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.

熊胆对肿瘤细胞p53蛋白表达的影响

目的探讨熊胆对p53基因特性不同的肿瘤细胞p53蛋白表达的影响。方法采用Western blot和RT-PCR分析方法检测熊胆对6个癌细胞株p53蛋白表达影响。结果Western blot分析可见,有p53基因突变的HaCaT、KUMA3、KUMA4和KUMA6细胞株p53蛋白表达增加,而无p53基因突变的HCT116和KUMA5细胞株p53蛋白表达没有变化。经RT-PCR分析p53mRNA结果,所有细胞株都没有p53mRNA量的变化。结论熊胆对癌细胞株p53蛋白表达的影响是依其所具有的p53基因特性不同而不同的,即熊胆只作用于突变型p53蛋白。

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.

DETECTION OF POINT MUTATION OF p53 GENE BY SILVER STAINING PCR-SSCP IN PARAFFIN-EMBEDDED MALIGNANT FIBROUS HISTIOCYTOMA

Silver staining PCR-SSCP method was used to detect point mutation of p53 gene in paraffin-embedded malignant fibrous histiocytoma (MFH) tissues. The abnormal shifting of the single-stranded DNA (ssDNA) was identified in 9 out of 16 cases (56.3%). The positive figure of SSCP was 1, 4, 4, 3 in exon 5, 6, 7, 8, respectively. The mutant of p53 protein was detected by microwave oven treatment and ABC immunohistochemistry. Positive nuclear staining was observed in 10 cases (62.5%). The positive coincidence rate was 90.0% between SSCP and p53 protein expression. The mutation of p53 gene was not correlated with the subtypes of MFH. Our results indicate that detection of point mutations with silver staining PCR-SSCP is convenient, rapid and reliable in the screening of point mutation of genes.

Lack of correlation between p53 codon 72 polymorphism and anal cancer risk

AIM:To investigate the potential role of p53 codon 72 polymorphism as a risk factor for development of anal cancer. METHODS:Thirty-two patients with invasive anal carcinoma and 103 healthy blood donors were included in the study.p53 codon 72 polymorphism was analyzed in blood samples through polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing. RESULTS:The relative frequency of each allele was 0.60 for Arg and 0.40 for Pro in patients with anal cancer, and 0.61 for Arg and 0.39 for Pro in normal controls. No significant differences in distribution of the codon 72 genotypes between patients and controls were found. CONCLUSION:These results do not support a role for the p53 codon 72 polymorphism in anal carcinogenesis.

Cloning and sequencing of a DNA fragment encoding N37 apoptotic peptide derived from p53

Objective It was reported that p53 apoptotic peptide (N37) could inhibit p73 gene through being bound with iASPP,which could induce tumor cell apoptosis. To further explore the function of N37,we constructed the cloning plasmid of DNA fragment encoding p53 (N37) apoptotic peptide by using DNA synthesis and molecular biology methods. Methods According to human p53 sequence from the GenBank database,the primer of p53(N37) gene was designed using Primer V7.0 software. The DNA fragment encoding p53 (N37) apoptotic peptide was amplified by using self-complementation polymerase chain reaction (PCR) method and cloned into the pGEM-T Easy vector. The constructed plasmid was confirmed by endonuclease analysis and sequencing. Results The insertion of objective DNA fragment was confirmed by plasmid DNA enzyme spectrum analysis. p53(N37)gene was successfully synthesized chemically in vitro. The sequencing result of positive clone was completely identical to the human p53(N37) sequence in GenBank using BLAST software (http://www.ncbi.nlm.nih.gov/cgi-bin /BLASTn). Conclusion The cloning of DNA fragment encoding p53(N37) apoptotic peptide was constructed by using DNA synthesis and pGEM-T Easy cloning methods. With the constructed plasmid,we could further investigate the function of N37 peptide.

THE DETECTION OF P53 GENE IN HUMAN CERVICAL CARCINOMA WITH AND WITHOUT HUMAN PAPILLOMAVIRUS INFECTION

The special primers Of p53 exon 7 as wed as HPV16 E6 and E7 ORFs (Opening Rending Frame) were used with PCR, PCR-SSCP technique, and 35 specimens of cervical carcinoma were examined. The results were as follows: ① HPV16 E6, E7 DNA was found in 25/35 specimens (71. 4%),which proved again HPV16 Infection an important event in cervical carcinogenesis. However only 11/35 (31.42% ) bad E6 and E7 ORFs simultaneously, 3/35 (8. 57%) and 11/35 (31. 42% ) had only E6 or E7 respectively. ② No mutation and LOH (Loss of Heterozygote) of p53 exon 7 were found in allof 35 specimens. Additionally in the present study, we developed a non-isotopic PCR-SSCP method.

PCR扩增石蜡包埋小鼠组织p53基因突变热点片段

目的 扩增石蜡包埋小鼠组织 p5 3基因第 5、7外显子。方法 用 3种方法提取DNA ,比较提取效果 ,用聚合酶链式反应 (PCR)扩增相应片段。结果 用二甲苯脱蜡 ,蛋白酶K消化 12 0小时可获得较好的DNA提取物。结论 用该提取物为模板扩增小鼠p5

Infrequent p53 gene mutation and expression of the cardia adenocarcinomas from a high-incidence area of Southwest China

INTRODUCTIONAdenocarcinomas of the cardia are the lesionsarising from the proximal stomach or within 3 cm ofthe gastroesophageal junction.These cancerstended to be advanced at the time of presentation,usually with poor prognosis.In recent decade,the incidence of adenocarcinoma of gastric eardiaand esophagus are increasing steadily,while therehas been a decrease in the proportion of the cancersarising from the distal stomach.The

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.

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.

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。

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.

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.

Relationship between tumor suppressor gene p53 and tumors of adipose tissue

OBJECTIVE: To investigate the relationship between p53 gene and tumors of adipose tissue at the level of protein and gene. METHODS: Immunohistochemical LSAB, PCR-SSCP and DNA sequencing were used in 82 cases. RESULTS: p53 protein is expressed only in liposarcomas, in which the positive staining rate was 48.08% (25/52). In different subtypes of liposarcomas, the positive staining rate in well differentiated liposarcomas was 30.00% (9/30), which is much lower than that of the poorly differentiated liposarcomas (P ATC) were detected by DNA sequencing. Another heterozygotic cosense mutation may exist at exon 6 codon 221 of p53 gene (GAG-->GAA). CONCLUSIONS: The data suggest that the p53 protein has a relationship with development, differentiation and malignancy of liposarcoma. Detecting the level of p53 protein expression may be valuable in evaluating the level of differentiation and malignancy of liposarcoma. There appear point mutation on exon 8, 6 of p53 gene.

脑星形细胞瘤P^(53)抗癌基因突变的研究

目的研究Ｐ５３抗癌基因突变与脑星形细胞瘤恶性程度的关系。方法采用非同位素ＰＣＲ－ＳＳＣＰ分析法检测了３１例星形细胞瘤患者Ｐ５３抗癌基因的突变情况。结果（１）９例存在突变，突变区域分布于第５～８外显子中，以第６外显子突变率最高，第９外显子未测得突变病例。（２）２例存在多点突变，其中１例同时存在第５、６外显子突变，另１例同时存在第６、７外显子突变。（３）病理级别为Ⅰ、Ⅱ级组的突变率为０，Ⅲ级组的突变率为３６．４％，Ⅳ级组的突变率为５０％。Ⅲ、Ⅳ级组与Ⅰ、Ⅱ级组的突变率比较，均存在极显著差异（Ｐ＜０．０１）。

皮肤鳞状细胞癌P^(53)基因突变研究

采用聚合酶反应—单链构象多态技术对 18例皮肤鳞状细胞癌P53 基因 5— 9外显子突变进行了研究。结果表明 ,突变病例为 5/ 18例 ( 2 8% ) ,其中G11/ 7例 ,G2 2 / 7例 ,G32 / 4例P53基因突变倾向分布于G2 +G3级。

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。

STUDY OF ECK GENE EXON-3 FROM HUMAN NORMAL TISSUE AND BREAST CANCER CELL LINE

Objective To establish a method cloning the exon 3 of eck gene from normal tissue and ZR 75 1 cell line (a human breast cancer cell line)and study whether these genes exist mutant. Methods Designed a pair of specific primers and amplified the exon 3 of eck gene fragment from the extracted genomic DNA derived from normal epithelial cells from skin tissue and ZR 75 1 cell line respectively by PCR technique. Transformed the E.coil. JM109 with recombinant plamids constructed by inserting the amplified fragments into medium vector pUCm T and sequenced these amplified fragments after primary screening of endonuclease restriction digestion and PCR amplification. Results ① Obtained the genomic DNA of human normal epithelial cells and ZR 75 1 cell line respectively. ② Obtained the amplified fragments of human exon 3 of eck gene through PCR technique. ③ Obtained the cloning vectors of exon 3 of eck gene of human normal epithelial cells and ZR 75 1 cell line respectively. ④ ZR 75 1 cell line exists mutation of nucleotides. Conclusion Successfully established the method of cloning the human exon 3 of eck gene and found some mutations in the detected samples. This study lays a foundation for further studying the function of eck gene in tumorgenesis.

二苯乙烯苷对小鼠肝细胞色素P450的影响

目的观察二苯乙烯苷(TSG)对小鼠肝微粒体细胞色素P450(CYP)的影响。方法将昆明种雄性小鼠分为空白组、TSG低剂量组和TSG高剂量组,TSG灌胃3、5、7d后分别麻醉处死小鼠,取肝脏通过荧光实时逆转录聚合酶链反应(real time RT-PCR)检测小鼠肝脏组织CYP相关基因mRNA的表达。结果 TSG作用第3、5、7天时均能够抑制CYP1A2和CYP3A4mRNA表达;TSG呈时间依赖性的增加CYP2E1mRNA表达;TSG作用7d能够显著抑制CYP4A14mRNA表达。此外,TSG对CYP2B10、3A11和3A25mRNA表达无显著性影响。结论 TSG对CYP1A2、CYP2E1、CYP3A4和CYP4A14有显著影响,对CYP2B10、3A11和3A25无明显作用。