Analysis of the complete hepatitis B virus genome in patients with genotype C chronic hepatitis and hepatocellular carcinoma

Hepatitis B virus (HBV) genotype C and the basic core promoter (BCP) mutations were reported to be associated with the development of hepatocellular carcinoma (HCC). In this study the full sequences of HBV genomes were analyzed in order to find the other predictors of HCC development. We determined the full sequences of HBV genomes in 24 genotype C carriers who developed HCC (HCC group) at the beginning of follow-up and at the time of HCC diagnosis, and 20 patients who did not develop HCC (non-HCC group) served as a control. The number of nucleotide and amino acid substitutions in most regions was higher in the HCC group than in the non-HCC group, and the following substitutions and deletions were found more frequently in the HCC group than in the non-HCC group: G1317A and T1341C/A/G in the X promoter region were detected in 13 and six of the HCC cases, four and none of the non-HCC cases, respectively; and pre-S2 deletion was detected in eight HCC and none of the non-HCC cases. Compared with the wild type X promoter, the mutant type X promoters, M1 (G1317A), M2 (T1341C), and M4 (T1341G) showed increases in activity of 2.3, 3.8, and 1.4 times, respectively, in HepG2 cells. Substitutions and deletion of nucleotides of the HBV genome, especially the pre-S2 deletion and G1317A and T1341C/A/G mutations may be useful markers for predicting the development of HCC. ( Cancer Sci 2007; 98: 1921–1929)     

乙肝病毒相关的肝癌p16异常甲基化

Objective： To investigate the potential linkage between high rate of p16 methylation and hepatitis B virus （HBV） infection, methylation status of p16, HBV infection markers in serum and HBV-DNA replication level in cancerous and non-cancerous tissue of 32 cases of hepatocellular carcinomas （HCC） with HBV infection and 12 HCCs without HBV infection were examined. Methods： p16 methylation was detected with methylation-specific polymerase Chain reaction （PCR）, and HBV markers were examined with real-time PCR and immunologic method. Results： Methylation of p16 promoter was found in 31 （70.5%） of 44 cancerous tissues of HCC, 2 （16.7%） of 12 HCC without HBV infection, 29 （90.6%） of 32 HCCs with HBV infection marker, p16 methylation was detected in 5 （83.3%） of 6 HCCs positive for HBsAg and HBeAg, 17 （94.4%） of 18 HCCs positive for HBsAg and negative for HBeAg, 7/8 （87.5%） of HCCs positive for other HBV infection markers, such as HBsAB, HBcAb, HBeAb. p16 methylation products were also found in non-cancerous tissues of 4 cases of HCCs with HBV infection, not detected in non-cancerous tissues without HBV infection. HBV-DNA was detected in cancerous tissues of 29/32 （90%） HCCs with HBV infection. Surprisingly, Methylation product of p16 promoter was found in all cases （29/29） of HCCs with detectable HBV-DNA in neoplastic tissue. Conclusion： Persistent HBV infection may promote p16 hypermethylation, suggesting that HBV, via enhancing the aberrant methylation of p16, indirectly involved in development of HCC.     

Pre-S mutant surface antigens in chronic hepatitis B virus infection induce oxidative stress and DNA damage

Ground glass hepatocytes (GGHs) are the historic hallmarks for the hepatocytes in the late and non-replicative stages of hepatitis B virus (HBV) infection. We have identified type I and type II GGHs that contain two mutant types of large HBV surface antigens (HBsAg) with deletions over the pre-S1 and pre-S2 regions, respectively. These pre-S mutant HBVsAg accumulate in endoplasmic reticulum (ER), resulting in strong ER stress. Type II GGHs often appear in hepatic nodules in the late phases of HBV infection and proliferate in clusters, suggesting that these mutant pre-S1/S2 HBsAg may be involved in HBV-related hepatocarcinogenesis, associated with ER stress. In this study, we investigated the potential genomic instability imposed by pre-S mutant HBsAg. Based on the analysis of comet assays, we found that the pre-S1 and pre-S2 mutant HBsAg caused oxidative stress and DNA damage. The DNA repair gene ogg1 was greatly induced by over-expression of pre-S mutant HBsAg. Induction of the DNA repair gene ogg1 was also detected in the pre-S2 HBsAg transgenic mice, as well as the type II GGHs from patients with hepatocellular carcinoma, strongly suggesting that the pre-S mutant HBsAg contributes to the oxidative DNA damage to hepatocytes. In addition, the mutation rates in the X-linked hprt gene were enhanced in mouse hepatoma ML1-4a cells, which constitutively expressed the pre-S1/S2 HBsAg. These results indicate that pre-S1/S2 mutant HBsAg, which make up GGHs, induce oxidative DNA damage and mutations in hepatocytes in the late stages of HBV infection.     

TP53 R249S mutation, genetic variations in HBX and risk of hepatocellular carcinoma in The Gambia

In regions with high prevalence of chronic hepatitis B virus (HBV) infection and dietary aflatoxin B(1) (AFB(1)) exposure, hepatocellular carcinomas (HCCs) often contain TP53 mutation at codon 249 (R249S). Furthermore, a C-terminal truncated HBx protein expressed from hepatocyte integrated HBV is associated with HCC development. This study evaluates the association between R249S and HBX status in relation to HCC in West African population. HBX (complete or 3'-truncated) and HBS genes were assessed by PCR in cell-free DNA (CFDNA) from plasma of subjects recruited in a hospital-based case-control study (325 controls, 78 cirrhotic patients and 198 HCC cases) conducted in The Gambia. These samples had been previously analyzed for R249S and HBV serological status. Complete HBX sequence was frequently detected in CFDNA of HCC-R249S positive (77%, 43/56) compared with HCC-R249S-negative cases (44%, 22/50). Conversely, the proportion of 3'-truncated HBX gene was significantly higher in HCC-R249S negative than positive cases (34%, 17/50, compared with 12%, 7/56) (χ(2) = 12.12; P = 0.002; distribution of R249S negative and positive according to HBX status). Occult HBV infection (detected by PCR) was present in 24% of HCC previously considered as negative by HBV serology. Moreover, HBV mutation analysis revealed that double mutation at nucleotides 1762(T)/1764(A) was associated with diagnosis of cirrhosis or HCC {cirrhosis: odds ratio (OR): 9.50 [95% confidence interval (CI) 1.50-60.11]; HCC: OR: 11.29 [95% CI 2.07-61.47]}. These findings suggest that in HCC from The Gambia, complete HBX sequences are often associated with the presence of TP53 R249S mutation.     

HBV感染与肝癌组织CDKN2A基因甲基化关系的研究

目的:探讨HBV感染和CDKN2A异常甲基化之间的潜在关联及其相互作用在肝癌形成过程中的作用。方法:对44例肝癌患者癌组织及癌旁组织CDKN2A甲基化状况、血清HBV感染标志物等到进行了检测分析。血清HBV、HCV感染标志物采用全自动化学发光分析仪进行检测。采用酚、氯仿法抽提组织DNA,用CDKN2A甲基化特异性引物进行PCR扩增,并检测特异性产物。患者按HBV感染情况进行分组,统计分析。结果:CDKN2A甲基化产物在肝癌组织中的总检出率为70.5%(31/44)。其中,HBV相关肝癌组和非病毒相关肝癌组CDKN2A甲基化率为90.6%(29/32)和16.7%(2/12),P=0.000。对HBV相关肝癌进一步分组分析,HBsAg /HBeAg 、HBsAg /HBeAg-及HBcAb /HBcAb-三组,三组之间差异无统计学意义(P=0.568)。经非参数相关和logistic回归分析,发现:CDKN2A甲基化与性别、肿瘤分化程度无明显相关(R2=0.11,P=0.650),而与HBV感染,特别是HBsAg密切相关(R2=0.37,P=0.02)。结论:在肝癌形成过程中,HBV可能通过促进抑癌基因,如CDKN2A等的异常甲基化,使CDKN2A失活,肝细胞正常的生长调控机制失常,形成肝癌,这也可能是HBV导致肝癌形成的机制之一。     

Aberrant cyclin A expression and centrosome overduplication induced by hepatitis B virus pre-S2 mutants and its implication in hepatocarcinogenesis

Ground glass hepatocytes harboring hepatitis B virus (HBV) pre-S2 mutants have been recognized as pre-neoplastic lesions of hepatocellular carcinoma (HCC). The pre-S2 mutants accumulated in endoplasmic reticulum (ER) can induce ER stress, upregulate cyclin A and promote hepatocyte proliferation. Notably, cyclin A was aberrantly detected in the cytoplasm, instead of nucleus, of pre-S2 mutant-transgenic mice livers, thereby raising the potential role of cytoplasmic cyclin A in HBV hepatocarcinogenesis. In this study, we confirmed that cyclin A was detected in the cytoplasm in the majority of HBV-related HCC tissues. In vitro, the pre-S2 mutant-initiated ER stress could induce cytoplasmic cyclin A mediated via cleavage by the calcium-dependent protease μ-calpain, resulting in an N-terminal truncated product which was preferentially located in the cytoplasm. The aberrant cyclin A expression subsequently induced centrosome overduplication, and this effect was abolished by calpain-specific inhibitors or RNA interference targeting to cyclin A. Overall, our data indicate that HBV pre-S2 mutant may elicit aberrant cyclin A expression and centrosome overduplication through ER stress induction and thereby represent a potential mechanism for the chromosome instability in HBV hepatocarcinogenesis.     

Microsatellite instability mutator phenotype in hepatocellular carcinoma in non-alcoholic and non-virally infected normal livers

Microsatellite instability (MSI) seems to be a rare event in hepatocarcinogenesis and might actually be associated with the progression of hepatocellular carcinoma (HCC) in which the liver is often the site of chronic hepatitis or cirrhosis. The aim of this work was to define the MSI phenotype in HCC affecting exclusively normal livers to avoid slippage errors due to cirrhosis. One hundred and sixty-four patients with HCC affecting non-cirrhotic livers were operated on in our hospital between 1984 and 2001. We analyzed 37 patients selected for low alcohol consumption and the absence of HBV or HCV infection. All the livers were histologically normal. MSI was analyzed according to the criteria defined during the conference consensus workshop for colorectal cancer. High MSI (MSI-H > 30%) was found in 6 (16%) and low MSI (MSI-L < 30%) in 10 (27%) of the 37 HCCs. None of the 10 microsatellite markers tested were altered in the remaining 21 tumors (57%). Immunohistochemistry showed that normal amounts of hMLH1 and hMSH2 were present both in MSI-H and in MSI-L HCCs. MSI-H was significantly associated with more aggressive histological tumor features and a shorter median delay before recurrence. Thus, we have found a small subgroup of HCC tumors which can be considered as a new clinical/histological entity.     

Relationship between serum and histochemical markers for hepatitis B virus and rate of viral integration in hepatocellular carcinomas in Japan

The relationship between serological and histochemical markers of hepatitis B virus (HBV) and viral integration in hepatocellular carcinomas (HCCs) in Japan was investigated. Special attention was paid to the exclusion of false-positive results due to bacterial contamination of autopsy materials. Of 85 patients with HCC, 23 were positive for serum HBsAg, 18 were positive for serum HBV antibodies and the remaining 44 were negative for serum HBV markers. Among the 23 HCCs from HBsAg carriers, 19 (82.6%) had integrated HBV DNA in the tumor DNA. In contrast, of the 18 HCCs from HBV antibody-positive patients, only one was positive for HBV DNA integration; this particular HCC was from a longterm HBsAg carrier who became a non-carrier by seroconversion at a later age. Of 44 HCCs from patients negative for serum HBV markers, 3 had integrated HBV DNA. Of these 3 cases, 2 were, however, histochemically positive for HBsAg in non-cancerous portions of the liver. Thus, our present results show a high integration rate of HBV DNA in HCCs of carrier patients and an extremely low rate of viral integration in HCCs of noncarrier patients, The latter finding contrasts strongly with previous results gained from analysing materials from patients in Europe and Africa. No histological differences were apparent between HCCs with or without viral integration.     

启东肝癌高发区乙肝病毒流行株全基因分析

目的：通过对江苏启东地区乙型肝炎病毒（HBV）全基因序列的分析，探讨该地区肝癌高发的分子病毒学病因。方法：以蛋白酶K消化后，酚／氯仿抽提7例肝炎和7例肝癌患者血清中DNA。应用聚合酶链反应（PCR），扩增血清中HBV基因全长，克隆至T载体后行全自动测序。以PHYLIP软件构建的系统进化树判断HBV的基因型；以Clastal W软件对序列进行突变分析。结果：14例标本中有12例为C基因型，2例为B基因型。肝癌和肝炎组中HBV基因型类别分布无差异。有5例HBV发生了PreS2的缺失突变，其中肝癌标本4例（57．1％），肝炎标本仅1例（14．3％）。HBV基因组中常见的点突变为PreS1区的nt．3116及核心启动子区的nt．1762／1764，发生率高达78．6％（11／14）。与肝炎组相比，肝癌中点突变发生率显著增高的位点为前C区nt．1899G→A的突变（P=0．01）及核心启动子区nt．1653C→T的突变（P=0．05）。结论：启东地区HBV以C基因型为主；启东HBV基因组中可能存在着与肝癌相关的点突变和缺失突变。     

Molecular signature in three types of hepatocellular carcinoma with different viral origin by oligonucleotide microarray

Chronic infection with hepatitis B or C virus (HBV or HCV) is the most clearly established risk factor for hepato-cellular carcinoma (HCC). One type of HCC (non-B, non-C HCC) also appears to develop in patients negative for both HBV and HCV. Using a supervised learning method, we investigated gene expression in 11 non-B, non-C HCCs with high-density oligonucleotide microarrays, and compared the patterns of gene expression with those of HBV-infected HCCs (B-type HCCs) and HCV-infected HCCs (C-type HCCs) in the previous dataset. Our gene selection identified 112 and 64 genes that were differentially expressed in non-B, non-C HCC in comparison with B- and C-type HCCs, respectively. In both gene selections, we found that the false discovery rate, the percentage of genes identified by chance, was less than 5%. Additionally, in combination with the previous data, our present data revealed a set of genes specific to each type of B- and C-type HCCs and non-B, non-C HCC. Among these, an interferon-induced gene, IFI27, was differentially expressed among all three types of HCCs, and this result was confirmed by RT-PCR. Thus, our present study provides a framework to characterize the molecular features in the three subtypes of HCC with different viral origin.     

MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma

The identification of target mRNAs is a key step for assessing the role of aberrantly expressed microRNAs in human cancer. MiR-221 is upregulated in human hepatocellular carcinoma (HCC) as well as in other malignancies. One proven target of miR-221 is CDKN1B/p27, whose downregulation affects HCC prognosis. Here, we proved that the cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 is also a direct target of miR-221. Indeed, downregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to miR-221 transfection into HCC-derived cells and a significant upregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to antimiR-221 transfection. A direct interaction of miR-221 with a target site on the 3' UTR of CDKN1C/p57 mRNA was also demonstrated. By controlling these two CDKIs, upregulation of miR-221 can promote growth of HCC cells by increasing the number of cells in S-phase. To assess the relevance of these studies in primary tumors, matched HCC and cirrhosis samples were assayed for miR-221, for CDKN1B/p27 and CDKN1C/p57 expression. MiR-221 was upregulated in 71% of HCCs, whereas CDKN1B/p27 and CDKN1C/p57 proteins were downregulated in 77% of cases. A significant inverse correlation between miR-221 and both CDKN1B/p27 and CDKN1C/p57 was found in HCCs. In conclusion, we suggest that miR-221 has an oncogenic function in hepatocarcinogenesis by targeting CDKN1B/p27 and CDKN1C/p57, hence promoting proliferation by controlling cell-cycle inhibitors. These findings establish a basis toward the development of therapeutic strategies aimed at blocking miR-221 in HCC.     

肝癌患者C型乙型肝炎病毒前S基因突变研究

目的探讨肝癌患者C型乙型肝炎病毒(HBV)前S(pre-S)基因的突变情况。方法自肝癌患者血清中提取HBV基因组DNA,扩增其pre-S基因片段并进行序列分析,用MEGA3.1和C lustalx1.8作为所测基因进行分型,并对比pre-S基因序列。结果所测样品中,C型占56.0%,B型占20.0%,B/C混合感染型占24.0%。肝癌患者C型HBV前S区普遍存在基因突变。肝癌患者C型HBV pre-S1区域以个体化的点突变为主,而pre-S2区域则存在大量的缺失突变。结论 Pre-S基因是肝癌患者C型HBV的高变区。     

广西乙肝病毒/黄曲霉毒素B1双暴露人肝细胞癌中PTEN mRNA表达及突变的初步研究

目的探讨乙肝病毒/黄曲霉毒素B1双暴露相关肝细胞性肝癌(HCC)中PTEN基因的突变特点及其mRNA的表达水平。方法 108例HCC来自广西不同地区样本,按照乙肝病毒与黄曲霉毒素的暴露情况,分为4个亚组:A组:HBV(+)/AFB1-DNA(+),共48例;B组:HBV(+)/AFB1-DNA(-),共27例;C组:HBV(-)/AFB1-DNA(+),共19例;D组:HBV(-)/AFB1-DNA(-),共14例。采用PCR联合基因直接测序法,检测108例肝癌组织中PTEN基因第4、5、8外显子的突变情况。同时采用RT-PCR法检测其基因mRNA的表达状况。结果①PTEN基因第4、5、8外显子测序结果未发现发生在外显子上的突变。但在第4外显子与第4内含子交界处有61例发生大片段的缺失,缺失率为56.4%。②PTEN缺失率在A、B、C、D组中分别为60.4%、62.9%、47.3%和46.6%,其差异在4个亚组间均无统计学意义(P>0.05)。③PTEN mRNA在4个亚组中的表达半定量灰度值分别为:A组:0.54±0.13;B组:0.59±0.16;C组:0.97±0.16;D组:0.92±0.13。其中,A、B组分别与C、D组相比,其差异均具有统计学意义(PAC=0.002,PAD=0.032,PBC=0.000,PBD=0.011)。结论在广西乙型肝炎病毒/黄曲霉毒素B1的双暴露肝细胞癌中,PTEN mRNA的表达下调是一个常见的分子生物学事件,PTEN mRNA表达下调可能主要与HBV感染有关。AFB1对PTEN mRNA的表达下调可能有协同作用。     

肿瘤-睾丸抗原SSX1和SSX4的mRNA在肝细胞肝癌中的表达

目的 检测肿瘤-睾丸抗原(Cancer-Testis antigens，CT)SSX1和SSX4基因mRNA在肝细胞肝癌(HCC)中的表达，探讨CT抗原SSX1 和SSX4 mRNA在HCC中表达有无特异性。 方法 应用逆转录-聚合酶链反应(RT-PCR)方法对35例HCC患者癌组织和相应的癌旁组织SSX1和SSX4基因表达进行检测，对 其中6例RT-PCR扩增产物的目的片段进行DNA序列测定。 结果 35例HCC患者中，SSX1和SSX4的表达率分别为81％(27／35)和73％(23／35)；癌旁组织、12例肝硬化和15例正常组织未 检测到SSX1和SSX4的表达。6例DNA序列测定结果显示RT-PCR产物为SSX1和SSX4基因的cDNA。SSX1和SSX4的表达与患 者年龄、性别、肿瘤大小、分化程度、血清甲胎蛋白(AFP)水平、乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)感染无显著相关性(P >0．05)。 结论 SSX1和SSX4在HCC中呈高特异性表达，是制备HCC疫苗理想的靶向分子，多种CT抗原联合表达为多效价疫苗治疗HCC 提供了理论基础。     

乙型肝炎病毒前S2区缺失突变与肝癌相关性研究

目的研究肝细胞肝癌（hepatocarcinoma,HCC）患者乙型肝炎病毒（Hepatitis B virus,HBV）前S2区缺失突变情况。方法应用PCR法扩增HBV前S区基因,应用DNAMAN软件对所测基因进行分析。结果 23.08%（6/26）的肝癌患者发现存在preS2区的缺失突变,缺失位点为nt 3223～nt 3268,缺失长度为30～45 bp。肝癌患者缺失突变发生率显著高于HBV无症状携带者。结论 HBV preS2区的缺失突变可能与肝癌的发生密切相关,该突变的发生机制仍有待进一步研究。     

肿瘤-睾丸抗原SSX1和SSX4的mRNA在肝细胞肝癌中的表达

目的检测肿瘤-睾丸抗原(Cancer-Testis antigens,CT)SSX1和 SSX4基因 mRNA 在肝细胞肝癌(HCC)中的表达,探讨 CT 抗原 SSX1和 SSX4 mRNA 在 HCC 中表达有无特异性。方法应用逆转录-聚合酶链反应(RT-PCR)方法对35例 HCC 患者癌组织和相应的癌旁组织 SSX1和 SSX4基因表达进行检测,对其中6例 RT-PCR 扩增产物的目的片段进行 DNA 序列测定。结果 35例 HCC 患者中,SSX1和 SSX4的表达率分别为81%(27/35)和73%(23/35);癌旁组织、12例肝硬化和15例正常组织未检测到 SSX1和 SSX4的表达。6例 DNA 序列测定结果显示 RT-PCR 产物为 SSX1和 SSX4基因的 cDNA。SSX1和 SSX4的表达与患者年龄、性别、肿瘤大小、分化程度、血清甲胎蛋白(AFP)水平、乙型肝炎病毒(HBV)和丙型肝炎病毒(HCV)感染无显著相关性(P>0.05)。结论 SSX1和 SSX4在 HCC 中呈高特异性表达,是制备 HCC 疫苗理想的靶向分子,多种 CT 抗原联合表达为多效价疫苗治疗 HCC提供了理论基础。     

The association of hepatocellular carcinoma in childhood with hepatitis B virus infection

Eleven cases of hepatocellular carcinoma (HCC) in childhood were investigated by immunohistochemistry for association with hepatitis B virus (HBV) infection. Seven of 11 cases (64%) demonstrated positivity for hepatitis B surface antigen (HBsAG), whereas all 11 were negative for hepatitis B core antigen (HBcAG). Cirrhosis was absent in all cases, and other causes for HCC in childhood were not found. All children with HBV-associated HCC died within 6 months of diagnosis. The median survival time of these children was 2 months. Only one child with HCC of trabecular subtype without HBV association is still living after 18 months. However, this child has metastases and a local recurrence. Three other children with HCC of fibrolamellar subtype are free of disease after 2, 5, and 6 years, respectively. The high number of cases of HBV-associated HCC shows the important role of HBV infection as an etiologic factor for the development of childhood HCC in middle Europe.     

广西乙肝病毒／黄曲霉毒素B1双暴露人肝细胞癌中PTENmRNA表达及突变的初步研究

目的探讨乙肝病毒／黄曲霉毒素B1双暴露相关肝细胞性肝癌（HCC）中PTEN基因的突变特点及其mRNA的表达水平。方法108例HCC来自广西不同地区样本,按照乙肝病毒与黄曲霉毒素的暴露情况,分为4个亚组：A组：HBV（＋）／AFB1-DNA（＋）,共48例;B组：HBV（＋）／AFB1-DNA（-）,共27例;C组：HBV（-）／AFB1-DNA（＋）,共19例;D组：HBV（-）／AFB1-DNA（-）,共14例。采用PCR联合基因直接测序法,检测108例肝癌组织中PTEN基因第4、5、8外显子的突变情况。同时采用RT—PCR法检测其基因mRNA的表达状况。结果（1）PTEN基因第4、5、8外显子测序结果未发现发生在外显子上的突变。但在第4外显子与第4内含子交界处有61例发生大片段的缺失,缺失率为56.4％。②PTEN缺失率在A、B、C、D组中分别为60．4％、62．9％、47．3％和46．6％,其差异在4个亚组间均无统计学意义（P〉0．05）。@PTENmRNA在4个亚组中的表达半定量灰度值分别为：A组：0．54±0．13;B组：0．59±0．16;C组：0．97±0．16;D组：0．92±0．13。其中,A、B组分别与C、D组相比,其差异均具有统计学意义（PAC=0．002,PAD=0．032,PBC=0．000,PBC=0．011）。结论在广西乙型肝炎病毒／黄曲霉毒素B1的双暴露肝细胞癌中,PTENmRNA的表达下调是一个常见的分子生物学事件,PTENmRNA表达下调可能主要与HBV感染有关。AFB1对PTENmRNA的表达下调可能有协同作用。     

P53 mutations may be related to tumor invasiveness of human hepatocellular-carcinoma in china

A combined polymerase chain reaction (PCR) with the enzyme HaeIII restriction analysis and DNA sequencing have been employed to study the mutations at codon 249 of p53 gene in two human hepatocellular carcinoma (HCC) cell lines and 28 surgical specimens of HCC. 14 of the 28 HCC samples (50%) had p53 point mutations at codon 249. All of point mutations at codon 249 in 10 cases sequenced are AGG to AGT transversion. p53 gene mutated more frequently in invasive HCCs than that in non-invasive HCCs. This suggested that the codon 249 was a mutational hotspot of p53 gene in human HCCs in China, and p53 mutations may be related to tumor invasiveness of human HCC.