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

IM To study the relationship between Nras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS The Nras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reactionsingle strand conformation polymorphism (PCRSSCP) and immunohistochemistry.RESULTS Thirteen cases of HCCs were p53 positive (448%), which showed a rather high percentage of p53 gene mutation in Guangxi. The aberrations at Nras codon 2-37 were found in 7931% of HCCs and 8077% of adjacent nontumorous liver tissues. More than 2 point mutations of Nras gene were observed in 22 cases (7586%). Twelve cases (4137%) of HCCs showed both Nras gene mutation and p53 gene expression.CONCLUSIONS Nras gene and p53 gene may be involved in the carcinogenesis and the development of HCC. That 38% of HCCs with Nras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.

纤维支气管镜刷检细胞H－ras基因检测对肺癌的诊断价值

为探讨纤维支气管镜直视下毛刷脱落细胞中Ｈ－ｒａｓ基因突变在肺癌中的诊断价值，采用在纤维支气管镜毛刷脱落细胞中应用ＰＣＲ－ＲＦＩＰ方法（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ－ｒｅｓｔｒｉｃｔｉｏｎｆｒａｇｍｅｎｔｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍｓ）检测Ｈ－ｒａｓ基因，人突变。对５０例肺癌及２０例肺部炎症患者支气管上皮脱落细胞进行了检测，结果肺癌中Ｎ－ｒａｓ基因突变为５４％（２７／５０）．鳞癌为５９．１％（１３／２２）．腺癌为５３．８％（７／１３），小细胞肺癌为３３．３％（２／６），肺透明细胞癌１００％（２／２），未能分型的肺癌４２．９％（３／７），肺炎组１０％（２／２０）。肺癌组的Ｈ－ｒａｓ基因突变明显高于肺炎组，二者有明显的差异（Ｐ＜０．０１）。研究肺癌Ｈ－ｒａｓ基因突变有较高的阳性，在肺癌与非肺癌组中有明显的差别，有希望成为肺癌诊断标记物。

p53 gene mutations in primary gastric cancer

AIMS p53 gene is one of the focuses in the study of tu- mour suppressor genes.So far,there is still controversy about the relationship between p53 alterations and clinicolpathological parameters of gastic cancers such as macroscopic classifica- tion,stage,degree of differentiation,depth of tumour invasion and lymphonod metastasis.Tamura has reported that p53 gene mutations mainly occur in the aneuploid tumours.But in China, nothing is reported in this field of study.Our aim is to analyze the relationship between p53 gene mutations and these param- eters including DNA ploidy in Chinese primary gasrtic cancers. METHODS Mutations of the p53 gene in exon5-8 were examined in 20 cases of primary gasric cancer by PCR-SSCP (Polymerase-chain-reaction-single-strand-conforma- tion-polymorphism)analysis. RESULTS Mutations were detected in 8(40%)cases:2 cases in exon5-6,2 cases in exon7,4 cases in exon8.These mutations were detected from stage 0 to stage Ⅲ No significant association was found between p53 gene mutations and the clinicopathological parameters such as macroscopic classifico- tion,degree of histological differentiation,depth of tumour in- vasion and lymphonod metastasis.In addition,66.7%(6 of 9) of aneuploid tumours had p53 mutations and only 18.2%(2 of 11)of diploid tumours had mutations. CONCLUSIONS These results suggest that p53 gene muta- tions are related to DNA ploidy alterations and that p53 gene is one of the important turnout suppressor genes in human gastric cancer.

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

AIM To study the significance of p53 gene in hepatocarcinogenesis through analyzing codon 249 mutations of p53 gene in non neoplastic liver tissues. METHODS Codon 249 mutation was detected using single stranded conformational polymorphism analysis and allele specific PCR in liver tissues from 10 cases of chronic hepatitis, 5 cases of cirrhosis and 20 cases of HCCs. RESULTS The detection rate of codon 249 mutation in chronic hepatitis, cirrhosis and pericancerous tissues was 70% (7/10), 100% (5/5) and 70% (14/20), respectively by AS PCR. These mutations could not be detected by SSCP analysis. The detection rates were 65% (13/20) and 45% (9/20) in cancerous tissues by AS PCR and SSCP analysis. CONCLUSION Codon 249 mutations of p53 gene were very popular in non neoplastic liver tissues though the number of those mutant cells was only in subsection. Those mutations in cancerous tissues might take place in the stage before the formation of tumor.

Codon 249 mutations of p53 gene in development of hepatocellular carcinoma

AIM To investigate the mechanisms of codon 249 mutation of p53 gene in the formation of hepatocellular carcinoma (HCC). METHODS Codon 249 mutation accompanied by loss of heterozygosity (LOH) and its effect on translation and transcription were studied using SSCP, IHC and RT PCR/slot hybridization. RESULTS Codon 249 mutations were detected in 32 9%, LOH detected in 68 4% among the HCC patients. Mutations of condon 249 were accompanied by LOH in 90%. The positive rates of p53 protein and mRNA were 91 3% and 95 7%, in mutational group, both were significantly higher than those in the non mutational group (91 3% vs 19 1% and 95 7% vs 40 4%, respectively, both P <0 01). The translation of p53 gene was strongly related to its transcription by correlation analysis ( r =0 8208). CONCLUSIONS LOH might play an important role in hepatocarcinogenesis of codon 249 mutation, which could increase both transcription and translation of p53 gene. The increased expression of p53 protein mainly depend on the increased transcription of p53 gene.

The point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province,a non HCC prevalent area in China

AIM: In hepatocellular carcinoma (HCC) prevalent areas of China, the point mutation of p53 exon7 is highly correlated with Hepatitis B virus(HBV) infection and aflatoxin B intake. While in non-HCC-prevalent areas of China, these factors are not so important in the etiology of HCC. Therefore, the point mutation of p53 exon7 may also be different than that in HCC-prevalent areas of China. The aim of this study is to investigate the status and carcinogenic role of the point mutation of p53 gene exon7 in hepatocellular carcinoma from Anhui Province, a non-HCC-prevalent area in China. METHODS: PCR PCR-SSCP and PCR-RFLP were applied to analyze the homozygous deletion and point mutation of p53 exon7 in HCC samples from Anhui, which were confirmed by DNA sequencing and Genbank comparison. RESULTS: In the 38 samples of hepatocellular carcinoma, no homozygous deletion of p53 exon7 was detected and point mutations of p53 exon7 were found in 4 cases, which were found to be heterozygous mutation of codon 249 with a mutation rate of 10.53%(4/38). The third base mutation(G-T) of p53 codon 249 was found by DNA sequencing and Genbank comparison. CONCLUSION: The incidence of point mutation of p53 codon 249 is lower in hepatocellular carcinoma and the heterozygous mutation of p53 exon7 found in these patients only indicate that they have genetic susceptibility to HCC. p53 codon 249 is a hotspot of p53 exon7 point mutation, suggesting that the point mutation of p53 exon 7 may not play a major role in the carcinogenesis of HCC in Anhui Province, a non-HCC-prevalent area in China.

人卵巢癌P53基因的突变

目的：了解人卵巢癌中Ｐ５３基因突变的特点及其与临床分期的关系。方法：采用聚合酶链反应单链构像多态性分析及序列分析，对６０例卵巢癌组织及癌旁组织Ｐ５３基因外显子５、６、７进行了筛查。结果：所有癌旁组织均未发现突变，癌组织有９例发生了Ｐ５３基因的突变，并对其中５例进行了序列分析，证实了其突变的存在。５例测序标本中有４例发生了Ｇ∶Ｃ→Ａ∶Ｔ的转换突变。９例阳性标本分别为临床Ⅰ期２例，临床Ⅲ期６例，临床Ⅳ期１例。结论：人卵巢癌中Ｐ５３基因的突变是自发性突变的结果。Ｐ５３基因改变可能发生于卵巢癌早期，并持续存在于肿瘤发展的全过程，是卵巢癌发生发展的危险因素之一。推测２４５位密码子是我国卵巢癌患者的一个突变热点。

Detecting K-ras and p53 gene mutation from stool and pancreatic juice for diagnosis of early pancreatic cancer

OBJECTIVE: To explore new methods for the early diagnosis of pancreatic cancer through detection of K-ras and p53 mutations in pancreatic juice and stool. METHODS: 201 patients in PUMC Hospital from 1994 - 2000 and 60 control individuals were enrolled in this study. K-ras point mutation was detected by PCR-RFLP while p53 mutation was detected by PCR-SSCP. RESULTS: K-ras mutation was found in pancreatic juice in 87.8% (36/41) of pancreatic cancer patients and 23.5% (4/17) of benign pancreatic disease patients. In 261 stool specimens, amplification found mutations successfully in 235 patients (90%). K-ras mutation was found in stool in 88% (66/75) of pancreatic cancer patients, 51.1% (24/47) of benign pancreatic disease patients and 19.6% (9/46) of normal individuals. p53 mutation was found in pancreatic juice in 47.4% (18/38) of pancreatic cancer patients and 12.5% (2/16) of benign pancreatic disease patients. p53 mutation was found in stool in 37.1% (23/62) and 19.1% (4/21) of chronic pancreatitis patients. CONCLUSION: K-ras mutation in pancreatic juice has higher diagnosis sensitivity and specificity, and therefore may be used as a supplement in the diagnosis of pancreatic cancer. Detection of K-ras mutation combined with p53 mutation in stool can aid in the screening of pancreatic cancer.

肺癌中P^(53)基因DNA水平突变与核蛋白的水平突变比较

目的 研究肺癌 P53基因 DNA水平和蛋白水平的突变。方法 应用 PCR- SSCP银染技术检测了 5 4例肺癌标本。结果 2 2例 DNA水平突变阳性 ,突变率为 41%。 32例在核蛋白水平突变 ,突变率 5 9%。其中 2 2例 DNA突变阳性者 ,核蛋白突变均阳性。而 32例 DNA水平突变阴性者 ,核蛋白突变阳性者 9例 ,占 2 8%。P53 DNA突变与核蛋白的突变之间有显著相关性 ,并且两者都与患者的淋巴结转移有关 (P<0 .0 1)。结论 PCR- SSCP是一种高灵敏快速检测 P53是否存在异常的有效方法。但是 ,P53突变阳性患者预后较差。

肺癌患者P^(53)基因突变的研究

作者应用多聚酶链反应（PCR）结合限制性内切酶位点多态性及单链构象多态性（SSCP）DNA序列分析技术，对20例肺癌患者的肿瘤组织及正常肺组织P53基因进行了研究。结果显示：15例患者的肿瘤组织中，P53基因存在各种类型的基因突变，为P53基因突变在肺癌的致病机制提供了一定的参考资料，并探讨了P53基因突变与肺癌患者的临床分期及病理组织学分型之间的相关性。

上皮性卵巢癌中P^（53）基因点突变的初步研究

为研究肿瘤抑制基因Ｐ５３与卵巢癌的关系，用多聚酶链反应－单链构象多态（ＰＣＲ－ＳＳＣＰ）银染技术检测了３０例上皮性卵巢癌中Ｐ５３基因第５～８外显子的点突变。结果；ＳＳＣＰ阳性１２例，阳性率为４０％，第５、６、７外显子分别为１、２、９例。Ｐ５３基因点突变率在各临床分期、组织学分级、组织学类型之间未发现明显差异，说明Ｐ５３基因是突变在卵巢癌的发展过程中可能一直起着重要作用。

不同肺腺癌细胞系N－ras、p53基因突变核酸测序分析

应用聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎＰＣＲ）和ｄｓＤＮＡｃｙｃｌｅｓｅｑｕｅｎｃｉｎｇｓｙｓｔｅｍ技术对体外培养的人肺腺癌细胞系ＬＴＥｐ－ａ＿２和ｈＬＡ中Ｎ－ｒａｓ癌基因及ｐ５３抑癌基因外显子５、７进行核酸序列测定分析。结果表明Ｎ－ｒａｓ突变热点第１２、１３、６１密码子未见异常。ｐ５３抑癌基因第１５４密码子均发现ＧＧＣ→ＧＴＣ突变（Ｇｌｙ→Ｖａｌ变异）。经光盘检索（美国Ｓｉｌｖｅｒｐｌａｔｔｅｒ公司提供的ＣＯ－ＲＯＭＭＥＤＩＬＩＮＥ）分析了１９８８～１９９３年间的专题文献，尚未见肺癌ｐ５３基因第１５４密码子突变的报道。

特异引物双扩增即时PCR与传统测序法检测肠癌、肺癌患者K-ras基因突变的比较

目的 以特异引物双扩增即时PCR技术K-ras检测试剂盒（下称ADx-K-ras即时PCR试剂盒）和Sanger DNA测序法同时检测结直肠癌和肺癌患者K-ras基因,以了解K-ras基因突变频率和突变类型,比较ADx-K-ras即时PCR试剂盒和Sanger DNA测序法用于肿瘤K-ras基因体细胞突变检测的临床价值.方法 收集临床肿瘤病理石蜡切片样品827例,其中肠癌样品583例,肺癌样品244例,提取DNA后,对K-ras基因第12和13密码子进行PCR扩增后,使用ADx-K-ras即时PCR试剂盒进行检测,与此同时,将PCR扩增后的产物进行Sanger DNA测序.两种方法对K-ras基因第12和13密码子的检测结果进一步进行各个突变类型的数目和突变率的统计对比.结果 ADx-K-ras即时PCR试剂盒对827例样品检测都得到了明确的结果,检测成功率为100%,Sanger DNA测序成功检测了677例,成功率为81.9%.583例肠癌样品中ADx-K-ras即时PCR试剂盒检测出突变192例,突变检出率为32.9%,Sanger DNA测序成功的样品533例,检出突变160例,突变检出率为30.0%.244例肺癌样品中ADx-K-ras即时PCR试剂盒检出突变26例,突变检出率为10.7%,Sanger DNA测序成功的样品144例,检出突变12例,突变检出率为8.3%.肠癌中第12密码子第2位的GGT→GAT最常见,占全部突变的35.1%（66/188）,其次是第13密码子第2位的GGC→GAC,26.6%（50/188）,第12密码子第2位的GGT→GTT,18.6%（35/188）,第12密码子第1位的GGT→GCT最少见,1.6%（3/188）.肺癌中第12密码子第1位的GGT→GTT最常见,占全部突变的40.9%（9/22）,同样第12密码子第1位的GGT→GCT最少见,占全部突变的4.5%（1/22）.结论 肠癌中K-ras突变率明显高于肺癌.对于甲醛固定石蜡包埋样品而言,ADx-K-ras即时PCR试剂盒对样品的DNA质量的耐受性较好,检测成功率高于Sanger DNA测序,可替代Sanger DNA测序法成为临床上对肿瘤K-ras基因体细胞突变检测的实用方法.