p53蛋白三维空间结构改变与人骨肉瘤的关系

目的 研究骨肉瘤中p53基因突变及其蛋白三维结构的改变,旨在分析p53基因突变的分子机制及其与骨肉瘤发生、发展的关系.方法 应用PCR-SSCP、DNA序列分析以及相关分析软件重构p53蛋白三维结构等技术对43例骨肉瘤进行研究.结果 将16例p53基因突变病例分为A组(p53基因DNA结合位点发生突变或缺损组)与B组(非DNA结合位点突变组或同义突变),A组的远处转移率明显高于B组(P<0.01),组织学分化低于B组(P<0.05),且中位生存时间(10.3个月)低于B组(34.3个月).结论 p53基因碱基的突变可引起相应氨基酸的改变,导致p53蛋白三维空间构象改变.p53基因DNA结合位点的突变可影响其蛋白的生物学功能,并促进骨肉瘤的发生、发展.     

人类恶性畸胎瘤PA-1细胞的p53基因结构、功能状态及其意义

目的:探讨人类恶性畸胎瘤PA-1细胞株p53基因的结构、功能状态及其意义。方法:采用DNA碱基序列分析、FASAY（酵母细胞中单个等位基因的功能分析）及Western blot（蛋白印迹分析）等方法对经20余年407-445继代培养的PA-1细胞株p53的基因结构、功能状态进行了研究。结果:RT-PCR产物DNA定向序列分析显示具有野生及突变2个带（p53密码子239突变）,FASAY结果也显示p53一个等位基因是有活性的（野生的）,而另一个是非活性的（突变的）。Western blot未检测出突变的p53基因蛋白,而p21蛋白的表达水平比正常成纤维细胞低。结论:人卵巢恶性畸胎瘤PA-1细胞株经20年的继代培养后,一个p53等位基因发生错义突变,另一个仍然是野生型的。仅一个p53等位基因发生突变,不足以引起细胞的染色体不稳定性。因此,研究PA-1细胞稳定核型的维持结构,在确定有关染色体的稳定性与不稳定性的基因的方面是非常重要的。     

人恶性畸胎瘤PA-1细胞染色体特性及其影响因素的探讨

目的:探讨人类恶性畸胎瘤PA-1细胞株染色体特性及其影响因素。方法:采用G带核型分析、DNA碱基序列分析及Western blot(蛋白印迹分析)等方法对经20余年407-445继代培养的PA-1细胞株染色体核型及p 53 基因状态进行了研究。结果:PA-1细胞株80%以上仍然保持近乎二倍体的核型,30代以后的细胞由于第15号染色体与20号染色体间的相互易位形成了M1及M2标识染色体。RT-PCR产物DNA定向序列分析显示具有野生及突变两个带(p53密码子239突变),Western blot 未检测出突变的p53基因蛋白,而p21蛋白的表达水平比正常成纤维细胞低。结论:人卵巢恶性畸胎瘤PA-1细胞株经20年的继代培养后,一个p53 等位基因发生错义突变,另一个仍然是野生型的。仅一个p53 等位基因发生突变,不足以引起细胞的染色体不稳定性。     

p53基因第8外显子在喉癌中的缺失和点突变

目的 探讨抑癌基因p53第8外显子突变在喉鳞癌分子发病机理中所起的作用。方法 应用聚合酶链反应－单链构象多态性（PCR－SSCP）银染技术和DNA直接测序法,检测喉鳞癌新鲜组织中抑癌基因p53基因第8外显子的突变情况。结果 60例喉鳞癌组织中,15例发生迁移率异常的SSCP区带。在SSCP阳性样本中随机抽出4例进行测序分析,发现两个标本在288位密码子缺失一个A,两个标本在292位密码子上缺失一个A。285、287密码子上共发生3次G→T的颠换,286密码子第3位碱基发生A→T的颠换。结论 喉鳞癌p53基因点突变与碱基缺失常常同时并存。p53基因第8外显子突变在喉癌的发生中可能起着重要作用。     

p53与肿瘤的形成,治疗和预后

p53基因作为抑制基因在约50％的人类肿瘤中被检测出来,其蛋白产物p53蛋白与特异性DNA序列结合并激活转录。转录激活导致p53增加,进而诱导蛋白p21、WAF1／CIP1产生,通过p21与。cyclin－cdk复合物结合来抑制cdk活性,从而达到控制细胞从GI期进入S期的目的．也可通过p21与PCNA结合抑制DNA复制。p53在肿瘤形成中的作用50－55％的人类肿瘤中发现有p53突变。这些突变完全淘汰不能与特异DNA序列结合的P53蛋白。P53的细胞周期抑制功能需要p53转录活性,至少有些p53的凋亡活动不需要有赖于p53的基因产物。这可能意味着,被选p53靶基因的…     

肝癌的肿瘤抑制基因研究

肝癌的肿瘤抑制基因研究进展迅速。杂合性丢失分析资料显示,染色体8p、13q、16q和17p区域存在与肝癌发生和演进关系密切的肿瘤抑制基因。肝癌中常见TP53基因突变,且多为第249密码子第3碱基G→T颠换。TP53基因这种特异性点突变代表肝癌的特征,可能和黄曲霉素B1摄入有关,但在肝癌中并没有普遍性。肝癌中染色体13q缺失的最小重叠区域为13q14,在肝癌组织中还同时发现伴随等位基因丢失的存留RBl等位基因突变以及和RBl等位基因丢失高度相关的p110~(RBl)蛋白丢失。因此,RBl基因为肝癌的又一重要肿瘤抑制基因。     

p53研究的新进展

肿瘤抑制基因p53是目前研究最为广泛和系统的抑癌基因之一。野生型p53(wt-p53)参与了DNA损伤修复、细胞周期调控、细胞凋亡及抑制血管生成等过程。p53基因的突变使上述功能丧失,从而导致肿瘤的形成。在大约50％人类肿瘤中可发现p53基因的突变,且几乎可见于各种类型的肿瘤细胞中。本文就目前p53研究及相关基冈治疗的进展作一综述。     

p53基因249M和273H突变在肝癌细胞系HepG2中与热休克蛋白70定位关系的研究

目的:定点突变合成p53基因突变子,构建绿色荧光蛋白表达载体,进而观察其在HepG2细胞中与内源性热休克蛋白70的共定位关系.方法:以野生型p53为模板,采用PCR体外定点突变技术通过重叠延伸法2次PCR扩增得到目的基因片段,进一步将其克隆到绿色荧光蛋白载体pEGFP-C3中.将构建好的载体转染到HepG2 细胞中,利用免疫荧光染色法检测内源性热休克蛋白70的表达,利用荧光显微镜观察hsp70与p53的共定位关系.结果:测序结果显示片段插入正确,在预期位点发生了点突变,249位氨基酸由AGG突变为AGC,273位氨基酸由CGT突变为CAT.载体成功构建.转染后观察到热休克蛋白70与273H p53共同定位于肝癌细胞系HepG2的胞质,而wt p53与249M p53均定位于细胞核.结论:热休克蛋白70与不同点突变的p53在肝癌细胞系HepG2中的共定位关系与之前我们在肝癌组织中发现的hsp70与p53共定位关系不同,这些提示在hsp70与p53的共定位关系和相互作用上存在某种尚未阐明的机制.     

乳腺癌细胞动力学指标与相关基因表达、突变的关系

目的 探讨乳腺癌细胞动力学及凋亡与相关基因表达、突变的关系。方法 采用流式细胞术检测54例乳腺癌DNA指数(DI)、S期细胞比例(SPF)、细胞增殖指数(PI)及细胞凋亡指数(AI)，免疫组织化学法检测原癌基因c—erbB-2、Bcl-2、抑癌基因p53，增殖细胞核抗原PCNA、Ki67及托普DNA酶Ⅱ(TopoⅡ)的表达，PCR—SSCP法检测p53点突变及突变部位、类型；结果 高DI、异倍体率、SPF、PI、AI与c—erbB-2、p53、PCNA、Ki67、TopoⅡ高表达相关。低DI、SPF、PI、AI与Bcl-2高表达相关。高DI、SPF、PI、AI与p53高突变相关，高AI与p53突变类型之一——杂合性缺失(LOH)相关。结论 乳腺癌细胞动力学及凋亡的异常与相关基因的异常表达及突变密切相关。     

肝细胞癌病人中p53基因突变状况探讨

目的 了解肿瘤抑制基因p53在肝细胞癌中的突变情况,探讨乙型肝炎病毒（HBV）感染与p53基因突变之间的关系。方法 提取50例有乙型肝炎病毒感染史肝癌患者手术样本中的DNA,用聚合酶链式反应（PCR）扩增5－9外显子,作单链构象多态性（SSCP）分析。结果 p53基因突变率超过26％,突变主要分布于5－8外显子,5、6、7、8外显子分别有3、3、4、3例,另仍4个可疑突变。结论 p53基因突变可能是肝细胞癌的病因之一,而乙型肝炎病毒感染在中国肝癌患者p53基因突变中可能起到比较重要的作用。     

Multistep carcinogenesis in anaplastic thyroid carcinoma: a case report

We previously established an anaplastic thyroid carcinoma cell line (KOA2) that had double mutations: an N-ras mutation and a p53 gene mutation. To clarify multistep carcinogenesis, we analysed surgical material from the patient from whom KOA2 was derived for abnormalities in the N-ras and p53 genes. The resected material had two histologically different lesions: a follicular neoplasm and an anaplastic carcinoma. The N-ras mutation was observed in both lesions, but the p53 gene mutation only in the anaplastic lesion. These facts indicate that an N-ras mutation may induce follicular neoplasm and a subsequent p53 mutation may have caused the follicular neoplasm to transform to anaplastic carcinoma in this patient. This report suggests direct evidence for multistep carcinogenesis in anaplastic thyroid carcinoma.     

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。     

Analysis of K-ras, N-ras, H-ras, and p53 in lung neuroendocrine neoplasms.

This study screened 11 samples of typical carcinoid (TC), 4 samples of atypical carcinoid (AC), 1 sample of large cell neuroendocrine carcinoma (LCNEC), and four metastases for point mutations in exons 5 to 8 of the p53 gene, and exons 1 and 2 of the K-ras. H-ras, and N-ras genes using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) and direct sequencing and by immunohistochemistry for p53. Exon 1 of K-ras was mutated in two samples of low-grade AC and a metastasis from one of these tumors (GAT12 and AGT12, respectively). No mutations in N-ras or H-ras were found. Mutations in exons 5 and 8 of the p53 gene were identified in a high-grade AC and a LCNEC. Positive immunostaining for p53 was present in three samples, with only one genotypic mutation shown (LCNEC). In conclusion, point mutations of the p53 gene were infrequent in these pulmonary neuroendocrine tumors, did not correlate in all samples with immunostaining, and were associated with the higher-grade tumors. Second, the presence of K-ras mutations seems to be associated with the higher-grade carcinomas. Third, N-ras and H-ras mutations were not found with these pulmonary neuroendocrine tumors.     

用显微切割技术定点检测诱发大鼠肺癌发生发展各阶段p53、K-ras基因突变与表达

目的　探讨p5 3、K ras基因突变、蛋白表达在 3 甲基胆蒽 (3 methylcholanthrene ,MCA)和二乙基亚硝胺 (diethylinitrosamine ,DEN)诱发大鼠肺鳞癌发生演进中的作用 ,及其突变与蛋白表达的关系。方法　将大鼠诱发肺癌石蜡标本连续切片 ,切片用于HE染色确定肺癌发生发展的病变阶段 ,及免疫组织化学 (SP法 )检测各阶段p5 3、K ras蛋白表达 ,并用于显微切割 ,定点对位分别切割由正常支气管黏膜上皮细胞演变成癌细胞 ,癌浸润、转移各阶段病灶的主质 ,提取DNA ,用聚合酶链反应 单链构象多态性 (PCR SSCP)检测各阶段p5 3、K ras基因的突变。结果　 30例正常支气管黏膜上皮未检测到p5 3、K ras基因突变及其蛋白表达。在 32例支气管黏膜增生和鳞状化生、2 1例不典型增生、12例原位癌、4 3例浸润癌及 17例转移癌组织中 ,p5 3基因突变率分别为 3 1% ,2 8 6 % ,30 0 % ,5 1 2 % ,5 2 9% ;p5 3蛋白阳性表达率分别为 0 ,4 2 9% ,5 0 0 % ,6 0 5 % ,6 4 7% ;不典型增生阶段与增生、鳞状化生阶段相比 ,p5 3基因突变率及蛋白表达率增高 ,差异均有显著性意义 (P <0 0 2 5 ,P <0 0 0 5 ) ,p5 3基因突变及蛋白阳性表达高度相关 (P <0 0 0 5 ,Pearson′sR =0 5 996 )。K ras基因突变率分别为 0 ,4 8% ,8 3% ,9 3     

p^53基因与Rb基因在卵巢癌组织中突变的初步分析

为了解卵巢癌组织中抗癌基因Ｐ＾５３与Ｒｂ基因的突变情况，我们把ＰＣＲ单链构象多态（ＰＣＲ－ＳＳＣＰ）银染技术及ＰＣＲ－ＤＮＡ直接测序方法应用于５７例卵巢癌组织细胞的检测，结果发现ｐ＾５３基因可能的突变率为３２％，Ｒｂ基因可能的突变率为２１％，不同分化程度肿瘤的Ｐ５３及Ｒｂ基因突变率未见明显区别，结论：ＰＣＲ－ＳＳＣＰ角染技术是筛查基因突变简便，敏感，无核素污染的最佳方法，而ＰＣＲ－ＤＮＡ直接测序仪     

No consistent pattern of mutations in p53 and ras genes in liver tumors of rat treated with the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX)

The frequency of point mutations in p53 (exons 4-7) and in Ki-ras, Ha-ras, and N-ras (exons 1 and 2) and the expression of p53 protein were evaluated in the liver tumors of Wistar rats of a 104-week carcinogenicity study on 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product in drinking water. Mutations were analyzed in 16 hepatocellular adenomas, 7 hepatocellular carcinomas, 23 cholangiomas, and 2 cholangiocarcinomas of the MX-treated animals and one hepatocellular carcinoma and cholangiocarcinoma in control animals using PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) or PCR-TGGE (temperature gradient gel electrophoresis) and direct sequencing. The expression of the p53 protein (wild-type and mutated protein) was detected by immunohistochemistry (CM5 antibody). The expression of p53 and that of the proliferating cell nuclear antigen (PCNA, 19 A2) were also evaluated in livers of female animals exposed to MX for 1 week, 3 weeks, or 18 weeks. Altogether, four mutations were found in p53 in three tumors, in two hepatocellular adenomas, and one cholangiocarcinoma, all in females receiving the highest MX dose (6. 6 mg/kg/day) of the study. Three of the mutations were G:C --> A:T transitions and one was an A:T --> T:A transversion. The mutations were scattered at different codons and positions of the codon. One hepatocellular adenoma contained two p53 mutations. All cholangiomas and cholangiocarcinomas, but no hepatocellular adenomas and carcinomas, overexpressed the p53 protein. MX treatment did not induce p53 expression at any age in the liver or alter the expression of the PCNA in the liver of younger animals. The p53 protein was overexpressed in hyperplastic bile ducts in aged rats but not in bile ducts of younger rats (up to 24 weeks). No mutations were observed in either Ki-ras, Ha-ras, or N-ras of the liver tumors. These data suggest that point mutations in p53, Ki-ras, Ha-ras, and N-ras are not involved in the MX-induced liver carcinogenesis in rats.     

亚硝基诱发大鼠胃腺癌发生发展过程中p53,ras基因表达和突?…

目的 探讨Ｐ５３、ｒａｓ基因在亚硝基胍诱发大鼠胃腺癌发生发展过程中的作用。方法 用免疫组织化学、聚合酶链反应－单链构象多态怀分析（ＰＣＲ－ＳＳＣＰ）技术对大鼠正常腺胃粘膜、癌前病变和胃腺癌组织中ｐ５３、ｒａｓ基因的表达和突变进行检测。结果Ｐ５３蛋白在癌组织 的了性表达率为５０％,正常和癌前病缲为阴性;ｒａｓ蛋白癌前病变组织中阳性率为４４％,在癌组织中为２３％。癌组织中ｐ５３基因突变率为４５％,非     

Overexpression of inducible nitric oxide synthase and accumulation of 8-OHdG in nasopharyngeal carcinoma

Aims:  Nitric oxide (NO), produced by inducible NO synthase (iNOS), has been suggested to cause oxidative stress, leading to 8-hydroxydeoxyguanosine (8-OHdG) accumulation and subsequent transversion mutation of DNA. The aim was to evaluate iNOS expression and the status of oxidative stress in nasopharyngeal carcinoma (NPC).
Methods and results:  Seventy-three cases of NPC were investigated to examine the immunohistochemical expression of iNOS, 8-OHdG and latent membrane protein-1 (LMP-1) and Epstein–Barr virus-encoded small RNA (EBER) expression using in situ hybridization. iNOS mRNA expression and p53 gene mutations were also assessed. Overexpression of iNOS, LMP-1 and EBER was observed in 62 (84.9%), 28 (38.4%) and 53 (72.6%) cases respectively. p53 gene mutation was found in 10 of 73 (13.7%) cases. Immunohistochemical iNOS expression was associated with the 8-OHdG labelling index, iNOS mRNA expression and p53 gene alteration ( P  < 0.0001, P  = 0.016 and 0.0082 respectively).
Conclusions:  Our present findings suggest that the expression of iNOS induces oxidative stress in NPC. Although the presence of p53 mutation was associated with iNOS overexpression, the type of acid–base change of p53 was transition, but not transversion, which suggests that the p53 gene is not the direct target of DNA damage by 8-OHdG accumulation.     

Analysis of p53 and mdm2 proteins in malignant fibrous histiocytoma in absence of gene alteration: prognostic significance

TP53 and MDM2 genes and their protein expression were evaluated in frozen and paraffin-embedded tissue from 27 patients with malignant fibrous histiocytoma to elucidate the relationship between them, their implication in tumor progression mechanisms and their possible diagnostic-prognostic value in malignant fibrous histiocytoma. Single-strand conformation polymorphism analysis and direct sequencing of polymerase chain reaction-amplified DNA were used to establish two TP53 mutations (7.4%): a point mutation and a 63-bp duplication. Amplification of the MDM2 gene was observed in two tumors (7.4%) by means of Southern-blot analysis, one of them also carrying the TP53 point mutation. Immunohistochemical and Western-blot techniques were used to study nuclear accumulation of p53 and mdm2 proteins: 11 cases (40.7%) with p53 protein expression and thirteen cases (48.1%) with mdm2 protein expression were detected. We confirmed overexpression of mdm2 protein in eight of ten cases (80%) with p53 protein expression without TP53 gene mutation. Statistical analysis shows that simultaneous co-expression of p53 and mdm2 in malignant fibrous histiocytoma is significantly correlated with survival in absence of gene alteration in contrast to the lack of statistical correlation with survival of p53 protein expression alone. Received: 13 April 1999 / Accepted: 14 July 1999     

Myoepithelial carcinoma arising in an adenomyoepithelioma of the breast: case report with immunohistochemical and mutational analysis

Adenomyoepithelioma (AME) of the breast is an uncommon tumor characterized by biphasic proliferation of both epithelial and myoepithelial cells. In rare instances, the epithelial, the myoepithelial or both components of an AME may become malignant. Described herein is the case of a 69-year-old woman who presented with myoepithelial carcinoma of the breast in an AME. Malignancy of myoepithelial component (MEC) was evidenced by the presence of cytological atypia, high mitotic rate, necrosis and local invasion. Immunohistochemical study demonstrated strong expression of P53 and phosphorylated extracellular signal-regulated kinase 1/2 in MEC. Laser capture microdissection technique and mutational analysis further revealed point mutation of the p53 gene (T-->G transversion at codon 270) in this population, but not in glandular epithelial cells or adjacent normal ductal epithelium. No mutations in exons 1 and 2 of the K-, H-, and N-ras genes were identified in any of the neoplastic component. To the authors' knowledge this is the first report of a mutation in the p53 gene in a malignant AME of the breast.