International Research Program, Special Cancer Research); Smoking Research Foundation.
*Correspondence to: Department of Pathology, Graduate School of
Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 1130033, Japan. Fax: (81) 3-5812-3346. E-mail: [email protected]
Received 26 November 1998; Revised 1 February 1999
SHIMIZU ET AL.
188
tion method with a T7 sequencing kit (Pharmacia). When a
mutation was identified, the PCR, sub-cloning and sequencing
were repeated using the original genomic DNA to confirm the
results.
RESULTS
Exons 5 to 8 of the p53 gene of 31 cases of HCC were amplified
and sequenced. As shown in Table I, 15 cases (48%) showed
mutations of the p53 gene. A total of 17 mutations were observed,
all point mutations of the mis-sense type, except for 2 non-sense
mutations (cases 13 and 29). Double mutations were observed in 2
cases, otherwise they were single. Out of 17 mutations, 10 were
clustered at codon 249 of exon 7, being G-to-T transversions at the
third letter of the codon (Fig. 2). The other 7 mutations comprised 2
transitions and 5 transversions. In no cases were there mutations in
the normal liver tissues.
A close correlation between codon-249 mutation and the patients’ residential area was observed. As shown in Table I and
Figure 3, cases with codon-249 mutation were mainly from the
southern part of the Jiang-su province, living in Haimen, Qidong
and Nanton along the Yangsu river. In this area, 9 out of 16 cases
(56%) showed this hot-spot mutation. On the other hand, among
FIGURE 2 – Typical sequencing results showing a point mutation at
the 3rd base of codon 249. Cases 21 and 26 are shown with a normal
sequencing result.
FIGURE 1 – Location of Jiang-su province in China.
TABLE I – PATIENTS’ DATA AND RESULTS OF p53-GENE MUTATION ANALYSIS
Case
number
Age/gender
Address
HBsAg
1
2
3
4
5
6
7
8
9
10
11
12
13
43 M
48 M
65 M
38 F
52 M
67 M
39 M
51 M
50 M
56 M
49 M
39 M
40 M
Haian county
Jinsha city
Haian county
Tai county
Jinsha city
Rugao county
Jinsha city
Rugao city
Dafeng county
Sheyang county
Tai county
Rugao county
Rugao county
unknown
⫺
⫺
⫺
⫹
⫺
⫹
unknown
unknown
unknown
unknown
⫹
⫹
14
15
39 M
48 M
Rugao county
Rugao county
unknown
⫹
16
17
18
19
20
21
22
23
24
25
26
27
28
29
40 M
50 M
41 F
47 M
35 F
37 M
56 M
41 F
43 M
38 M
51 F
40 F
37 M
35 M
Qidong city
Qidong city
Nanton city
Nanton city
Haimen city
Haimen city
Haimen city
Haimen city
Haimen city
Haimen city
Haimen city
Haimen city
Haimen city
Haimen city
⫹
unknown
⫹
unknown
⫹
⫹
⫹
⫹
⫺
⫹
⫺
⫹
⫹
⫹
30
31
39 M
57 M
Haimen city
Haimen city
⫹
⫹
Codon (exon)
Oligonucleotide change
Amino-acid change
Background liver status
(liver cirrhosis)
Northern part
278 (8)
159 (5)
CCT = CTT
GCC = GTC
Pro = Leu
Ala = Tyr
166 (5)
249 (7)
TCA = TAA/TCA
AGG = AGT
Ser = stop
Arg = Ser
218 (6)
GTG = GCG
Southern part
249 (7)
AGG = AGT
Val = Ala
Arg = Ser
249 (7)
278 (8)
AGG = AGT
CCT = CTT/CCT
Arg = Ser
Pro = Leu
249 (7)
249 (7)
AGG = AGT/AGG
AGG = AGT/AGG
Arg = Ser
Arg = Ser
157 (5)
249 (7)
249 (7)
GTC = TTC
AGG = AGT/AGG
AGG = AGT
Val = Phe
Arg = Ser
Arg = Ser
166 (5)
249 (7)
249 (7)
249 (7)
TCA = TAA/TCA
AGG = AGT/AGG
AGG = AGT/AGG
AGG = AGT
Ser = stop
Arg = Ser
Arg = Ser
Arg = Ser
not available
⫺
⫺
⫺
⫹
⫺
⫹
⫹
⫺
⫺
⫺
⫹
⫺
⫺
⫹
⫹
⫺
⫹
⫹
⫺
⫹
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫹
p53 CODON-249 MUTATIONS IN HCC IN CHINA
189
FIGURE 3 – Relation between HCC patients’ place of residence in Jiang-su province and p53-mutation status.
the cases from the northern part of the province, only one case out
of 15 (8%) showed the codon-249 mutation.
DISCUSSION
The significant finding of this study is the difference in frequency
of codon-249 mutations in HCCs between the northern and the
southern part of the Jiang-su province. Inhabited by the same ethic
group, with a similar way of life and a similar climate, the 2 areas
had marked variation in their p53 status, which correlated with the
dietary aflatoxin contamination. Thus, the results were in direct
agreement with the high levels of AFB1-albumin adducts detected
in the serum of residents in this southern part (IARC, 1993) and the
low levels in the northern part (Yu et al., 1989). More specifically,
they support the idea that AFB1 causes codon-249 mutations of the
p53 gene observed in HCCs.
Fujimoto et al. (1994) reported a difference in p53-mutation
patterns in HCC patients from Qidong and Beijing. However, with
the distance between these areas, people have different ways of life
and the climate is very different. We here report the p53-mutation
status of HCCs occurring in a restricted small area in China. A
similar study described by Ozturk et al. (1991) for HCCs in
southern Africa showed significant different frequencies of codon249 mutations in HCCs between Mozambique and South Africa.
Our report confirms and extends the validity of these earlier
reports. Since various factors are associated with carcinogenesis
and the mutational pattern of important genes, similarity of patient
background is important.
There are some findings contrary to the idea that AFB1-induced
hepatocarcinogenesis is related to specific mutations in the p53
gene (Fujimoto et al., 1992; Hulla et al., 1993). However, such
observations were made using animal cells or experimental carcinogenesis in non-human primates or rats. Although the region
harboring codon 249 is conserved among various species, the
nucleotide sequence is not identical in human and in animal p53,
one possible reason why codon-249 mutations are not observed in
aflatoxin-induced liver tumors in animals. Experimental animal
models are important for understanding human disease and elucidating mechanisms, but genetic changes are not necessarily shared
with humans. Consequently, data from human material are necessary for various reasons to obtain the definite results. Although the
present study suffered from insufficient data regarding virus status
and AFB1 blood levels, it did point to a pronounced relation
between AFB1 exposure and p53 hot-spot mutations in HCCs.
With regard to other etiological factors, hepatitis-B-virus infection may be important for the patients in this area (Campbell et al.,
1990), since their average age (45 years old) was relatively young
and HBsAg positivity was high. Furthermore, HCCs in this area
appear frequently in non-cirrhotic liver (cirrhosis present in only
44%). Thus, HCC patients may be exposed to HBV infection in
early life. Furthermore, since cases from the southern part have
higher positivity in HBsAg than those from the north (Table I),
HBV infection might have played some role in the generation of
codon-249 hot-spot mutations of p53.
ACKNOWLEDGEMENTS
We thank Dr. Shun-Zang Yu (School of Public Health, Shanghai
Medical University) and Dr. Shinkan Tokudome (Department of
Public Health, Nagoya City University) for helpful discussions.
190
SHIMIZU ET AL.
REFERENCES
AGUILAR, F., HUSSAIN, S.P. and CERUTTI, P., Aflatoxin B1 induces the
transversion of G to T in codon 249 of the p53 tumor suppressor gene in
human hepatocytes. Proc. nat. Acad. Sci. (Wash.), 90, 8586–8590 (1993).
ARMSTRONG, B., The epidemiology of cancer in the People’s Republic of
China. Int. J. Epidemiol., 9, 305–315 (1980).
BRASH, D.E., RUDOLPH, J.A., SIMON, J.A., LIN, A., MCKENNA, G.J., BADEN,
H.P., HALPERIN, A.J. and PONTEN, J., A role for sunlight in skin cancer:
UV-induced p53 mutations in squamous cell carcinoma. Proc. nat. Acad.
Sci. (Wash.), 88, 10124–10128 (1991).
BRESSAC, B., KEW, M., WANDS, J. and OZTURK, M., Selective G to T
mutations of p53 gene in hepatocellular carcinoma from southern Africa.
Nature (Lond.), 350, 429–431 (1991).
CAMPBELL, T.C., CHEN, J., LIU, C., LI, J. and PARPIA, B., Nonassociation of
aflatoxin with primary liver cancer in a cross-sectional ecological survey in
the People’s Republic of China. Cancer Res., 50, 6882–6893 (1990).
CHALLEN, C., LUNAC, J., WARREN, W., COLLIER, J. and BASSENDINE, M.F.,
Analysis of the p53 tumor-suppressor gene in hepatocellular carcinomas
from Britain. Hepatology, 16, 1362–1366 (1992).
COURSAGET, P., DEPRIL, N., CHABAUD, M., NANDI, R., MAYELO, V., LECANN,
P. and YVONNET, B., High prevalence of mutations at codon 249 of the p53
gene in hepatocellular carcinomas from Senegal. Brit. J. Cancer, 67,
1395–1397 (1993).
FUJIMOTO, Y., HAMPTON, L.L., LUO, L.D., WIRTH, P.J. and THORGEIRSSON,
S.S., Low frequency of p53 gene mutation in tumors induced by aflatoxin
B1 in nonhuman primates. Cancer Res., 52, 1044–1046 (1992).
FUJIMOTO, Y., HAMPTON, L.L., WIRTH, P.J., WANG, N.J., XIE, J.P. and
THORGEIRSSON, S.S., Alterations of tumor suppressor genes and allelic
losses in human hepatocellular carcinomas in China. Cancer Res., 54,
281–285 (1994).
HOLLSTEIN, M., SIDRANSKY, D., VOGELSTEIN, B. and HARRIS, C.C., p53
mutations in human cancers. Science, 253, 49–53 (1991).
HOLLSTEIN, M.S., WILD, C.P., BLEICHER, F., CHUTIMATAEWIN, S., HARRIS,
C.C., STRIVATANAKUL, P. and MONTESANO, R., p53 mutations and aflatoxin
b1 exposure in hepatocellular-carcinoma patients from Thailand. Int. J.
Cancer, 53, 51–55 (1993).
HSIEH, D.P.H. and ATKINSON, D.N., Recent aflatoxin exposure and mutation
at codon 249 of the human p53 gene: lack of association. Food Addit.
Contam., 12, 421–428 (1995).
HSU, I.C., METCALF, R.A., SUN, T., WELSH, J.A., WANG, N.G. and HARRIS,
C.C., Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature (Lond.), 350, 427–428 (1991).
HULLA, J.E., CHEN, Z.Y. and EATON, D.L., Aflatoxin B1-induced rat hepatic
hyperplastic nodules do not exhibit a site-specific mutation within the p53
gene. Cancer Res., 53, 9–11 (1993).
HUSSAIN, S.P. and HARRIS, C.C., Molecular epidemiology of human cancer:
contribution of mutation spectra studies of tumor suppressor genes. Cancer
Res., 58, 4023–4037 (1998).
INTERNATIONAL AGENCY FOR RESEARCH ON CANCER. IARC monographs on
the evaluation of the carcinogenic risk of chemicals to humans. Some
naturally occurring substances: food items and constituents, heterocyclic
aromatic amines and mycotoxins. IARC Monograph 57, 309–395 (1993).
KRESS, S., JAHN, U.R., BUCHMANN, A., BANNASCH, P. and SCHWARZ, M.,
Mutations in human hepatocellular carcinomas from Germany. Cancer
Res., 52, 3220–3223 (1992).
MURAKAMI, Y., HAYASHI, K., HIROHASHI, S. and SEKIYA, T., Aberrations of
the tumor-suppressor p53 and retinoblastoma genes in human hepatocellular carcinomas. Cancer Res., 51, 5520–5525 (1991).
ODA, H., NAKATSURU, Y. and ISHIKAWA, T., A mutational hot spot in the p53
gene is associated with hepatoblastomas. Int. J. Cancer, 60, 786–790
(1995).
OZTURK, M. and OTHERS, p53 mutations in hepatocellular carcinoma after
aflatoxin exposure. Lancet, 338, 1356–1359 (1991).
SHEU, J.C., HUANG, G.T., LEE, P.H., CHUNG, J.C., CHOU, H.I., LAI, M.Y.,
WANG, J.T., LEE, H.S., SHIN, L.N., YANG, P.M., WANG, T.H. and CHEN, D.S.,
Mutations of p53 gene in hepatocellular carcinoma in Taiwan. Cancer Res.,
52, 6098–6100 (1992).
SOINI, Y. and 16 OTHERS, An aflatoxin-associated mutational hot spot at
codon 249 in the p53 tumor suppressor gene occurs in hepatocellular
carcinomas from Mexico. Carcinogenesis, 17, 1007–1012 (1996).
TAKESHIMA, Y., SEYAMA, T., BENNETT, W.P., AKIYAMA, M., TOKUOKA, S.,
INAI, K., MABUCHI, K., LAND, C.E. and HARRIS, C.C., p53 mutations in lung
cancers from non-smoking atomic-bomb survivors. Lancet, 342, 1520–
1521 (1993).
TORNALETTI, S., ROZEK, D. and PFEIFER, G.P., The distribution of UV
photoproducts along the human p53 gene and its relation to mutations in
skin cancer. Oncogene, 8, 2051–2057 (1993).
YU, S.Z., Primary prevention of hepatocellular carcinoma. J. Gastroenterol.
Hepatol., 10, 674–682 (1995).
YU, S.Z., CHEN, Z.Q., LIU, Y.K., HUANG, Z.Y. and ZHAO, Y.F., The aflatoxins
and contaminated water in the etiological study of primary liver cancer. In:
S. Natori, K. Hashimoto and Y. Ueno (eds.), Mycotoxins and phycotoxins,
pp. 37–44, Elsevier, Amsterdam (1989).
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