鼻咽癌中KLF6基因突变的检测

背景与目的：鼻咽癌（nasopharyngeal carcinoma,NPC)高发于中国南方和东南亚地区,其发病是一个多因素,多步骤的过程,与遗传因素,EB病毒感染和环境因子有关,但分子机理仍不清楚,KLF6基因编码一种广泛表达的核转录因子,在某些前列腺癌肿瘤中发现有高频的等位基因的丢失和基因突变,本研究通过对KLF6基因在NPC肿瘤中的突变检测,探讨KLF6基因与NPC发病的关系。方法：采用PCR-直接测序分析方法。在19例散发性NPC组织细胞和3个NPC细胞系（CNE1,CNE2,SUNE1）中对KLF6编码区和拼接位点进行检测。结果：在3你散发性NPC肿瘤组织DNA中检测到KLF6基因的3个不同位置的错义改变,分别为GAG→TG（Glu75Val),AGG→AGG（Ser136Arg)和AGA→AAA（Arg243Lys),在50个随机正常个体（100条染色体）和3个NPC细胞系中没有发现这3个位点的碱基变异,结论：这3个变异可能是KLF6基因的新的突变位点,在某些NPC发病中可能有作用。     

人前列腺癌组织中p53基因突变和p21表达缺失

目的 :探讨人前列腺癌组织中p53基因的突变和p2 1WAF1/CIP1　　　蛋白表达的缺失。方法 :采用p53基因突变的PCR SSCP分析及DNA测序测定 2 0例患者前列腺癌标本 ,同时检测p2 1WAF1/CIP1　　　　蛋白的表达。结果 :在 2 0个前列腺癌标本中 ,4例发现有p53基因的点突变 ( 2 0 % ) ,这 4例标本的前列腺癌细胞中均伴有p2 1WAF1/CIP1　　　　蛋白的表达缺失。结论 :人前列腺癌的发生和p53基因的突变有关 ,而这些p53基因异常可导致p2 1WAF1/CIP1　　　　蛋白的表达缺失。     

前列腺癌可能存在多克隆起源

目的 :探寻人类前列腺癌发生和多发的细胞克隆来源。方法 :采用病理切片上病灶显微切割、P5 3基因突变的PCR SSCP分析及DNA测序测定 3例患者前列腺癌根治切除术标本中癌灶和高分级PIN灶组织 ;同时还采用免疫组织化学方法检测了p2 1WAF1/CIP1蛋白的表达。结果 :在人前列腺癌灶和高分级PIN灶组织中均发现有p5 3基因的点突变 ,同 1例患者标本中癌灶和高分级PIN灶所含点突变均在不同位点上。含p5 3基因的点突变的癌灶和高分级PIN灶组织中均未见有p2 1WAF1/CIP1蛋白的表达。结论 :人前列腺癌发生和多发可能有多细胞克隆来源 ,其p2 1WAF1/CIP1蛋白的表达系P5 3基因依赖性途径     

50例乳腺癌患者BBCA1基因exon11突变研究

[目的]检测50例乳腺癌患者BRCA1基因exon11突变情况及突变位置,探讨BRCA1突变与乳腺癌的关系。[方法]采取50例乳腺癌全血标本为实验组,28例非癌乳腺全血标本为对照组。应用PCR和DNA直接测序法检测所有标本BRCA1基因exon11的突变情况。[结果]28例非癌乳腺组织BRCA1基因exon11未检出突变,50例乳腺癌中有6例发生基因突变。占总例数的12．0％。6例中2例发生多个位点突变,19号标本5个突变位点：2685T→C,2201C→T。2731C→T,3232A→G,3667A→G;30号标本2个突变位点：2685T→C;204T→A。8个位点为错义突变：2532T→C,2685T→C2例,2731C→T,3232A→G,3667A→G2例,2041T→A。3个位点为同义突变：2630T→G2例,2201C→T。发现两个新位点2201位和2731位。[结论]BRCA1基因exon11突变与乳腺癌的发生关系密切,对其进行检测可能对乳腺癌的患病风险评估及早期诊断具有重要意义。     

鼻咽癌组织中PIK3GA基因热点突变区的突变筛查

背景与目的:近期研究发现磷脂酰肌醇激酶-3催化亚单位α基因(phosphatidylinositol 3-kinase catalytic alpha polypeptide gene,PIK3CA)在多种肿瘤中存在高频的体细胞突变,并且突变常发生在PIK3CA的第9外显子和20外显子两个热点突变区.对这两个热点突变的研究表明,这些突变能增强该酶的活性、有助于肿瘤细胞的侵袭、对抗凋亡等.因此提示PIK3CA基因是与多种肿瘤发生、发展相关的癌基因.本研究旨在检测鼻咽癌组织中PIK3CA两个热点突变区的突变,以及该基因的变异与鼻咽癌发病的关系.方法:在鼻咽癌组织及患者外周血中筛查PIK3CA的突变热点区第9外显子和第20外显子.对46例散发性鼻咽癌组织标本采用PCR产物克隆测序,对与之匹配的46例鼻咽癌患者外周血标本和3个鼻咽癌细胞系(CNE1,CNE2,SUNE1),则将PCR产物直接测序.结果:在46例鼻咽癌组织标本中在PIK3CA热点突变区第9号外显子检出2例突变(4.3%):1例为T1563G(521Asn→Lys);另1例为A1646G(549Asp→Gly).在46例鼻咽癌标本第9外显子中18例检出A1634C-G1658C-del 1659T"复合突变",进一步研究表明该"复合突变"可能为22号染色体上同源区域的序列.在对PIK3CA另外一个突变热点区第20外显子的检测中,46例鼻咽癌组织标本中都没检测到突变.另外,在3个鼻咽癌细胞系和46例鼻咽癌匹配外周血DNA标本用PCR-直接测序法均未检测到第9外显子和第20外显子突变.结论:PIK3CA基因第9外显子与第20外显子鼻咽癌中较少发生突变;克隆测序检测体细胞突变具有更高的敏感性,通过该方法在第9外显子发现的"复合突变"可能是22q11.2的Cat Eye Syndrome region高度同源区域的序列而非PIK3CA基因座的变异.     

人前列腺癌组织中p53基因突变和p21表达缺失

目的：探讨人前列腺癌组织中p53基因的突变和p21^WAF1/CIP1蛋白表达的缺失。方法：采用p53基因突变的PCR－SSCP分析及DNA测序测定20例患者前列腺癌标本,同时检测p21^WAF1/CIP1蛋白的表达。结果：在20个前列腺癌标本中,4例发现有p53基因的点突变（20％）,这4例标本的前列腺癌细胞中均伴有p21^WAF1/CIP1蛋白的表达缺失。结论：人前列腺癌的发生和p53基因的突变有关,而这些p53基因异常可导致p21^WAF1/CIP1蛋白的表达缺失。     

食管癌组织中抑癌基因APC,MCC突变的研究

应用聚合酶链反应（ＰＣＲ）扩增与直接测序方法，分析抑癌基因ＡＰＣ、ＭＣＣ在食管癌中的变化，应用ＰＣＲ扩增，发现１／１０的食管癌组织有ＡＰＣ基因第１１外显子缺失，１／１０的食管癌组织有ＭＣＣ基因第１２外显子缺失，并发现１例食管癌旁癌组织有ＭＣＣ基因基因第１２个外显子缺失。ＰＣＲ直接测序发现：２／１０的食管癌标本有ＡＰＣ基因第１１外显子突变，２／７的食管癌组织有ＭＣＣ基因第１２外显子突变。以上研究证实     

乳腺癌中P16基因缺失与突变的研究

背景与目的:了解乳腺癌与p16基因的关系.材料与方法:采用PCR和DNA测序方法,对33例乳腺癌患者肿瘤组织标本的p16基因进行检测,研究p16基因的缺失和突变情况.结果:所有标本均未检出纯合缺失,10例标本进行了外显子2的序列测定,也未发现点突变.结论:p166基因的纯合缺失和点突变可能与乳腺癌的发生无关.     

非小细胞肺癌组织化疗前后表皮生长因子受体基因的突变

目的:探讨非小细胞肺癌(non-small cell lung cancer,NSCLC)患者化疗前、后肿瘤组织中表皮生长因子受体(epidermal growth factor receptor,EGFR)基因外显子19和21的突变状况。方法:提取31例NSCLC患者化疗前、后肿瘤组织标本中的基因组DNA,采用巢式PCR技术扩增EGFR基因外显子19和21,并进行测序分析。结果:6例患者化疗前、后EGFR基因发生突变,其中4例为19号外显子发生缺失突变,2例为2l号外显子发生替代突变,且化疗前、后的突变状况一致。女性患者突变率(2/3)高于男性(4/28)(P=0.029),非吸烟者的突变率(4/9)高于吸烟者(2/22)(P=0.043)。结论:NSCLC组织EGFR基因外显子19和21突变在化疗前、后无明显改变。     

肝癌DKK1基因的表达及突变分析

目的:探讨中国地区人肝癌发生发展过程中有无DKK1基因突变。方法:Northernblot方法分析12例肝癌患者癌和癌旁肝组织DKK1mRNA表达水平。采用PCRSSCP方法,检测20例肝癌患者(包括癌和癌旁肝组织)及8种人肝癌细胞株中有无DKK1基因突变,并采用DNA测序对PCRSSCP突变分析结果加以验证。结果:Northernblot显示12例肝癌患者中7例存在癌组织DKK1mRNA高表达而癌旁肝组织微弱表达或不表达。突变检测发现20例肝癌患者及8种人肝癌细胞株中仅1例肝癌患者存在DKK1基因的同义突变(单核苷酸多态现象)。结论:DKK1在中国人肝癌中存在高表达,但未发现突变发生,提示DKK1基因参与肝癌的癌变过程可能并不依赖于该基因的突变。     

Analysis of human prostate cancers and cell lines for mutations in the TP53 and KLF6 tumour suppressor genes

A recent report suggests that the KLF6 gene encoding the Krüppel-like factor 6 protein is a frequently mutated, putative tumour suppressor gene in prostate cancer. The aims of the present study were to confirm these initial findings by determining the frequency of exon2 KLF6 mutations in a cohort of European prostate cancer patients, and to investigate whether there was evidence for mutational inactivation of both the KLF6 and TP53 tumour suppressor loci in some tumours. We examined 32 primary prostate tumours and three prostate tumour cell lines for mutations by PCR amplification and direct dideoxy sequencing (KLF6), and by oligonucleotide microarray (p53GeneChip) analysis and dideoxy sequencing (TP53). Whereas TP53 mutations typical of prostate cancer were found at a frequency consistent with the literature, no KLF6 mutations were found in any of the tumour samples nor in the three prostate cancer cell lines.     

Difference in the role of loss of heterozygosity at 10p15 (KLF6 locus) in colorectal carcinogenesis between sporadic and familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer patients

OBJECTIVES: To clarify the role of the KLF6 (Kruppel-like factor 6) locus in multistep colorectal carcinogenesis, we analyzed loss of heterozygosity (LOH) at 10p15 (KLF6 locus) and mutations of the KLF6gene in 298 colorectal tumors at various pathological stages of sporadic and familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) patients. METHODS: 10p15 LOH was analyzed using KLF6M1 and KLF6M2, and KLF6 gene mutation was analyzed using PCR-SSCP and sequencing. RESULTS: It was found that the frequencies of LOH (sum of M1 and M2) were 4% in adenomas, 0% in intramucosal carcinomas, 35% in invasive carcinomas, and 33% in liver metastases in sporadic cases. Invasive carcinomas from FAP patients showed only 6% LOH, and invasive carcinomas from HNPCC patients exhibited 0% LOH. Mutation analysis of the KLF6 gene in 298 colorectal tumors detected no somatic mutations. CONCLUSIONS: The present data suggest that LOH of the KLF6 locus at chromosome 10p15 contributes to the invasion step from an intramucosal carcinoma to an invasive carcinoma specifically in sporadic colorectal carcinogenesis, but is rarely involved in the carcinogenesis of FAP and HNPCC cases. Moreover, the absence of somatic mutations suggests the uncertainty of the KLF6 gene as a classical tumor suppressor gene at the lost 10p15 region in colorectal carcinogenesis.     

Frequency and characterization of p53 mutations in primary and metastatic human prostate cancer

We have studied the frequency of mutations in the p53 gene in human prostate cancer. The investigated material consisted of 20 primary-tumor tissue specimens, obtained by transurethral resection and tissue specimens of 15 lymph-node metastases, obtained at total prostatectomy. The applied methods encompassed immunohistochemistry on frozen sections, using the monoclonal antibody PAb 1801, and single-strand conformation polymorphism (SSCP) analysis, after amplification of single exon sequences by PCR, on exons 5 to 8 of the p53 gene. The mutations, leading to aberrantly migrating bands in the PCRSSCP analysis, wereidentified by direct sequencing of the PCR product. Immunohistochemical and PCR-SSCP analysis were completely confirmative. In the primary tumors, mutations were found in 10% of the specimens (codons 232 and 273), and in lymph-node metastases in 15% of the specimens (codons 248 and 273). In one case (codon 273), the same mutation was found both in the primary tumor and in the lymph-node metastasis. Our results show that p53 mutations are infrequent in both primary and metastatic prostate tumors. In addition, they indicate that there is no strict correlation between p53 mutation and tumor metastasis.     

Targeted inhibition of the KLF6 splice variant, KLF6 SV1, suppresses prostate cancer cell growth and spread

Prostate cancer is a leading cause of cancer death in men. Risk prognostication, treatment stratification, and the development of rational therapeutic strategies lag because the molecular mechanisms underlying the initiation and progression from primary to metastatic disease are unknown. Multiple lines of evidence now suggest that KLF6 is a key prostate cancer tumor suppressor gene including loss and/or mutation in prostate cancer tumors and cell lines and decreased KLF6 expression levels in recurrent prostate cancer samples. Most recently, we identified a common KLF6 germ line single nucleotide polymorphism that is associated with an increased relative risk of prostate cancer and the increased production of three alternatively spliced, dominant-negative KLF6 isoforms. Here we show that although wild-type KLF6 (wtKLF6) acts as a classic tumor suppressor, the single nucleotide polymorphism-increased splice isoform, KLF6 SV1, displays a markedly opposite effect on cell proliferation, colony formation, and invasion. In addition, whereas wtKLF6 knockdown increases tumor growth in nude mice >2-fold, short interfering RNA-mediated KLF6 SV1 inhibition reduces growth by approximately 50% and decreases the expression of a number of growth- and angiogenesis-related proteins. Together, these findings begin to highlight a dynamic and functional antagonism between wtKLF6 and its splice variant KLF6 SV1 in tumor growth and dissemination.