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).