Codon 249 mutations of p53 gene in non-neoplastic liver tissues

Ｓｕｂｊｅｃｔｈｅａｄｉｎｇｓｌｉｖｅｒ;ｐ５３ｇｅｎｅ;ｃｏｄｏｎ２４９ｍｕｔａｔｉｏｎ;ｌｉｖｅｒｎｅｏｐｌａｓｍｓ;ｈｅｐａｔｉｔｉｓ,ｖｉｒａｌ;ｌｉｖｅｒｃｉｒｈｏｓｉｓ;ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎＡｂｓｔｒａｃｔＡＩＭ...     

The mutation of codon 249 in the p53 gene is not specific in Japanese hepatocellular carcinoma

ABSTRACT— Hepatocellular carcinoma samples obtained from 59 patients at surgical resection were examined for mutations of the third base at codon 249 of the p53 gene, using the polymerase chain reaction and oligonucleotide hybridization techniques. This point mutation, which is frequently observed in HCC cases from Southern Africa and Quidong in China, was not recognized in either 60 hepatocellular carcinomas or 53 noncancerous liver tissue samples from Japan. Thirty-four of 45 patients (75.6%) were positive for the hepatitis C virus, which was a higher rate than that for hepatitis B virus infection (9 of 55; 16.4%). The exposure to aflatoxin B1 was not considered to be remarkable. These results suggest that the point mutation of the third base at codon 249 is not common in Japanese patients, and it is suggested that numerous other factors affect the mutation of the p53 gene and the development of hepatocellular carcinoma.     

Detection of p53 codon 249 mutation in Nigerian patients with hepatocellular carcinoma using a novel evaluation of cell-free DNA: Original Article

Objectives: This case-control study was done to determine the association and prevalence of p53 codon 249 mutation using cell-free DNA in the plasma of patients with hepatocellular carcinoma (HCC) in South-Western Nigeria. Method: Eighty-five adults with HCC and seventy-seven age and gender matched controls without evidence of liver disease or malignancy involving any part of the body, were recruited. Plasma DNA was analyzed for p53 codon 249 by restriction fragment length polymorphism. Patient evaluation was done by means questionnaire interview, clinical examination, laboratory and radiological tests. The prevalence of the p53 codon 249 mutation was expressed as a percentage amplifiable DNA samples analyzed from HCC patients while that of controls was expressed in the same way. Fisher’s exact test or the student t-test where appropriate were used to assess statistical significance of prevalence between both groups as well as comparison of some characteristics in the HCC cases between those who had codon 249 mutation and those who did not. Associations between the various parameters assessed were determined by odds ratio and significant difference was specified at p < 0.05. Results: p53 codon 249 mutation was present in 6 (7.6%) of the 79 samples from the HCC patients with amplifiable plasma DNA while none (i.e. 0%) of the 73 samples with amplifiable plasma DNA from the controls had this mutation. This prevalence is significantly higher among HCC patients than controls (0.029). The mutation was also found to be significantly associated with HCC (odds ratio = 2.00; 95% C I: 1.70 – 2.35). Conclusion: The prevalence of the p53 codon 249 mutation from plasma DNA of hepatocellular carcinoma patients is significantly higher than among controls in South-Western Nigeria and the presence of this mutation is significantly associated with HCC in this region.     

Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas

AIM:To study the relationship between N-ras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS:The N-ras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemistry.RESULTS:Thirteen cases of HCCs were p53 positive (44.8%), which showed a rather high Cpercen-tage of p53 gene mutation in Guangxi. The aberrations at N-ras codon 2-37 were found in 79.31% of HCCs and 80.77% of adjacent non-tumorous liver tissues. More than 2 point mutations of N-ras gene were observed in 22 cases (75.86%). Twelve cases (41.37%) of HCCs showed both N-ras gene mutation and p53 gene expression.CONCLUSION:N-ras gene and p53 gene may be involved in the carcinogenesis and the development of HCC.That 38% of HCCs with N-ras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.     

原发性胃癌p53基因突变

目的 p53基因是当前抑癌基因研究中的热点之一。迄今,有关 p53基因异常与胃癌临床病理学参数如大体类型、临床分期、组织分化程度,浸润深度及淋巴结转移之间的关系尚无定论。Tumura 报告p53基因改变主要发生于异倍体瘤,国内尚无报道。本实验目的主要是分析中国人原发性胃癌 p53基因突变与这些病理参数,包括 DNA 倍体之间的关系。方法用聚合酶链式反应—单构象多态分析(PCR—SSCP)技术对20例原发性胃癌 p53基因外显子5—8突变进行检测。结果 8例(40%)发生了突变,其中2例发生在外显子7,4例发生在外显子8。0至Ⅲ期均有突变存在。66.7%(6/9)的异倍体瘤检测到了p53突变,而二倍体瘤中只有18.2%(2/11)发生了 p53突变。结论 p53基因突变与胃癌临床病理参数如大体类型、分期、组织分化程度、浸润深度及淋巴结转移之间无明显关系,而与胃癌 DNA 倍体改变有关。     

肝细胞癌抑癌基因p53突变

目的分析重庆地区肝细胞癌ｐ５３基因突变谱．方法住院肝细胞癌患者２０例，皆经病理证实，长期在重庆地区居住，其中早期小肝癌４例，中期６例，晚期１０例．采用ＰＣＲ－ＳＳＣＰ，ＰＣＲ直接测序技术分析ｐ５３基因５，６，７和８外显子突变．结果ｐ５３基因总的突变率为４０％．其中外显子５和６各占１０％，外显子７占２０％，未发现外显子８的突变；测序证实外显子７为第２４９位密码子Ｇ→Ｔ的颠换突变．突变病例多为晚期肿瘤．结论重庆地区肝细胞癌存在明显的ｐ５３基因突变，反映了该地区肝癌与黄曲霉毒素和ＨＢＶ或ＨＣＶ病毒有关     

Aflatoxin sufferer and p53 gene mutation in hepatocellular carcinoma

Ａｆｌａｔｏｘｉｎｓｕｆｅｒｅｒａｎｄｐ５３ｇｅｎｅｍｕｔａｔｉｏｎｉｎｈｅｐａｔｏｃｅｌｕｌａｒｃａｒｃｉｎｏｍａＤＥＮＧＺｈｕｏＬｉｎａｎｄＭＡＹｕｎＳｕｂｊｅｃｔｈｅａｄｉｎｇｓＡｆｌａｔｏｘｉｎＢ１;ｇｅｎｅｓ,ｐ５３;ｍｕｔａｔｉｏｎ;...     

肝细胞癌p53及nm23-H1 mRNA表达的意义

目的探讨ｐ５３,ｎｍ２３Ｈ１与原发性肝细胞癌（ＨＣＣ）发生发展的关系．方法运用原位分子杂交技术对４９例ＨＣＣ中ｐ５３和ｎｍ２３Ｈ１基因ｍＲＮＡ进行检测,并结合临床病理特征进行分析．结果ｐ５３ｍＲＮＡ杂交阳性２３例,占４６９％;ｐ５３ｍＲＮＡ过表达与肿瘤的肝内转移．包膜侵犯及Ｅｄｍｏｎｄｓｏｎ分级相关（Ｐ＜００５）;ｎｍ２３Ｈ１ｍＲＮＡ阳性表达２７例,占５５１％;ｎｍ２３Ｈ１ｍＲＮＡ表达与肿瘤肝内转移及ＴＮＭ分期呈负相关（Ｐ＜００５）;同时发现ｐ５３ｍＲＮＡ过表达和ｎｍ２３Ｈ１ｍＲＮＡ低表达在ＨＣＣ肝内转移中具有协同作用．结论ｐ５３和ｎｍ２３Ｈ１参与ＨＣＣ的发生发展,ｐ５３过表达及ｎｍ２３Ｈ１低表达提示ＨＣＣ肝内转移．     

Aflatoxin and p53 abnormality in duck hepatocellular carcinoma

Recent studies have revealed that a point mutation at codon 249 in the p53 gene predominates in hepatocellular carcinoma (HCC) cases from Southern Africa and China, where infection with hepatitis B virus (HBV) and contamination of aflatoxin B₁ in food are risk factors for HCC. This unique mutation from G to T at the third base in codon 249 observed in human HCC cases is suggested to be linked to aflatoxin exposure. Six ducks with HCC, five of which were fed a diet containing aflatoxin B₁ for 1–2 years, were analysed for the presence of point mutations at this codon of the p53 gene by polymerase chain reaction and direct nucleotide sequencing. None of the six ducks with HCC showed the change at this codon regardless of duck hepatitis B virus infection. This suggests that aflatoxin B₁ itself might not be involved in the unique mutation at codon 249 in hepatocar-cinogenesis, or that other factors coincident with aflatoxin may be responsible for this unique mutation.     

肝细胞癌mdm2基因表达及其与p53基因突变的关系

目的研究ｍｄｍ２基因在原发性肝细胞癌（ＨＣＣ）中的表达并探讨其与ｐ５３基因突变的关系．方法用银染ＰＣＲＳＳＣＰ法检测ｐ５３基因第５～８外显子的突变,原位杂交检测ｍｄｍ２基因ｍＲＮＡ的表达,ＳＡＢＣ法检测ｍｄｍ２蛋白的表达．结果３９３％（１１／２８）的病例有异常的电泳迁移率．ｐ５３基因突变与肿瘤的大小、分化及转移无关．原位杂交显示９例ＨＣＣ出现ｍｄｍ２基因ｍＲＮＡ增加,７例ＨＣＣ可检测到ｍｄｍ２蛋白表达,ｍｄｍ２基因表达与ＨＣＣ的大小、分化及是否转移无关．Ⅰ～Ⅱ级ＨＣＣ中ｍｄｍ２阳性表达率（１３３％）明显低于Ⅲ～Ⅳ级ＨＣＣ中的阳性表达率（５３８％）．１１例有ｐ５３基因突变的ＨＣＣ中,只有３例出现ｍｄｍ２基因表达,另外６例有ｍｄｍ２过表达的ＨＣＣ未见ｐ５３基因突变．ｐ５３基因突变的ＨＣＣ与ｐ５３基因无突变的ＨＣＣ相比,ｍｄｍ２基因表达阳性率无显著差别．结论ｐ５３基因突变和ｍｄｍ２基因表达在原发性ＨＣＣ的发病中起重要作用．ｍｄｍ２基因表达与ＨＣＣ的恶性程度相关．ｍｄｍ２基因表达与ｐ５３基因是否突变无关．     

肝细胞癌肿瘤抑制基因p53过度表达及点突变的研究

目的检测重庆地区肝细胞癌 p53突变发生率,并进一步探讨 p53突变与肝细胞癌临床病理及相关危险因素的关系.方法应用一种敏感的 ARF 免疫组化和 PCR、银染 PCR-SSCP 方法检测本地区38例肝细胞癌(HCC)组织中肿瘤抑制基因p53的过度表达及点突变.结果 16例有P53蛋白过度表达(41.2%),7例有 p53基因249位密码子点突变(18.4%),2例249位密码子外第7外显子点突变.9例 p53基因有突变的肝癌中8例 P53蛋白阳性,两者符合率为88.9%.p53基因蛋白过度表达和点突变与 HCC 分化和转移有关.本组 HCC p53基因突变率与该地区黄曲霉素(AFB1)含量及乙型肝炎病毒(HBV)感染分布一致.结论该结果提示 p53基因突变与 AFB1和 HBV 等环境因素的协同作用有关,其中 AFB1主要与 p53基因249位密码子特异型突变有关,而 HBV 可能在散发型突变中发挥重要作用.     

High levels of p53 protein expression do not correlate with p53 mutations in hepatocellular carcinoma

To determine the relationship between p53 altered expression and p53 mutations in hepatocellular carcinoma (HCC), we analysed p53 protein immunohistochemically and assessed the presence of mutations in exons 4-8 of the p53 gene using SSCP assay in 117 HCCs corresponding to 78 patients. We also determined the relationship of p53 expression with cellular proliferation by immunostaining with monoclonal antibodies to Ki-67. We found significant levels of p53 protein expression in 23.1% of the 117 cases studied, but identified mutations in only 12 cases (10.3%). Only four of the p53-positive cases had mutations in the regions analysed. Six of the cases that displayed mutations at p53 gene were negative for immunohistochemical analysis (IHC) and two cases showed positive immunoreactivity in the cytoplasm of the cell. In conclusion, strong IHC reactivity for p53 protein is not an indicator of the presence of p53 gene mutations at exons 4-8 in HCC. Thus, p53 loss of function in HCC should be evaluated both by p53 mutation analysis and p53 protein expression, as both give complementary information about p53 status.     

肝细胞癌p53基因249密码子热点突变的研究

目的 研究肝细胞癌(HCC)中p53基因249密码子(p53 E7 cd249)点突变情况。方法 用PCR法及HAEⅢ限制性片段长度多态性分析(HAEⅢ/RFLP)检测河南豫东地区38例HCC石蜡包埋组织及2例肝细胞癌株中p53 E7cd249点突变情况,DNA测序证实。选取广西桂西南地区的10例HCC作对照。结果 来自河南豫东地区的HCC p53 E7 cd249点突变率为10.5％(4/38),对照组广西桂西南地区的HCC p53 E7 cd249点突变为40％(4/10),二者相比具有显著性差异(P<0.05)。2例肝细胞癌株中均未发现HCC p53 E7 cd249点突变。结论 河南豫东地区HCC中p53基因E7 cd249点突变为非高发事件;p53 E7 cd249点突变可能发生在肝细胞癌变的晚期。     

抑癌基因PTEN及p53在肝细胞肝癌中表达的免疫组化研究

为探讨肝细胞肝癌组织中抑癌基因PTEN及p53蛋白的表达情况及临床病理意义。应用免疫组织化学技术检测了41例肝细胞肝癌及其相应的癌旁组织中PTEN和p53蛋白的表达情况。41例癌旁组织PTEN全部阳性表达,肝细胞肝癌组织中PTEN阳性表达率39％,阳性信号显示于胞浆中。p53阳性表达率51％,PTEN蛋白在肝细胞肝癌组织中的阳性表达与组织分化程度明显相关,高分化癌的阳性率为73％,低分化癌阳性率27％。肝细胞肝癌细胞中存在较高比例的PTEN蛋白阴性表达,说明在肝细胞肝癌的发生发展中PTEN基因失活起着重要作用,它的阳性表达可能有一定的预后意义。     

p53 gene mutations and expression of p53 and mdm2 proteins in invasive breast carcinoma

The aim of the study was to analyzep53 gene mutations and the expression of p53 and mdm2 proteins in 31 randomly selected invasive breast carcinomas. The results were then correlated with tumor grade, stage, estrogen receptor status, nodal status, and DNA ploidy. The expression of the proteins p53 and mdm2 was determined immunohistochemically using formalin-fixed, paraffin-embedded material. Screening for p53 mutation involved analysis of the highly conserved regions of thep53 gene (exons 5–9) by the polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) technique. PCR products with band shifts were directly sequenced. Immunohistochemical staining of p53 was positive in 9 cases (29.0%), only 2 of which showed ap53 gene mutation. These were identified as a CG transversion at the second position of codon 278 in exon 8 and an AG transition at the second position of codon 205 in exon 6. A third case with a mutation was observed (CT transition, position 1 of codon 250 in exon 7) that did not show p53 immunohistochemically. Of the 9 p53-positive tumors, 2 were moderately differentiated (grade II). The remaining tumors were poorly differentiated (7/9). By contrast, p53-negative carcinomas were well differentiated (grade I) in most cases (P=0.02). DNA cytometry in 8 of the 9 p53-positive carcinomas revealed an aneuploid stem line. The majority of the p53-negative tumors were diploid (P=0.01). Mdm2 oncoprotein was detected in 10 tumors (32.2%), 4 of which were p53-positive, including the 3 with mutations. The grading of the mdm2-positive tumors was moderate or poor, G1 carcinomas were always noted to be mdm2-negative (P=0.04). Overexpression of p53 protein is a complex mechanism and does not merely indicate the detection of mutations in thep53 gene. This study has shown that p53 expression correlates with tumor grade and DNA ploidy. Mdm2 expression was also associated with the tumor grade. Immunohistological demonstration of the p53 protein alone is insufficient as a basis for comment on the functional state of thep53 gene and gene product. The interrelation between recognition of the p53 protein and gene mutation needs more careful assessment to define their roles in the control of neoplasia.     

p53 point mutations in primary human gastric carcinomas

Summary p53 point mutations in primary gastric carcinomas were analyzed by performing cDNA deoxynucleotide sequencing of the gene. Out of 16,9 (56.3%) primary gastric carcinoma cases, including early cancer, showed one or more p53 point mutations in their open-reading frame, and 4 out of 9 cases had a p53 point mutation within highly conserved domains. The characteristics of the p53 mutation spectrum observed in primary tumors were (a) frequent mutation at an A:T pair (50%, 7 out of 14 mutations), (b) high transversion incidence (29%, 4 out of 14 mutations), (c) no transition at CpG, and (d) no G:C to T:A transversion. Our results suggest that p53 mutation is a common event in gastric carcinoma occurring from the early stage of progression with its specific mutation spectrum.Abbreviation PCR-SSCP polymerase chain reaction single-strand conformation polymorphism     

The Pro variant of the p53 codon 72 polymorphism is associated with hepatocellular carcinoma in Moroccan population

Aim: Codon 72 polymorphism of the p53 gene has been implicated in cancer risk, and it has been suggested that it may have an impact on the clinical outcome of the disease. Our objective was to evaluate the association between p53 polymorphism at codon 72 and hepatocellular carcinoma (HCC) in the Moroccan population. Methods: Genomic DNA was extracted from peripheral blood cells of 96 patients with HCC and 222 controls without HCC matched for age, gender and ethnicity. Codon 72 polymorphism of p53 was identified by PCR-restriction fragment length polymorphism, confirmed by sequencing. Results: Patients with HCC had higher frequencies of Pro/Pro (13.5% vs. 6.3%, P < 0.02) than controls and consequently a 2.3-fold increased risk of liver cancer development (odds ratio [OR], 2.304; 95% confidence interval [CI], 1.014-5.234). In addition, we found a significant association between the p53Arg72Pro polymorphism and the female gender in HCC. Men with Pro/Pro genotype had a 1.57-fold increased risk for HCC, whereas the corresponding genotype in women had a 4.4-fold increased risk of HCC (OR, 4.4; 95% CI, 1.18-16.42). No correlation between the polymorphism and HCC risk was found when comparing the hepatitis C virus (HCV)-positive cases to HCV-positive controls. However, HCV-negative subjects and Pro/Pro genotype had a 3.31-fold increased risk for HCC. Conclusion: These results provide evidence that p53 polymorphism at codon 72 is a modifier of hepatocarcinogenesis, especially in women and HCV-negative subjects.     

Microsatellite instability in human hepatocellular carcinoma: relationship to p53 abnormalities

ABSTRACT— Aims/Background: Microsatellite instability was sought in 10 human hepatocellular carcinomas (HCCs) to determine whether defective DNA mismatch repair might be implicated in the multiple genetic alterations observed in the p53 tumor suppressor gene in some of these patients' tumors. Methods: Genomic DNA from HCCs and adjacent non-tumorous livers was subjected to PCR with primers for nine microsatellites, and PCR products were resolved in a denaturing gel. Microsatellite instability was defined as the presence of band shifts or additional bands for at least two microsatellite sequences in an HCC compared to the nontumorous liver tissue from the same patient. Results: Microsatellite instability was detected in four of ten HCCs. Three of these four HCCs did not have p53 exon mutations. However, one HCC had microsatellite instability as well as multiple p53 exon mutations and multiple intron alterations. Four other patients with multiple p53 intron alterations in HCC (compared to their own nontumorous liver), three of whom also had a mutation in the exons, had no microsatellite instability. Conclusions: Defective DNA mismatch repair, as indicated by microsatellite instability, might have played a role in hepatocarcinogenesis in four of the ten patients, but in general it was not associated with p53 alterations. In one of the ten patients, defective DNA mismatch repair might have been the cause of multiple mutations in both the coding and intron sequences of the p53 gene.     

血吸虫病患者肝癌细胞p53、p16蛋白表达的相关性研究

目的:探讨血吸虫病合并原发性肝细胞癌(hepatocellular carcinoma,HCC)与p53、p16蛋白的基因之间的相关性及其作用机制。方法:58例HCC患者分为两组:1组(HCC合并血吸虫病组)23例和2组(HCC不合并血吸虫病组)35例。采用免疫组织化学方法检测所有患者的p53、p16蛋白表达。结果:p53蛋白阳性表达率在1组中和2组中分别为73.9%(17/23)和31.4%(11/35),两组比较差异有显著性意义(P<0.01);而p16蛋白阳性率在1组和2组中分别为34.8%(8/23)和28.6%(10/35),两组比较差异无显著性意义(χ2=0.25,P>0.05)。p53蛋白及p16蛋白同时阳性仅7例,阳性率为12.1%(7/28);其中1组为13.0%(3/23),2组为11.4%(4/35),两组比较差异无显著性意义(χ2=0.052,P>0.05)。结论:患有日本血吸虫的肝细胞癌患者的癌组织中有较高的p53基因突变率,说明血吸虫感染对肝细胞癌组织细胞中p53突变蛋白的过量表达有促进作用;同时,血吸虫患者肝细胞癌患者肝组织细胞中p16肿瘤抑制基因-p16基因的丢失普遍。p16蛋白表达缺失,其与p53突变蛋白的过量表达同时存在,两者共同促进肿瘤的增长,可能是HCC恶性增殖的原因之一。     

Loss of heterozygosity and analysis of mutation of p53 in hepatocellular carcinoma

Abstract Thirty-six hepatocellular carcinoma (HCC) tissues obtained from 34 patients were classified according to histological diagnosis into six well-differentiated HCC, 20 moderately differentiated HCC and 10 poorly differentiated HCC. High molecular weight DNA was prepared from each tumour and the corresponding non-tumour tissue. Loss of heterozygosity (LOH) on chromosomes 4q, 5q, 10q, 11p, 16q, 17p, mutation of the p53 gene and polymorphism of intron 25 of the retinoblastoma (RB) gene were simultaneously analysed. The patients were composed of three cases of small HCC (the diameter of which was < 3 cm) and 31 cases of advanced HCC. Twenty-nine of 34 (85.3%) patients analysed had been exposed to hepatitis B virus and/or hepatitis C virus. The frequencies of LOH on seven chromosomes were 57.9% in 17p13.3, 45.1% in 17p, 45.1% in 11p, 41.9% in 5q, 41.9% in 16q24, 29.0% in 4q, 25.8% in 10q in advanced HCC (four of well differentiated, 18 of moderately differentiated and nine of poorly differentiated carcinoma). In contrast, LOH was observed on 4q, 5q, 16q and 17p in 33% (1/3) of the small HCC (two of well differentiated and one of moderately differentiated carcinoma). The mutation of the p53 genes and polymorphism of the RB gene were present in 25.8% (8/31) and 12.9% (4/31) of the advanced tumours, respectively, but the mutation was not found in small HCC. LOH on every chromosome and the p53 mutation were observed more frequently in more advanced tumours, and the genetic changes accumulated with the increase of the histopathological grade. These findings suggest that the accumulation of genetic changes in multiple tumour suppressor genes is involved in the progression of HCC.