食管脱落上皮细胞中p53基因突变的检测

目的 探讨在食管拉网细胞中进行食管癌基因检测的方法及可行性,了解食管癌前期病变细胞中p53基因的突变与癌变的关系。方法 对四川省盐亭县1982年进行食管癌普查的食管拉网脱落细胞涂片标本,应用聚合酶链反应-单链构象多态性分析(PCR-SSCP)方法,检测其p53基因第5外显子及第7外显子的突变情况。结果 全组48例标本中,食管正常上皮和重度不典型增生上皮各24例,p53基因检测均获成功。食管重度不典型增生上皮细胞中有5例检测到突变,均为p53基因第7外显子突变,而第5外显子未检测到突变;正常食管鳞状上皮拉网脱落细胞中未发现突变。检测到突变的5例食管上皮重度不典型增生者,有3例分别在10年、12年和14年后转变为食管癌。结论p53基因突变的食管上皮不典型增生细胞具有明显的癌变趋势;对食管拉网脱落细胞涂片标本进行微量DNA的提取、扩增和基因检测,可作为研究食管癌前期病变的一种新方法。     

从贮存20年的微量细胞中提取DNA并做PCR分析的实验研究

目的:探讨从二十年前贮存的食管拉网微量细胞涂片中提取DNA并进行食管癌基因检测的方法和可行性,比较P53基因的突变在食管癌前期病变和正常食管上皮中的差异。方法:利用螯合型的离子交换树脂Chelex100作为介质,一步法从食管拉网脱落细胞提取DNA,应用PCR-SSCP方法和PCR-RFLP方法进行p53基因第5外显子及第7外显子的突变检测。结果:48例标本P53基因检测均获成功。食管上皮重度不典型增生细胞中发现有5例突变发生,均为p53基因第7外显子,而第5外显子未发现突变;正常食管鳞状上皮拉网脱落细胞中均未发现突变。检测到的5例突变中,其中有3例分别在10年、12年和14年后转变为食管癌。结论:Chelex100法能从微量细胞涂片中提取DNA,使对20年前食管拉网细胞涂片标本进行分子水平的检测成为可能。为食管癌前期病变的研究提供一种新的方法。p53基因的突变使食管上皮癌前期病变细胞具有了癌变趋势。     

从贮存20年的微量细胞中提取DNA并做PCR分析的实验研究

目的:探讨从二十年前贮存的食管拉网微量细胞涂片中提取DNA并进行食管癌基因检测的方法和可行性,比较P53基因的突变在食管癌前期病变和正常食管上皮中的差异.方法:利用螯合型的离子交换树脂Chelex100作为介质,一步法从食管拉网脱落细胞提取DNA,应用PCR-SSCP方法和PCR-RFLP方法进行p53基因第5外显子及第7外显子的突变检测.结果:48例标本P53基因检测均获成功.食管上皮重度不典型增生细胞中发现有5例突变发生,均为p53基因第7外显子,而第5外显子未发现突变;正常食管鳞状上皮拉网脱落细胞中均未发现突变.检测到的5例突变中,其中有3例分别在10年、12年和14年后转变为食管癌.结论:Chelex100法能从微量细胞涂片中提取DNA,使对20年前食管拉网细胞涂片标本进行分子水平的检测成为可能.为食管癌前期病变的研究提供一种新的方法.p53基因的突变使食管上皮癌前期病变细胞具有了癌变趋势.     

53例国人肾透明细胞癌中PBRM 1基因的突变分析

目的 分析Polybromo 1（PBRM 1）基因在国人肾透明细胞癌中的突变特点,探讨基因突变与临床病理特征和预后的关系。方法 选取53例肾透明细胞癌患者,利用PCR结合直接测序的方法检测PBRM 1基因2～5号外显子、15～17号外显子的突变情况。分析PBRM 1基因突变与临床病理特征和预后的相关性。结果 53例样本中检测出PBRM 1基因突变14例,突变率为26％。突变较集中分布在第15、17外显子,第15外显子5例,第17外显子6例。PBRM 1基因突变组和无突变组的中位无进展生存期分别为5个月（95％CI：1.6～8.4个月）和14个月（95％CI：9.7～18.3个月）,中位生存期分别为7个月（95％CI：2.6～11.4个月）和18个月（95％CI：5.6～30.4个月）,差异均有统计学意义（P＜0.05）。 PBRM 1基因突变与性别、年龄、发病部位、分期及疾病控制率等无关（P＞0.05）。结论 中国人肾透明细胞癌中PBRM 1基因突变率较高,突变主要位于第15、17外显子。PBRM 1基因突变可能是肾透明细胞癌患者预后不良的危险因素。     

P53基因突变与甲醛致癌研究

本文采用PCR和DNA序列分析对甲醛诱发的大鼠鼻腔鳞状细胞癌及其建立的肿癌细胞系P53肿瘤抑制基因突变进行了初步研究．结果显示：①所测11个大鼠鼻腔鳞癌有5个肿瘤的P53基因发生了点突变．而且是纯合性改变；②所测8株肿瘤细胞系有5株细胞的P53基因分别在高度保守区的第132，271位密码子发生了点突变，这种突变与细胞的致瘤性(裸鼠接种)有关．与细胞体外建株无关；     

肝细胞癌p53和Rb基因改变的分析

目的 研究肝细胞癌p53和Rb基因的突变特点和规律。探讨2种抗癌基因在肝癌发生过程中的作用及其与细胞学和预后的关系。方法 应用PCR－SSCP和PCR－RFLP法检测65例肝癌p53基因第5－8外显子和Rb基因17和21外显子的突变率。结果 肝癌p53基因突变率为50．8％,以缺失为主;Rb基因的突变率为38．5％;p53和Rb基因同时突变率为12．3％。结论 肝细胞癌中既有p53基因突变也存在Rb基因突变,两者在肝癌的发生中均有重要作用。p53和Rb基因同时突变与肝癌组织学分级密切相关。     

p53基因在5—氟尿嘧啶致大肠肿瘤细胞凋亡中的影响

目的 p53基因被认为是引起哺乳类动物细胞凋亡的基因,许多抗肿瘤药物是通过引起肿瘤细胞凋亡来发挥其治疗作用的,故p53基因有否突变与化疗药敏的关系较密切.本文通过研究p53基因与5-FU对大肠肿瘤细胞凋亡的影响,旨在探讨p53基因与化疗药物5-FU之间的关系.方法 对手术切除的大肠癌39例标本,用PCR-SSCP法进行p53基因第5～8外显子检测,同时制备肿瘤单细胞悬液,加入5-FU(10μg/ml)和甲酰四氢叶酸钙(5μg/ml,10μg/ml)用TDT法分析5-FU对大肠肿瘤细胞凋亡的影响.全组男性18例.女性21例.Dukes分期:A期4例,B期22例,C期8例,D期5例.结果 大肠癌p53基因突变率为43.6%(17/39例),p53基因突变与Dukes分期,性别,年龄,大体类型等关系不明显.在加入5-FU(10μg/ml)2h,4h,15h于荧光显微镜下均可见典型的调亡小体.药物作用15h后行TDT法分析发现:P53基因突变组细胞凋亡率为16.4±4.89%,而P53基因未突变组(野生型p53基因组)细胞调亡率为26.6±6.80%(P<0.01)结论 5-FU是通过引起肿瘤细胞凋亡来发挥其治疗作用的,p53基因的状态在5-FU作用于大肠肿瘤细胞引起凋亡的过程中起重要作用.     

非小细胞肺癌患者DNA修复基因XPD多态性与p53基因突变的相关性研究

[目的]研究非小细胞肺癌(NSCLC)患者DNA修复基因XPD多态性与p53基因突变的相关性。[方法]以PCR-RFLP方法分析60例NSCLC患者外周血XPD基因Lys751Gln多态;运用PCR-SCCP银染结合DNA序列分析法检测癌组织p53基因第5、6、7和8外显子突变情况。[结果]19例患者至少携带1个XPD751Gln等位基因;25例发生p53基因突变,突变率为42%;携带至少1个XPD751Gln等位基因的患者中p53基因突变率为52.6%,XPD751野生基因型中突变率为36.6%(P=0.27)。所有p53基因突变患者DNA序列分析结果显示,颠换27个,转换12个;突变类型分析显示,携带至少1个XPD751Gln等位基因的患者颠换发生明显占优势,颠换∶转换为14∶2,XPD751野生基因型颠换∶转换为13∶10(P=0.08)。[结论]NSCLC患者中DNA修复基因XPD多态性与p53基因突变发生无明显相关性。     

中国南北方食管癌患者中 p53基因外显子突变谱的差异研究

目的：探讨南北方食管癌患者中抑癌基因p53全部编码外显子基因突变谱的差异。方法各选取揭阳居民食管癌患者42例,佛山患者52例,常规提取DNA, PCR扩增p53第2-11外显子全部编码区和附近的一部分非编码区,扩增产物用DHPLC进行突变的筛查。筛查出的突变样本进行DNA纯化测序分析,测序结果在NCBI网站进行p53突变位点的比对和定位。结果高、低发区组中p53至少有1个外显子基因突变的突变率分别为63．8％（28／42）和61．5％（32／52）,两者比较差异无统计学意义（P＝0．607＞0．05）; p53有2个外显子基因突变的突变率分别为14．3％（6／42）和15．4％（8／52）,两者比较差异也无统计学意义（P＝0．881＞0．05）。结论高低发区间可能具有相似的环境致癌物质,通过相同的p53基因突变机制导致了两地食管癌的发生。     

p53基因在甲状腺癌中的突变研究

目的：为探讨p53基因突变与甲状腺癌的发生、发展及预后的关系。方法：应用PCR单链构象多态性（SSCP）分析技术,对p53基因第7,8外显子突变进行了检测和分析。结果：在37例甲状腺癌中有11例在第7.8外显子发生突变,突变率为29.9％。p53基因突变在复发的患者中显著高于未复发的患者;p53基因突变与转移、组织学类型和分化状况无显著差异。结论：p53基因的突变可能与甲状腺癌患者预后有关。     

Use of the single strand conformation polymorphism technique and PCR to detect p53 gene mutations in small cell lung cancer.

Recent studies have suggested that the p53 oncoprotein might function normally as a tumor suppressor. Mutations in highly conserved regions of the p53 gene have been observed in numerous types of tumors and tumor cell lines. To detect in a more sensitive manner p53 gene mutations in small cell lung cancer (SCLC) we utilized the single strand conformation polymorphism (SSCP) technique of Orita et al., (1989). Using PCR primers for the most highly conserved regions of the p53 gene, including exons 4-9, we have identified p53 mutations in 5 of 9 small cell lung cancer (SCLC) tumor DNA samples and in 1 SCLC cell line. None of the mutations seen in tumor DNA samples were present in normal DNA from the same patients, indicating that mutation of the p53 gene in these tumors was a somatic event. Of the six mutations observed, two were found in exon 7, three were found in the region encompassing exons 8 and 9, and one was found in the region encompassing exons 5 and 6. Nucleotide sequencing of one of the exon 7 mutations and one of the exon 8-9 mutations indicated that each was a C to T transition. In SCLC-6 the mutation resulted in substitution of serine for proline at amino acid 278 and in SCLC-4 substitution of tryptophan for arginine at amino acid 248, both nonconservative amino acid substitutions. Both of these changes are in regions of the p53 gene where mutations have been observed in other tumors. Two additional mutations were observed in SCLC cell lines using conventional PCR techniques. One of these is a mutation which results in altered splicing of the p53 pre-mRNA.     

Infrequent p53 gene mutations in medulloblastomas

Cytogenetic and molecular studies of medulloblastomas have demonstrated frequent loss of sequences from the short arm of chromosome 17, possibly implicating loss or inactivation of the p53 tumor suppressor gene. We amplified exons 5 through 8 of the p53 gene by the polymerase chain reaction technique. These segments, which encompass the regions usually mutated in human tumors, were sequenced to search for p53 mutations in 12 medulloblastoma tumors, 8 xenografts, and 3 permanent cell lines. Mutation of the p53 gene was found in only 1 of 3 cell lines tested and in none of the xenografts or primary tumors studied. Our results suggest that p53 is mutated in an unusual way or that a second tumor suppressor gene on the short arm of chromosome 17 is involved in the pathogenesis of medulloblastoma.     

Role of p53 mutations in endocrine tumorigenesis: mutation detection by polymerase chain reaction-single strand conformation polymorphism.

To elucidate the molecular basis for endocrine tumorigenesis, p53 mutations in human endocrine tumors were analyzed by using polymerase chain reaction-single strand conformation polymorphism. Exons 5 through 10 of the p53 gene were studied in genomic DNAs from 134 primary endocrine tumors and 6 human endocrine cancer-derived cell lines. Mutations were detected and identified in 4 endocrine tumors, including one parathyroid adenoma and three thyroid carcinoma cell lines. The sites of these mutations were in exons 5 (codon 151 and 152) and 7 (codon 248 and 255). In all of three tumor cell lines, but not in a parathyroid adenoma, the normal allele encoding the p53 gene was lost. However, p53 mutations were not found in any other endocrine tumors or cell lines. Based upon these results, we concluded that the p53 gene may play a role in the tumorigenesis of a limited number of parathyroid adenoma and thyroid cancers, and that the p53 mutation with an allelic loss of the p53 gene is an important factor in malignant tumorigenesis of the thyroid gland.     

Mutation of the p53 gene in a differentiated human thyroid carcinoma cell line, but not in primary thyroid tumours

The p53 gene has been implicated as a tumour suppressor, with mutations occurring in many carcinomas, such as colon, breast and lung. We have sequenced exons 5, 7 and 8 containing conserved gene regions in the only available differentiated thyroid follicular carcinoma cell line and found a mutation at position 273, Arg----His, with no normal allele present. The same mutation was also present in DNA from the tumour of origin. However immunohistochemical analysis of 129 human thyroid tumours using a panel of p53 antibodies was unequivocally negative. Southern blotting in 20 cases failed to demonstrate any deletion or rearrangement, and direct genomic sequencing of 20 carcinomas showed normal DNA sequence for exons 5, 7 and 8. Thus p53 abnormalities may not be important in human thyroid carcinogenesis, in contrast to colon, breast and lung. However, the FTC 133 cell line was only established after 132 unsuccessful attempts with other differentiated thyroid follicular tumours. Since this line and the corresponding tumour of origin have a p53 mutation, we propose that p53 mutation may confer on thyroid follicular tumour cells the ability to grow in culture. This has potential applications for the future development of thyroid carcinoma cell lines.     

Molecular analysis of the p53 alleles in primary hepatocellular carcinomas and cell lines.

We have examined p53 oncogene/anti-oncogene alleles in 10 different human hepatoma cell lines and 18 primary hepatocellular carcinomas. The p53 allele in these hepatoma cell lines appears to be a frequent target of mutation as demonstrated by Southern and Northern blotting, immunoprecipitation and Western blot analysis. In general, the steady state level of p53 specific RNA or protein in these hepatoma cell lines is higher than in normal liver. However, in three out of ten cell lines, normal-sized p53 mRNA cannot be detected. In contrast, the involvement of the p53 allele in primary hepatocellular carcinoma appears to be an exceedingly rare event. Steady state levels of p53 specific RNA in primary hepatomas are practically indistinguishable from those in normal adult liver. Using the polymerase chain reaction technique, we have amplified and subcloned exons 5, 6, 7 and 8 of p53 from 10 different hepatoma samples. DNA sequence analysis of these exon subclones reveals no apparent structural alterations. Finally, synthesis of p53 specific mRNA or protein in a HepG2 human hepatoblastoma cell line does not appear to be affected by gene expression and replication of human hepatitus B virus. Surprisingly, unlike many other kinds of human solid tumors, point mutations in p53 do not appear to be important in primary tumors of hepatocellular carcinomas.     

Murine p53 intron sequences 5-8 and their use in polymerase chain reaction/direct sequencing analysis of p53 mutations in CD-1 mouse liver and lung tumors.

Inactivating point mutations and small deletions in the p53 tumor suppressor gene have been found in human liver and lung tumor--derived cell lines and tumors. However, little evidence has been reported concerning inactivation or mutation of the p53 gene in mouse primary tumors. To examine CD-1 mouse liver and lung tumors for mutations in the p53 gene, we first sequenced p53 introns 5-8 so that polymerase chain reaction amplification and sequencing primers located within the introns could be prepared. Use of these primers prevented amplification of the mouse p53 pseudogene and allowed sequencing of exons 5-8 in their entirety as well as their intron-exon junctions. DNA isolated from CD-1 mouse tumors was amplified and directly sequenced using nested primers. Nine spontaneous hepatocellular carcinomas (HCCs) and 34 chemically induced HCCs (induced by single intraperitoneal injections of N-nitrosodiethylamine [DEN] [8 HCCs], 7,12-dimethylbenz[a]anthracene [DMBA] [8 HCCs], 4-aminoazobenzene [8 HCCs], and N-OH-2-acetylaminofluorene [10 HCCs]) were examined for mutations in exons 5-8 of the p53 gene. In addition, 12 spontaneous, 10 DMBA-induced, and 13 DEN-induced lung adenocarcinomas or adenomas were analyzed for mutations. No mutations were found in any of the tumors examined. However, a mutation was demonstrated at codon 135 in the positive-control plasmid LTRp53cG(val). The results of this study suggest that inactivation of p53 is unlikely to play a major role in murine lung or liver carcinogenesis. However, inactivation of p53 may occur at a very low frequency, or it may occur as a late event and therefore be present in only a very small number of the tumor cells, rendering it undetectable by this method. Lastly, although few p53-inactivating mutations are found outside of exons 5-8 in human tumors, it is possible that these murine tumors contained mutations outside of this region and were therefore missed by our approach.     

Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation.

We investigated the immunocytochemical staining and immunoblotting characteristics of 33 different p53 mutant proteins identified in lung cancer cell lines (18 small-cell lung cancer and 15 non-small-cell lung cancer) using monoclonal antibodies pAbs 240, 421 and 1801. The p53 mutants studied were representative of those found in lung cancer and included three deletions, four nonsense, seven splicing and 19 missense lesions. Control cell lines included six B-lymphoblastoid cell lines and two lung cancer cell lines without p53 mutations. Immunocytochemistry demonstrated 16 cell lines (48%) with definite overexpression of p53 protein (the high-expresser group of mutants), while in the remainder of cases either no p53 expression or low levels of p53 protein expression were found (the low-expresser group of mutants). The type of p53 mutation correlated with the expresser group. High expressers all had p53 missense mutations in exons 5-8, and immunocytochemistry identified 16/17 (94%) of these mutants. Several classes of p53 mutations occur in the low-expresser groups: deletions, splicing mutants, nonsense mutants and missense mutations outside of exons 5-8 all resulted in very low or undetectable levels of p53 protein. We conclude that there are low- and high-expression groups of p53 mutants in lung cancer and that the detection of protein expression in tumor cells by immunocytochemistry and immunoblotting is dependent upon the type of mutation of the p53 tumor-suppressor gene.     

Effect of wild-type and mutant E-cadherin on cell proliferation and responsiveness to the chemotherapeutic agents cisplatin, etoposide, and 5-fluorouracil

OBJECTIVES: The cell adhesion molecule E-cadherin acts as a tumor and invasion suppressor and regulates cell proliferation. The aim of the present study was to investigate the impact of wild-type (wt) E-cadherin and tumor-derived mutant E-cadherin variants on the proliferation rate of MDA-MB-435S mammary carcinoma cells and the sensitivity of the cells to the chemotherapeutic drugs cisplatin, etoposide and 5-fluorouracil (5-FU) and whether p53 is involved in the chemotherapeutic response. METHODS: Proliferation rate was measured by XTT cell viability assay in the presence or absence of chemotherapeutics. Chemosensitivity was also measured by colony formation assay. Expression of p53 was investigated by immunoblot analysis. The mutational hot spot region exon 5-8 of p53 was analyzed for mutations by denaturing high-performance liquid chromatography. RESULTS: The growth rate of MDA-MB-435S cells transfected with wt E-cadherin was reduced as compared with the parental cell line. In contrast, tumor-associated mutations of exons 8 or 9 of the E-cadherin gene interfere with the growth-suppressive function of E-cadherin. Cisplatin sensitivity of wt and mutant E-cadherin-expressing MDA-MB-435S cells was reduced as compared with E-cadherin-negative, parental MDA-MB-435S cells. In contrast, chemosensitivity of parental, wt or mutant E-cadherin-expressing MDA-MB-435S cells measured after etoposide or 5-FU exposure was found to be similar in all tested cell lines. Since p53 influences the sensitivity of cells to chemotherapeutic agents, we investigated whether the p53 expression level or mutation status were different in the nontransfected or E-cadherin-transfected MDA-MB-435S cell lines. We found that the p53 expression pattern and genomic background were similar in all cell lines and not affected by cisplatin. CONCLUSION: The results obtained in this study suggest that the expression and/or mutation of the E-cadherin gene influence the proliferation rate and drug sensitivity of tumor cells.     

Mutation of p53 gene in hepatocellular carcinoma in Taiwan.

To elucidate the role of p53 mutation in hepatocarcinogenesis in Taiwan, a hepatitis B viral infection hyperendemic area, exons 5 to 8 of the p53 gene in the tumor tissue of 61 hepatocellular carcinomas were amplified and sequenced. A total of 20 cases (32.8%) were found to have mutations; 36.6% (15 of 41) for the hepatitis B surface antigen positive group and 25.0% (5 of 20) for the hepatitis B surface antigen negative group. The corresponding normal liver showed no mutation. The mutation is widely distributed throughout exons 5 to 8. Only 4 cases (6.6%), all positive for hepatitis B surface antigen, had a specific hot spot mutation at codon 249 with G to T transversion. Our results show that scattered point mutations in p53 are not uncommon in hepatocellular carcinoma samples from Taiwan and may be important in the development of this cancer. However, the aflatoxin related specific mutation seems much less related to the genesis of hepatocellular carcinoma in Taiwan.