前列腺癌中P53基因突变及蛋白表达的研究

运用免疫组织化学ＬＳＡＢ法（链菌素－生物素标记法），结合ＰＣＲ－ＲＦＬＰ（ＰＣＲ限制性片段长度多态）、ＰＣＲ－ＳＳＣＰ（ＰＣＲ单链结构多态）技术，探讨了３４例前列腺癌穿刺标本中Ｐ５３蛋白表达及基因（５～８外显子）突变情况，发现３４例前列腺场中７例有Ｐ５３蛋白表达阳性，阳性率为２０．６％。２０例前列腺良性增生及１０例正常前列腺组织中Ｐ５３蛋白表达均为阴性（Ｐ＜０．０５），在前列腺癌高、中分化组及前列腺癌低、未分化组Ｐ５３蛋白表达阳性率分别为５．３％及４０％（Ｐ＜０．０５），在ＰＣＲ－ＲＦＬＰ中提示１例前列腺癌中有Ｐ５３突变，ＰＣＲ－ＳＳＣＰ中提示３例Ｐ５３基因突变（５～６外显子），一例无Ｐ５３基因扩增产物，高度怀疑为Ｐ５３基因的缺失，３４例前列腺癌中，ＣＴ及骨扫描提示有转移者为１０例，其中５例为Ｐ５３蛋白表达阳性，阳性率５０％，与非转移组阳性率比较Ｐ＜０．０５。本研究表明Ｐ５３基因突变参与前列腺癌的发生发展过程，与其转移的生物学行为关系密切。分析了以上不同方法在检测Ｐ５３基因突变中的价值。     

多药耐药基因在泌尿系统肿瘤细胞系中的表达

目的检测泌尿系统肿瘤多药耐药基因及其产物的表达。方法对肾颗粒细胞癌细胞系ＧＲＣ１，肾透明细胞癌细胞系ＲＣＣ９４９，膀胱移行细胞癌细胞系ＢＩＵ８７、Ｔ２４、ＥＪ，前列腺癌细胞系ＰＣ３ｍ和对照的白血病细胞系ＨＬ６０进行免疫细胞化学和逆转录聚合酶链反应（ＲＴＰＣＲ）检测。结果只有ＧＲＣ１同时表达Ｐ糖蛋白（ＰＧＰ）和ＭＤＲ１基因ｍＲＮＡ，其他肿瘤细胞系均无表达。结论泌尿系肿瘤细胞系多数无ＰＧＰ也没有ＭＤＲ１基因ｍＲＮＡ水平的表达，只有ＧＲＣ１例外     

糖皮质激素受体外显子9移码突变在狼疮肾炎发病及治疗中的意义

目的探讨下丘脑垂体肾上腺免疫轴稳定与否同狼疮肾炎（ＬＮ）发病间的关系。方法用放射免疫分析法及聚合酶链反应单链构象多态性（ＰＣＲＳＳＣＰ）、ＤＮＡ测序法分析观察３９例狼疮肾炎ＡＣＴＨ、皮质醇、糖皮质激素受体（ＧＲ）特性及其部分基因结构。结果ＡＣＴＨ、皮质醇在狼疮肾炎组及正常对照组无差别,ＧＲ位点在肾炎组明显低于正常组,但ＧＲ亲和性在两组之间无明显差别。３９例狼疮肾炎中有８例呈现ＧＲ基因外显子９单链构象多态性（ＳＳＣＰ）异常,其ＤＮＡ序列分析发现在ＧＲ基因的ｃＤＮＡ的第２４３９位点插入一个腺嘌呤核苷酸,此移码突变导致ＧＲ蛋白额外合成２０个氨基酸。结论ＧＲ异常可能与狼疮肾炎发病有关。     

Fas/APO-1 在泌尿系肿瘤细胞中的表达

为了探讨Ｆａｓ／ＡＰＯ１在泌尿系恶性肿瘤发生发展中的作用，采用免疫细胞化学方法对６种泌尿系肿瘤细胞，膀胱癌（Ｔ２４，ＥＪ，ＢＩＵ８７），肾癌（ＧＲＣ１，ＲＣＣ９４９），前列腺癌（ＰＣ３Ｍ）和一种原代培养正常肾间质成纤维细胞Ｆａｓ／ＡＰＯ１分子的表达进行了检测。结果显示原代培养正常肾间质成纤维细胞和Ｔ２４细胞系Ｆａｓ／ＡＰＯ１分子表达较强，ＰＣ３Ｍ属中等强度表达，ＲＣＣ９４９与ＢＩＵ８７属微弱表达，而ＥＪ及ＧＲＣ１则不表达。Ｆａｓ／ＡＰＯ１在泌尿系恶性肿瘤培养细胞中的表达强度偏低或完全不表达，可能参与了泌尿系恶性肿瘤的发生和发展。     

应用PCR—SSCP银染技术检测结直肠癌P53基因突变

应用ＰＣＲ－ＳＳＣＰ银染技术检测３０例结直肠癌Ｐ５３基因第５、６、７和８外显子的突变情况，结果发现１６例（５３．３３％）发生Ｐ５３基因突变，提示：（１）结直肠癌普遍存在Ｐ５３基因突变。（２）比较传统的同位素标记ＰＣＲ－ＳＳＣＰ技术，ＰＣＲ－ＳＳＣＰ银染技术不仅灵敏度相当，而且简便，快速，低耗，重复性好，尤其适于其样本筛选，可在临床实验室中推广。     

血小板衍生生长因子受体在前列腺良恶性病变中的表达及意义

生长因子及其受体表达失调可导致细胞增殖及恶变。为探讨血小板衍生生长因子受体（ＰＤＧＦＲ）表达与前列腺病变关系，用免疫组织化学ＳＰ法研究１４例正常前列腺（ＮＰ）、５６例前列腺增生症（ＢＰＨ）、３３例前列腺癌（ＰＣａ）组织中ＰＤＧＦＲ的表达。发现ＢＰＨ、ＰＣａ组织阳性率分别为７８６％、９３９％，明显高于正常组织的２１４％，Ｐ均＜００１。阳性染色分布于上皮、基质、血管平滑肌、炎症细胞的胞浆、胞膜及核膜。提示ＰＤＧＦＲ的高表达与ＢＰＨ、ＰＣａ和新生血管的发生、发展有密切关系，未发现ＰＤＧＦＲ的高表达在肿瘤的分期、分级中有差异。     

人大肠癌组织P53基因突变的初步研究

应用ＰＣＲ－ＳＳＣＰ技术对照分析１８例大肠腺癌及正常结肠粘膜组织Ｐ５３基因第５、６、７和８外显子。发现８例大肠癌组织出现了反映Ｐ５３基因突变的异常条带。结果提示Ｐ５３基因的突变与大肠癌发生和发展有关。ＰＣＲ－ＳＳＣＰ能在ＤＮＡ片段的不同部位检测ＤＮＡ多态性和点突变。该方法灵敏、快速，对大肠癌的基因诊断具有潜在性应用价值。     

大肠癌组织中结直肠癌缺失基因mRNA及增殖细胞核抗原的表达

目的 探讨结直肠癌缺失基因（ＤＣＣＧ）及增殖细胞核抗原（ＰＣＮＡ）与大肠癌的临床病理特征关系，方法 用逆转录ＰＣＲ及免疫组织化学染色方法，分别检测ＤＣＣＧｍＲＮＡ及ＰＣＮＡ在３３例大肠腺癌及正常粘膜组织中的表达状况。结果 （１）ＤＣＣＧｍＲＮＡ表达缺失率为６１％，ＰＣＮＡ指数为３％～９７％。（２）大肠癌组织ＤＣＣＧｍＲＮＡ表达缺失及及ＰＣＮＡ指数与肿瘤分化程度，浸润深度和转移情况有显著相关性（Ｐ〉     

P53基因突变结合术前肿瘤标志物检测对结直肠癌患者预后的评估

目的　进一步了解肿瘤标志物与基因检测在判断结直肠肿瘤患者预后中的价值。方法　对５４ 例结直肠癌患者,术前取血检测结直肠相关抗原血清癌胚抗原（ＣＥＡ） 、ＣＡ１９９ 水平,同时取手术切除标本,应用聚合酶链反应单链构像多肽性法（ＰＣＲＳＳＣＰ） 检测Ｐ５３ 基因第５ － ８ 外显子。结果　Ｐ５３ 基因突变率为４４－４ ％（２４／５４） ,在Ｐ５３ 基因突变患者中,ＣＥＡ阳性率明显高于Ｐ５３ 基因未突变者（ Ｐ＜ ０－０５）,而术前ＣＥＡ（ ＋） 及肿瘤标本Ｐ５３ 基因突变者多发生复发或远处转移（ Ｐ＜０－０５）。结论　术前ＣＥＡ（＋ ）及肿瘤标本中Ｐ５３ 基因突变者临床预后差,这不但说明了术前测定肿瘤标志物同样存在临床实用价值,而且对于术后如何制订合理的、优化的治疗方案、如何进一步有效地加强临床随访工作均有一定的参考价值。     

应用PCR－SSCP研究胆道肿瘤P53基因的突变

应用ＰＣＲ－ＳＳＣＰ（ＤＮＡ聚合酶链式反应－单链构象多态性）方法检测了１２例胆道肿瘤（胆管癌７例，胆囊癌３例，壶腹癌２例）的Ｐ５３基因第５、７外显子，结果发现２例胆管癌和１例胆囊癌Ｐ５３基因有突变。采用免疫组化方法对２２例胆管癌和８例癌旁组织Ｐ５３蛋白产物进行了检测，染色阳性者分别为１１例（５０％）和０。结果提示胆道肿瘤的发生与Ｐ５３基因的突变有关。     

Detection of human papillomavirus DNA and p53 gene mutations in human prostate cancer

The relationship between integration with human papillomavirus (HPV) and p53 gene mutations in tissues of prostate cancer was examined. Tissue samples analyzed were obtained by total prostatectomy (29 stage B cancer cases) and from autopsy (22 endocrine therapy-resistant metastatic disease cases). HPV DNA was detected in 8 of 51 (16%, 5 in stage B and 3 in autopsy cases) by polymerase chain reaction (PCR) using consensus primers on L1 region. Genotypes of HPV were entirely type 16. Structural abnormalities of p53 gene were detected in 7 of the 22 autopsy cases (32%) by PCR-single-strand conformation polymorphism analysis and direct sequencing. No p53 gene mutation was found in stage B cancer cases. Analysis of mutation spectra revealed clear differences between Japanese and Westerners. There was a significant difference in the mutation frequency between stage B and autopsy cases (P < 0.01, Fisher's exact test). One case showed both integration of HPV and p53 gene mutation in different cancer foci. However, the other cases revealed an inverse correlation between the presence of HPV DNA and p53 gene mutations. These data show that p53 genetic alteration is correlated with the progression of prostate cancer, in contrast to the integration of HPV that may occur in a relatively early stage. In conclusion, this study may indicate that either p53 gene mutation or the presence of HPV's oncogenic protein E6 is involved in the development of prostate cancer. © 1996 Wiley-Liss, Inc.     

大肠癌细胞p53基因突变与细胞凋亡及细胞倍性的关系

目的　了解ｐ５３ 基因突变、细胞倍性、细胞凋亡三者的关系,探讨突变型ｐ５３ 基因在肿瘤发生中的作用机制。方法　采用 Ｐ Ｃ Ｒ Ｓ Ｓ Ｃ Ｐ（ 单链构象多态性） 检测ｐ５３ 基因突变,用流式细胞仪 Ｆａｃｓｃａｎ 检测大肠癌细胞染色体倍性、凋亡率及其在细胞周期各相中的分布情况。结果　大肠癌标本ｐ５３基因突变率为５０ ％ ;其中突变组肿瘤细胞凋亡率（２２ ．１１ ％ ） 明显低于未突变组细胞凋亡率（４０ ．５７ ％ ） ,（ Ｐ＜ ０ ．０５） ;突变组异倍体细胞百分率（ 占７６ ．９ ％ ） 明显高于未突变组（ 占４６ ．２ ％ ） ,（ Ｐ＜ ０ ．０５） ;异倍体肿瘤细胞凋亡率为３４ ．０ ％ ,二倍体肿瘤细胞凋亡率为４０ ．７ ％ ,二者差异无显著性（ Ｐ＞ ０ ．０５） 。结论　ｐ５３ 基因突变使肿瘤细胞凋亡率下降,进而促进肿瘤发生、发展。ｐ５３ 基因突变多见于异倍体细胞,细胞凋亡率与细胞倍性无关。     

P53基因突变及蛋白表达与大肠癌发生及预后关系探讨

目的:探讨P53基因突变及蛋白表达与大肠癌发生及预后的关系.方法:以PCR-RFLP-SSCP技术,分析大肠癌及大肠息肉P53基因突变以及运用免疫组化技术检测P53蛋白表达与临床病理学因素及预后之间关系.结果:P53因基突变与P53蛋白表达之间是呈正相关.大肠癌P53突变率高于大场息肉(P<0.001)且与发病年龄、肿瘤原发部位以及肿瘤大体类型明显相关(P<0.05).应用COX模型分析大肠癌复发及死亡相关因素显示P53蛋白表达者复发及死亡危险系数均大于阴性者.结论:P53基因突变及P53蛋白表达可以作为一个预后的生物学指标.     

Loss of heterozygosity of M6P/IGF2R gene is an early event in the development of prostate cancer

BACKGROUND: The genetic events leading to initiation and/or progression of prostate cancer are not well characterized. The gene coding for the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) has recently been identified as a tumor suppressor in several types of cancer. The purpose of the present study is to determine whether the M6P/IGF2R gene is inactivated in human prostate cancer, and if so, whether this is an early or late transformational event. METHODS: In total, 43 patients with prostate cancer treated by radical prostatectomy, with archival material available for analysis, were assessed for loss of heterozygosity (LOH) in the M6P/IGF2R gene using six different gene-specific nucleotide polymorphisms. Regions of tumor, normal prostate and premalignant high-grade prostate intraepithelial neoplasia (PIN) were identified and cells were excised by laser capture microdissection (LCM). DNA segments were amplified using polymerase chain reaction (PCR). RESULTS: The M6P/IGF2R gene was polymorphic in 83.7% (36/43) of patients, and 41.7% (15/36) of these informative patients had LOH in the tumor tissue. In 11/15 patients with LOH in malignant tissue, high-grade PIN could be identified, and 63.6% (7/11) also had LOH in this premalignant tissue. CONCLUSIONS: This study is the first to find that the M6P/IGF2R gene is inactivated in prostate cancer. LOH in premalignant tissue as well suggests that mutation in the M6P/IGF2R gene is an early event in the development of prostate cancer, supporting the conclusion that it functions as a tumor suppressor gene in this disease.     

p53 oncogene mutations in three human prostate cancer cell lines

p53 gene structure and chromosome 17p alleles were studied in the three human prostate cancer cell lines, LNCaP, DU-145, and PC-3. Our laboratory has two separate culture lines of the LNCaP human prostate cancer cells. One strain, LNCaP-GW, had a mutation in one of two alleles at position 273 (arg > his). This mutation could not be detected in a second strain of LNCaP, LNCaP-ATCC. Immunohistochemical staining for P53 protein in the cell lines indicated that protein overexpression in LNCaP was heterogeneous, even in clonal isolates derived from LNCaP-GW that contained the codon 273 mutation in every cell. We also performed in vitro and in vivo growth analysis to compare the LNCaP-GW and LNCaP-ATCC cells. LNCaP-GW grew more rapidly than LNCaP-ATCC in vitro. However, LNCaP-ATCC formed tumors efficiently when inoculated into nude mice, whereas LNCaP-GW formed tumors much less efficiently. Consideration must be given to the notion that some of these p53 mutations arose during in vitro passage. We also confirmed published findings with two other human prostate cancer cell lines. In DU-145, two mutations were found in the p53 gene. A mutation at codon 274 (pro > leu) and a second mutation at codon 223 (val > phe) were present. PC-3 cells were hemizygous for chromosome 17p. The single copy of the p53 gene had a base pair deletion at codon 138 that generated a frame shift and a new in-frame stop codon at position 169. © 1993 Wiley-Liss, Inc.     

Association of codon 72 polymorphism of p53 with lower prostate cancer risk.

BACKGROUND: A common germline polymorphism of p53 produces a protein with an Arg to Pro change at codon 72. This Pro variant has altered biochemical properties suggesting altered cancer susceptibility. METHODS: A case control study with 115 men with prostate cancer and 181 community control male subjects was conducted. Demographics, family history of cancer, and blood were obtained. Codon 72 genotypes were determined using PCR. RESULTS: The Pro/Pro genotype was associated with a markedly lower risk of prostate cancer (OR = 0.23, CI = 0.07-0.79, P = 0.012). Similar reduction in risk was observed when the analysis was limited to Caucasian subjects (86% of total). Reduction in risk remained significant in a logistic regression model after correcting for age and family history of prostate cancer (OR = 0.14, CI = 0.03-0.71, P = 0.017). CONCLUSIONS: Men with the p53 codon 72 Pro/Pro genotype appear to be at reduced risk of prostate cancer.     

前列腺癌中p53和PTEN的表达对于临床分级及预后判断的作用分析

目的 探讨前列腺癌中p53和PTEN的表达对于临床分级及预后判断的作用.方法 采用免疫组织化学检测80例前列腺癌组织和癌旁组织以及40例正常组织中PTEN和p53蛋白表达水平,比较其表达水平和前列腺癌分级、预后等病理参数关系.结果 前列腺癌组织中p53表达率显著升高(P＜0.05),PTEN表达率显著降低(P＜0.05).不同分化程度、分期、转移和预后的癌组织中PTEN表达水平差异有统计学意义(P＜0.05).不同分化程度和预后的癌组织中p53表达水平差异有统计学意义(P＜0.05).p53和PTEN表达水平无相关性(P＞0.05).结论 凋亡相关基因p53和PTEN参与到了前列腺癌的分级并因此影响了患者的预后,但两者间无相关性.     

乳腺癌中p53基因DNA水平突变与核蛋白水平突变的比较

目的研究ｐ５３基因ＤＮＡ水平和蛋白水平的突变。方法分别采用多聚酶链反应单链构象多态分析银染方法和链酶亲和素生物素过氧化物酶复合物（ｓｔｒｅｐａｖｉｄｉｎｂｉｏｔｉｎｐｅｒｏｘｉｄａｓｅｃｏｍｐｌｅｘ,ＳＡＢＣ）免疫组化方法,检测了５４例乳腺浸润性导管癌标本。结果２２例在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５７％。其中２２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２８％。ｐ５３ＤＮＡ突变与核蛋白突变之间有显著相关性,并且两者都与患者的淋巴结转移以及雌、孕激素受体表达有关（Ｐ＜００１）。结论免疫组化是一种快速检测ｐ５３是否存在异常的有效方法。但是在蛋白水平突变阳性者中存在一定的假阳性。ｐ５３突变阳性患者预后较差     

Mutations within the tumour suppressor gene p53 are not confined to a late event in prostate cancer progression. a review of the evidence

Mutations in the p53 tumour suppressor gene are generally believed to be a late event in the progression of prostate cancer, and are associated with androgen independence, metastasis, and a worse prognosis. In this review, we examine the current literature available on p53 mutations and focus on stages A (T1) and B (T2) of prostate cancer. We report here that p53 mutations can be found in approximately one third of prostate cancers that are clinically localized to the prostate. In addition, high levels of p53 mutation are found in normal prostate tissue of prostate cancer patients, prostatic intraepithelial neoplasia, and benign prostatic hyperplasia. The limitations of techniques used to determine p53 mutations are discussed, as well as other modes of p53 loss in early stage prostate cancer.     

The relation of p53 protein nuclear accumulation and angiogenesis in human prostatic carcinoma

All neoplasms require angiogenesis and resulting neovascularity for growth beyond 1 mm(2). Quantitative microvessel density (MVD) has been shown to provide staging and prognostic significance in human prostate cancer (CaP). recently, it has been demonstrated that loss of the wild-type allele of the p53 tumour suppressor gene results in reduced expression of thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis. There is also an increased expression of vascular endothelial growth factor which promotes neovascularization. p53 gene mutation and MVD were investigated in men with prostate cancer. Sections from 103 radical prostatectomy cases were evaluated with immunohistochemistry to detect mutant p53 proteins. Quantitative MVD was performed on the cases exhibiting p53 positive staining and compared with negative fields of similar Gleason grade on the same histologic sections. Twenty of the 103 cases (19.4%) revealed positive p53 staining nuclei. In 19 of these 20 cases, the MVD in p53 positive areas was greater than corresponding control regions (overall P<0.0001). Extent of p53 abnormality, as well as MVD, correlated with pathologic stage. These data suggest that mutations of the p53 tumour suppressor gene may be associated with increased angiogenesis in CaP. In addition to providing staging and prognostic information, this relationship potentially has therapeutic implications.