子宫颈癌组织中HPV16癌基因及p53基因的检测

利用ＨＰＶ１６Ｅ６、Ｅ７基因特异性引物，ＰＣＲ技术及抗癌基因ｐ５３外显子７特异性引物，ＰＣＲ－ＳＳＣＰ技术对３５例进展期子宫颈癌组织进行研究，发现：（１）３５例标本中ＨＰＶ１６Ｅ６、Ｅ７ＤＮＡ的总检出率为７１．４２％（２５例），其中同时检出Ｅ６、Ｅ７为３１．４２％（１１例），另外８．５７％及３１．４２％（３例及１１例）仅分别检出Ｅ６、Ｅ７序列。（２）全组未见１例有ｐ５３基因外显子７的点突变及等位基因缺失。该结果说明ＨＰＶ１６与本地区妇女子宫颈癌发生有密切关系，并且癌组织中ＨＰＶ１６Ｅ６、Ｅ７亚基因的分布是不均一的，ｐ５３基因外显子７的改变并不常见。在实验中我们还建立了使用生物素标记的ｄＵＴＰ进行ＰＣＲ－ＳＳＣＰ的技术。     

A method to isolate DNA from small archival tissue samples for p53 gene analysis

The tumor suppressor gene p53 is involved in predisposition to a variety of human cancers, including those from Li–Fraumeni cancer family syndrome patients. Studies of inheritance of p53 germline mutations require confirmation of the mutation in the tumors from family members. These studies as well as other retrospective studies of tumor specific mutations, are often hampered by a lack of available fresh or frozen tumor tissue samples for DNA extraction to confirm the suspected p53 mutation. Here we describe a simple technique for DNA isolation that permits mutational analysis of p53 from minimal amounts of paraffin-embedded archival tissue samples. © 1993 Wiley-Liss, Inc.     

卵巢上皮性肿瘤p16抑癌基因突变与HPV感染的研究

采用聚合酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）技术，对同一卵巢上皮性肿瘤石蜡包埋组织中ｐ１６基因（第二外显子）突变及人乳头状瘤病毒（ＨＰＶ）感染进行相关性研究。并与正常卵巢组织进行对照。结果，２８例卵巢上皮性肿瘤组织中ｐ１６基因突变１５例，突变率为５３．６％（１５／２８），其中７例伴有ＨＰＶ１６型或ＨＰＶ１８型感染，占突变率的４６．７％。在卵巢上皮性肿瘤组ＨＰＶ１６、１８ＤＮＡ阳性率为５３．６％（１５／２８），对照组ＨＰＶ１６、１８ＤＮＡ阳性率为５．６（１／１８），二者比较有显著性差异。提示：卵巢上皮性肿瘤中ｐ１６基因突变与ＨＰＶ１６、１８型感染有关。ＨＰＶ１６、１８型感染与卵巢上皮肿瘤密切相关     

p53 Protein expression in laryngeal squamous cell carcinomas bearing wild-type and mutated p53 gene

We performed an immunohistochemical analysis to investigate the expression of p53 protein in a panel of 18 laryngeal squamous cell carcinomas, 15 primary tumours and three in relapse, previously analysed by us for the presence of p53 gene mutations. Dysplastic and/or normal surrounding mucosa was evaluated in 15 different tumours. The results of our study are the following: (1) expression of p53 protein was observed in one out of five tumours positive for p53 gene mutations (20%) and in 10 out of 13 (80%) negative cases; (2), p53 protein over-expression was frequently observed in normal and/or dysplastic mucosa surrounding either wild-type (7/11) or mutated p53 tumours (2/4); (3), p53 immunoreactive cells showed a pattern of distribution in normal and mildly/moderately dysplastic mucosa (basal layers), different from that in severely dysplastic mucosa (whole thickness). These data further support the hypothesis that p53 protein over-expression may be a marker of the earliest phases of multistep tumorigenesis in laryngeal squamous cell carcinoma.     

p53蛋白在宫颈鳞状上皮内病变与宫颈癌中的过度表达及其与HPV的关系

目的探讨宫颈鳞状上皮内病变（ＳＩＬ）和宫颈癌中抑癌基因ｐ５３蛋白表达水平以及与ＨＰＶ的关系。方法用免疫组化和ＰＣＲ－ＲＦＬＰ方法，对２６８例宫颈石蜡包埋组织（２９例慢性宫颈炎、６８例ＳＩＬ、１７１例宫颈癌）进行了ｐ５３蛋白水平及多型ＨＰＶ检测。结果宫颈癌ｐ５３蛋白的过度表达率及强阳性过度表达率高于ＬＳＩＬ和ＨＳＩＬ，ＨＰＶ１８阳性的ＳＩＬ及宫颈癌ｐ５３蛋白的过度表达率高于ＨＰＶ１６阳性和ＨＰＶ阴性者。结论抑癌基因ｐ５３蛋白过度表达率与宫颈癌的发生发展有关，与ＨＰＶ１８的内在联系尚有待于进一步研究。     

Absence of microsatellite instability in breast carcinomas with both p53 and c-erbB-2 alterations

Based on a previous finding that amplification of the c-erbB-2 oncogene and alteration of p53 are strongly associated in most aggressive breast tumours, the present study investigated whether microsatellite instability (MI) might also be associated with this tumour phenotype. Nine polymorphic microsatellite markers, including six dinucleotide, one trinucleotide, and two tetranucleotide repeats, were amplified from paired normal and tumour DNA samples of 15 breast tumours that overexpressed both c-erbB-2 and p53 and of 15 control breast tumours that overexpressed neither protein. All 30 breast tumours analysed exhibited a replication error-negative phenotype, with only one sample showing MI in one of the nine loci. This suggests that the genetic events underlying MI, which are critical in colorectal and gastric tumours, are not involved in the pathogenesis of c-erbB-2/p53 double-altered breast tumours and do not play a central role in breast tumour formation. Copyright © 1999 John Wiley & Sons, Ltd.     

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。     

Gastric adenoma — carcinoma sequence with special reference to p53 and Ki-ras gene alterations

With the aim of detecting the timing of p53 and Ki-ras gene alterations in the gastric adenoma-carcinoma sequence, 19 early gastric adenocarcinomas arising from adenomas were studied. Immunohistochemically, 5 adenocarcinomas were positive for p53; 3 focally and 2 diffusely. The p53 point mutations were detected in a focal area with p53 immunoreactivity in 2 of the 5 p53-positive adenocarcinomas. This indicated that p53 point mutations may play a less crucial part in malignant conversion of adenoma to adenocarcinoma in the stomach than in the colon. No Ki-ras gene mutations at codons 12 and 13 were detected in any lesion. These results suggest that the adenoma-carcinoma sequence in the stomach has a different mechanism from that in the colon.Supported in part by a Grant-in-Aid from the Ministry of Health and Welfare of Japan     

Expression of the GLUT1 glucose transporter, p63 and p53 in thyroid carcinomas

Only few studies have evaluated the usefulness of the GLUT1 and p63 status of thyroid carcinomas in revealing tumorigenesis. We studied GLUT1, p53, and p63 immunoexpression in a total of 86 cases of various thyroid carcinoma types to determine the biological significance of GLUT1 and p63 expression in thyroid carcinomas. GLUT1 was detected in six cases of anaplastic carcinoma and in one case of poorly differentiated carcinoma with membranous staining. p63 was detected in five cases of anaplastic carcinoma, in one case of poorly differentiated carcinoma, and in five cases of papillary carcinoma with nuclear positivity. p53 was detected in six cases of anaplastic carcinoma, in one case of poorly differentiated carcinoma, and in one case of follicular carcinoma with nuclear positivity. Five of seven cases of anaplastic carcinoma expressed all three of these markers. The results suggest that GLUT1, p63, and p53 are not expressed in well-differentiated thyroid carcinomas, and that they are usually expressed late in the course of thyroid tumor progression. These data strongly suggest that in anaplastic carcinomas, impairment of p53-mediated repression results in increased GLUT1 and p63 expression, and that this probably reflects the differential regulation of hypoxia-responsive pathways and basal/stem cell regulatory pathways.     

Expression of p53 protein in laryngeal squamous cell carcinoma and dysplasia: possible correlation with human papillomavirus infection and clinicopathological findings

In order to evaluate the expression of p53 protein in 28 premalignant and 40 malignant squamous cell proliferations of the larynx and its relationship to tobacco consumption, human papillomavirus infection and differentiation grade of the lesions, p53 expression was examined by means of a microwave post-fixation immunohistochemical method using the PAb 240 and PAb 1801 monoclonal antibodies. HPV infection was assessed by non-isotopic in situ hybridization (NISH) and polymerase chain reaction (PCR). A large proportion of carcinomas (77.5%) and dysplasias (61%) expressed p53. No difference was found between differentiation grades of the lesions regarding p53 detection (P>0.1), but moderate or intense p53 expression was more frequent in the carcinomas (P<0.05). A statistical correlation was found between cigarette consumption and both p53 detection and p53 staining intensity (P<0.05 in each case). HPV study revealed HPV 16 and 18 infection only in carcinomas. The frequency was 28% and the physical state of the virus as demonstrated by NISH was integration into the genome. We observed an inverse relationship between HPV infection and p53 expression (P=0.006). Our findings suggest that p53 overexpression is a common and early event which increases in frequency with progression of laryngeal squamous cell carcinoma. The expression of p53 is influenced by tobacco and high-risk types of HPV.     

Polymerase chain reaction-single strand conformation polymorphism analysis of the p53 gene in paraffin-embedded surgical material from human renal cell carcinomas

p53 tumour suppressor gene mutations were studied in 118 renal cell carcinomas using paraffin-embedded surgical material. Optimal results were obtained with analysis of exon lengths between 150 and 200 base pairs for polymerase chain reaction. Single strand conformation polymorphism and sequencing analysis revealed only two point mutations (2/118, 2%): one involving codon 135; TGCTTC (cysteinephenylalanine) and the other codon 175; CGCCAC (argininehistidine). Both of these cases were classified as granular cell subtype on microscopic observation. The data suggest that the p53 tumour suppressor gene is not related to tumour initiation, promotion, or progression of renal cell carcinomas. However, there is the possibility that granular cell type carcinomas may have a different genetic background from clear cell type renal neoplasms.     

Molecular analysis of p53 gene in laryngeal premalignant and malignant lesions. p53 protein immunohistochemical expression is positively related to proliferating cell nuclear antigen labelling index

This study was undertaken in order to investigate the molecular nature of the p53 gene in 19 laryngeal squamous cell carcinomas and dysplasias. Moreover, we have examined the possible relationship between proliferating cell nuclear antigen (PCNA) expression and p53 protein detection status in 42 laryngeal premalignant and malignant lesions in which 14 of the 19 samples used in the molecular study were included. p53 gene analysis was performed with the single-strand conformation polymorphism technique. PCNA was stained with the peroxidase/antiperoxidase immunohistochemical method using the monoclonal antibody PC-10. Data from previous work concerning p53 expression was used. We found that 9 of 12 of the immunohistochemically p53 positive (+) cases had mutations in exons 5 or 6. In the remaining immunohistochemically p53(+) and p53 negative (–) specimens there was no indication of sequence alterations. Furthermore, we did not observe any deletions in the chromosomal region 17p31.1 which encodes exons 4–8 of the p53 gene. The PCNA labelling index (LI) increased progressively with p53 protein detection status (percentage of cells immunohistochemically positive for p53). The difference between the group with the higher percentage of p53(+) cells and the others was statistically significant. These data show that although there is a discrepancy between immunohistochemical demonstration of p53 and molecular analysis, a large proportion of the former harbours the mutant form of the protein. In addition, p53 overexpression is positively correlated with PCNA LI, a finding which accompanies tumour progression.     

Correlation of p53 protein expression with gene mutation in gall-bladder carcinomas

The correlation of p53 protein expression and p53 mutation of 33 gall-bladder carclnomas was studied accordlng to the depth of invasion and grade of cytological atypia. Overexpresslon of p53 protein was detected by immunostaining in seven (70.00/) of 10 intramucosal and in 16 (69.6%) of 23 invasive carcinomas. p53 mutation was detected in five (71.4%) of the seven intramucosal carcinomas with overexpresslon and In eight (50.0%) of the 16 invasive cancers with overexpression and in one (10%) of the 10 non-overexpressing carclnomas at exons 5–8 by nested polymerase chain reactlon-single-strand conformatlon polymorphism. The overexpression of p53 protein was present In nine (56.3%) of 16 low-grade carcinomas and In 14 (82.3%) of 17 high-grade carcinomas. In cases of overexpresslon, p53 mutatlon was detectable in four (44.4%) of nlne lowgrade and in nlne (64.3%) of 14 high-grade carclnomas. In total, p53 mutation was verified In 56.5%0 (13123) of cases involving protein overexpression and in 10% (1/10) of cases of nonoverexpresslon. The sensltivity of p53 mutation was 56.5% (13/23), the specificity was 90.0% (9/10) and the validity was 1.47. in conclusion, our study indicates that p53 protein overexpression correlates well wlth gene mutatlon and that p53 alteration may be related to increaslng grade of cytologic atypla of carclnomas.     

人乳头瘤病毒与P 53协同致膀胱移行细胞癌关系的研究

目的 研究人类乳头瘤病毒（ＨＰＶ）６、１１、１６和１８型及Ｐ５３与膀胱移行细胞癌的关系。方法 采用聚合酶链反应（ＰＣＲ）方法检测了７５例膀胱移行细胞癌组织中ＨＰＶ的感染,免疫组化ＳＰ法检测Ｐ５３蛋白表达情况。结果 膀胱移行细胞癌组织中ＨＰＶ６、１１、１６和１８的阳性率分别为６．７％（５／７５）,５．３％（４／７５）,３３．３％（２５／７５）和６．７％（５／７５）。低危型ＨＰＶ（６或１１）阳性率为９．３％（７／７５）,高危型ＨＰＶ（１６或１８）阳性率为３４．７％（２６／７５）。同一膀胱癌组织中两种以上（包括两种）ＨＰＶ亚型感染８例,占１０．６％。ＨＰＶ６、１６和１８型之间感染阳性率在肿瘤有无转移组中差异显著（Ｐ〈０．０５）,ＨＰＶ１６、１８的阳性率在肿瘤病理分级中差异有极显著性（Ｐ〈０．０１）。ＨＰＶ ＤＮＡ型别     

Comparison of benign and malignant endometrial lesions for their p53 state, using immunohistochemistry and temperature-gradient gel electrophoresis

The aim of this study was to evaluate the presence and distribution of p53 alterations in pure endometrioid adenocarcinomas (n=120) of different grades and stages, as opposed to normal endometrium (n=13) and various risk groups of hyperplasia (n=39). All samples were initially analysed by immunohistochemistry with the monoclonal antibody Ab-6. Normal endometria were negative. With increasing degrees of malignancy, the number of cases with p53 accumulation rose and ranged from 9% to 18% in hyperplasia, through 25% in lowgrade carcinomas (G1), to 69% in high-grade carcinomas (G3). This increase was also seen when comparing tumours by stage. Of carcinomas in stage IA, only 17% showed p53 immunostaining, in contrast with 72% in stage IC. Of this material, 34 carcinomas and 8 hyperplasias were analysed for p53 mutations in exons 5–8 by means of polymerase chain reaction and temperature-gradient gel electrophoresis (TGGE). In none of 5 hyperplasia and 6 of 12 carcinomas showing p53 accumulation by immunohistochemistry, p53 mutations were detected by TGGE. In contrast, 4 of 22 carcinomas harboured mutant p53 but were negative by immunohistochemistry. Immunohistochemical and molecular investigations revealed that p53 alterations are related to the standard prognostic markers of endometrial cancer, i.e. grading and staging. TGGE, an indirect screening procedure for p53 mutations, is used to detect the type of p53 alteration and may provide additional insight into the complex figure of p53 abnormalities in the development and progression of malignant endometrial lesions.     

Relationship between abnormal p53 protein and failure to express p21 protein in human breast carcinomas

Alterations in the p53 protein are a common feature in most malignancies, including breast carcinomas. p53 protein alterations contribute to malignant transformation in several ways, through genomic instability and accumulation of additional genetic alterations in other genes, through alteration of the p53-dependent apoptotic pathway, and through downregulation of downstream effector proteins such as p21 (WAF1/CIP1), necessary for cell-cycle growth arrest. Cell-cycle arrest is needed to allow DNA repair after injury. This study examines the relationship between abnormalities in p53 protein and expression of p21 protein in 70 cases selected from a series of 212 sporadic human breast carcinomas. Immunohistochemistry (IHC) was used for detection of p53 and p21 protein expression. Constant denaturant gel electrophoresis (CDGE) was used for detection of mutations in exons 5–8 of the TP53 gene. A highly significant association was found between abnormalities in p53, scored as protein accumulation and/or mutations, and lack of p21 expression. p21 was also shown to be downregulated in samples without p53 alterations, indicating that other mechanisms are also involved in turning off this gene. © 1997 John Wiley & Sons, Ltd.     

Polymerase chain reaction

Summary The PCR, like recombinant DNA technology, has had an enormous impact in both basic and diagnostic aspects of molecular biology because it can produce large amounts of a specific DNA fragment from small amounts of a complex template. Recombinant DNA techniques create molecular clones by conferring on a specific sequence the ability to replicate by inserting it into a vector and introducing the vector into a host cell. PCR represents a form of in vitro cloning that can generate, as well as modify, DNA fragments of defined length and sequence in a simple automated reaction. In addition to its many applications in basic molecular biological research, PCR promises to play a critical role in the identification of medically important sequences as well as an important diagnostic one in their detection.     

Immunohistochemical detection of p53 in differentiated, poorly differentiated and undifferentiated carcinomas of the thyroid

In an attempt to find whether or not p53 immunoreactivity in the thyroid gland is restricted to undifferentiated carcinomas and to evaluate the putative prognostic usefulness of its detection, we investigated p53 immunoreactivity in a series of 14 benign thyroid lesions and 65 thyroid carcinomas (12 papillary; six minimally invasive follicular; four widely invasive follicular; 31 poorly differentiated and 12 undifferentiated tumours). Unequivocal nuclear immunostaining for p53 was observed in two widely invasive follicular carcinoma (20.0%), five poorly differentiated carcinomas (16.1%) and in 10 undifferentiated carcinomas (83.3%). The percentage of immunoreactive cells was much smaller in the former groups than in undifferentiated carcinomas. Despite a trend to a more aggressive behaviour of the p53 immunoreactive cases no significant differences in the outcome of patients with positive and negative tumours was found when the comparison was made within each category of carcinomas. We conclude that p53 immunoreactivity can be detected both in undifferentiated carcinomas and in some differentiated and poorly differentiated thyroid carcinomas. Larger series of cases are necessary to evaluate the prognostic usefulness of this finding.     

Mutations of the p53 gene in carcinomas of the urinary system

Deletion of p53, which is an anti-oncogene located on chromosome 17p, was reported to be present at a high incidence in tumor cells of colorectal carcinoma, as well as osteosarcoma of the familial cancer syndrome. Mutations of the p53 gene were investigated in 59 surgical specimens of primary carcinomas of the urinary system from 57 patients, using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis. The PCR products were sequenced using the dideoxy chain termination method or the DNA sequencer. The tumors examined were 20 transitional cell carcinomas (TCC) and 39 renal cell carcinomas (RCC). Mutations of the p53 gene were detected in 20.0% (4/20) of TCC and were present in 16.7% (1/6) of the tumors invading the muscular layer. In two patients with simultaneous double bladder TCC, the mutations were found only in the larger tumors. In RCC, mutations were detected in 7.7% (3/39) of patients. No significant correlation between the presence of the mutation and the clinicopathologic parameters was found in RCC except that the three tumors with p53 gene mutations were clear cell carcinomas. These results suggest that p53 gene mutations play a possible role in both carcinogenesis and progression of TCC, but the p53 gene mutations may not be significant in development of RCC.