PROGNOSTIC SIGNIFICANCE OF p53 ABNORMALITIES IN COLORECTAL CARCINOMA DETECTED BY PCR–SSCP AND IMMUNOHISTOCHEMICAL ANALYSIS

Abnormalities in the p53 tumour suppressor gene and in the expression of its protein are common in colorectal carcinoma. The prognostic significance of these p53 abnormalities was studied in 66 patients with colorectal cancer, followed for more than 10 years. Single-strand conformation polymorphism (SSCP) analysis was used to detect alterations in exons 5–8 of the p53 gene. Paraffin sections were examined immunohistochemically for p53 overexpression with the monoclonal antibody DO-7 (Dako) both with and without microwave antigen retrieval. Abnormalities of the p53 gene were found in 41 per cent of cases by SSCP analysis. Outcome was unrelated to SSCP abnormalities ( P =0·19), except for the Dukes' A and B subgroup, where decreased survival was found in cases with abnormal SSCP ( P =0·01). Overexpression of p53 protein was seen by immunohistochemistry in 47 per cent of cases without, and in 52 per cent of cases with microwave antigen retrieval. Immunohistochemical overexpression of p53 protein either with or without microwave antigen retrieval was an independent prognostic indicator of poor survival. These results suggest that for routine purposes, immunohistochemical detection of the p53 protein product may be more useful than SSCP analysis of the encoding p53 gene in identifying those at high risk of colorectal cancer recurrence and death.     

人肺癌组织中p53,Rb基因突变研究

为探讨肺癌发生的分子遗传学机理，采用聚合酶链反应及聚合酶链反应－单链构象多态性技术，对４１例人肺癌组织中ｐ５３基因外显子５～８及Ｒｂ基因外显子１４～１６、２２～２３进行了突变分析。结果显示：ｐ５３基因突变１６例（１６／４１，３９％），分布于外显子５～７；Ｒｂ基因异常４例（４／４１，９．８％），其中外显子１４～１６区域部分缺失和外显子２２～２３区域突变各２例；在９例小细胞肺癌标本中，７例发生ｐ５３及Ｒ５基因的突变，其中１例存在ｐ５３基因两个外显子突变，另１例同时存在ｐ５３及Ｒｂ基因的突变。对部分ｐ５３基因突变标本序列分析，均在１个或３个密码子上存在导致ｐ５３蛋白异常的单碱基置换或插入突变。以上结果表明：肺癌、特别是小细胞肺癌的发生可能与ｐ５３及Ｒｂ基因的突变有关。     

POTENTIAL FALSE-POSITIVE RESULTS WITH ANTIGEN ENHANCEMENT FOR IMMUNOHISTOCHEMISTRY OF THE p53 GENE PRODUCT IN COLORECTAL NEOPLASMS

Aberrant crypt foci (ACF) are putative precursor lesions of colon cancer, recently identified on the methylene blue-stained mucosal surface of human colon. No mutations in K- ras or p53 genes were found by non-radioactive single-strand conformation polymorphism analysis in 14 ACF collected from five patients. Using the more sensitive method of allele-specific polymerase chain reaction (PCR) for K- ras , 8 of 14 ACF were found to contain K- ras mutations, suggesting that mutated cells are present in minute clones in ACF. No dysplasia was observed in any of the ACF containing a mutated clone. The presence of K- ras mutations in ACF suggests that these lesions occur at a very early stage in human colorectal carcinogenesis.     

Microsatellite instability in sporadic colorectal carcinoma is not an indicator of prognosis

Fifty sporadic colorectal carcinomas diagnosed in 1991 were analysed for microsatellite instability at four loci. Five of 50 (10 per cent) tumours showed replication errors (RERs) at two or more loci and were classed as RER-positive (RER+). A further five showed RERs at one locus only. A significant association (Fisher exact test) was found between RER+ tumours and location in the proximal colon, exophytic shape, size >5 cm, histological margin, lymphoid reaction, and near-diploid DNA content. There was no significant difference for age, sex, grade, mucin production, p53 protein overexpression or Duke's stage. There was no significant difference in survival as measured over a 60-month follow-up period. The findings, though limited by the small case numbers involved, show an association between RER positivity in sporadic colorectal tumours and certain clinico-pathological features. They do not suggest a better clinical outcome for sporadic RER+ tumours. Copyright © 1999 John Wiley & Sons, Ltd.     

CHANGES IN Bcl-2 AND p53 EXPRESSION IN RECURRENT B-CELL LYMPHOMAS

The aim of this study was to investigate the changes involved in the evolution of nine cases of recurrent B-cell lymphomas. Using the polymerase chain reaction (PCR) on formalin-fixed, paraffin-embedded tissue from both the primary and the recurrent lymphoma of each case, monoclonality was demonstrated in every tumour. In all nine cases, the recurrent lymphoma was shown to belong to the same clone as the primary lymphoma. Eight of these cases were then investigated by immunohistochemistry for changes in Bcl-2 and p53 expression. Five out of eight of the primary lymphomas showed Bcl-2 overexpression. Two of the three cases initially negative for Bcl-2 expression became positive in the recurrence. One out of eight of the primary lymphomas was positive for p53 expression. Of the seven negative cases, one became positive for p53 expression in the recurrence. Both of the p53-positive cases showed high-grade histology. This study shows that Bcl-2 overexpression is probably an important early event in the development of B-cell lymphomas, although it may occur as a post-neoplastic event. p53 mutation is probably more important as a late event and may be associated with high-grade transformation.     

IN SITU ANALYSIS OF TISSUE DYNAMICS AND p53 EXPRESSION IN HUMAN GASTRIC MUCOSA

In situ tissue dynamics were studied in 12 cases of human gastric mucosa, including normal gastric body mucosa and gastric glands with intestinal metaplasia, obtained from gastrectomy specimens of adenocarcinoma. Cell proliferation was determined by Ki67 immunoreactivity. DNA fragmentation was studied in situ by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In addition, p53 expression was examined by both immunohistochemistry and mRNA in situ hybridization. In the oxyntic gastric glands, Ki67 immunoreactivity was observed exclusively in the proliferative zone and TUNEL-positive cells were present predominantly in the surface foveolar epithelium. In the gastric glands with complete intestinal metaplasia, Ki67-positive cells were present in the lower portion of the glands and TUNEL-positive cells in the superficial epithelium. In the gastric glands with incomplete intestinal metaplasia, TUNEL-positive cells were detected in the lower gastric glands adjacent to cells immunoreactive for Ki67; the proportion of these gastric glands with TUNEL-positive cells (40 out of 108 glands) was significantly higher than for oxyntic glands (94 out of 620 glands) or for glands with complete metaplasia (31 out of 254 glands). Relatively strong p53 immunoreactivity and mRNA hybridization were also observed in the proliferative and apoptotic areas of gastric glands with incomplete intestinal metaplasia. These results indicate that incomplete intestinal metaplasia is associated with increased cell turnover and p53 overexpression, possibly in response to various noxious or DNA-damaging stimuli.     

EXPRESSION OF bcl-2 AND p53 IN DE NOVO AND EX-ADENOMA COLON CARCINOMA: A COMPARATIVE IMMUNOHISTOCHEMICAL STUDY

The development of colorectal carcinoma from adenomas is recognized as the dominant mechanism of colon carcinogenesis. However, early colon carcinomas are being increasingly detected which have no adenomatous elements in their vicinity, and which, despite their small size, already show submucosal invasion. Such tumours (so-called ‘ de novo ’ carcinomas) have renewed consideration of the de novo colorectal carcinogenesis pathway. The goal of this study was to evaluate the expression of tumour suppressor gene p53 and apoptosis control gene bcl-2 in de novo carcinomas, compared with early carcinomas developing in the background of an adenoma (ex-adenoma). Fifty cases each of de novo and ex-adenoma carcinomas (pT1) were studied. p53 expression was significantly higher in the de novo carcinomas than in the ex-adenoma carcinomas (62 per cent vs. 42 per cent), while bcl-2 tended to be weaker in the de novo than in the ex-adenoma carcinomas. These differences support the concept that de novo carcinomas are a unique pathological entity, with a phenotype reflecting their more aggressive behaviour.     

Mutation of the transforming growth factor-β type II receptor gene in right-sided colorectal cancer: relationship to clinicopathological features and genetic alterations

The presence of inactivating mutations in the transforming growth factor-β (TGF-β) type II receptor (RII) gene in colon cancer suggests that it may behave like a tumour suppressor gene. RII is mutated in the majority of colon tumours exhibiting widespread microsatellite instability, a characteristic generally referred to as the replication error phenotype (RER+). We investigated the association between RII mutations and various clinicopathological variables and genetic alterations in a large series of sporadic adenocarcinomas arising in the proximal colon. RII mutations were found in 17 per cent (36/210) of right-sided tumours and in 86 per cent (32/37) of those displaying RER+. They were associated with the absence of lymph node invasion (P=0·04), poor histological differentiation (P=0·006), and with a trend for improved patient survival. Tumours with an RII mutation also showed non-significant trends for a lower incidence of p53 protein overexpression and of p53, K-ras, and APC gene mutation compared with tumours with normal RII. These results indicate that right-sided colorectal tumours containing RII mutations resemble those with the RER+ phenotype in terms of their clinicopathological features and genetic alterations. © 1998 John Wiley & Sons, Ltd.     

p53 OVEREXPRESSION AND HUMAN PAPILLOMAVIRUS INFECTION IN TRANSITIONAL CELL CARCINOMA OF THE URINARY BLADDER: CORRELATION WITH HISTOLOGICAL PARAMETERS

Seventy-nine transitional cell carcinomas (TCCs) of the urinary bladder (25 grade 1, 22 grade 2, and 32 grade 3 tumours) were examined for p53 overexpression by immunohistochemistry with a monoclonal antibody and for human papillomavirus (HPV) infection by the polymerase chain reaction (PCR). Positive immunostaining for p53 was detected in 40·5 per cent of the cases; the percentage of positive cases was significantly lower in low-grade (G1 and G2) TCCs than in high-grade (G3) tumours (10·6 per cent vs. 84·4 per cent; P <0·0001). The overall rate of HPV infection was 32·9 per cent; 20·3 per cent of the cases were positive for HPV 16, 3·8 per cent for HPV 18, and 8·9 per cent for both. Consensus primers as well as type-specific primers for HPV types 6, 11, and 33 failed to detect any additional case with HPV infection. The prevalence of HPV 16 and/or HPV 18 infection was significantly higher in low-grade than in high-grade tumours (44·7 per cent vs. 15·6 per cent; P =0·0061). p53-positive cases were more common among papillary, deeply infiltrating tumours, and HPV-positive cases among papillary, non-infiltrating lesions. According to these data, p53 overexpression and HPV 16/18 infection are common findings in bladder TCC and there appears to be an inverse correlation of p53 overexpression and of HPV infection with tumour aggressiveness. The possibility of different molecular pathways in superficial low-grade and in invasive high-grade tumours is suggested.     

EXPRESSION OF CYCLIN-DEPENDENT KINASE INHIBITOR p21WAF1/CIP1 IN NON-NEOPLASTIC MUCOSA AND NEOPLASIA OF THE STOMACH: RELATIONSHIP WITH p53 STATUS AND PROLIFERATIVE ACTIVITY

The expression of the p53-inducible cyclin-dependent kinase inhibitor p21^WAF1/CIP1 in non-neoplastic mucosa, adenoma, and adenocarcinoma of the stomach was examined immunohistochemically and its relationship with p53 expression and proliferative activity was analysed. In normal gastric mucosa as well as in intestinal metaplasia the epithelial cells at the surface which showed no proliferative activity expressed p21whereas the cells in the deep area of the glands expressing Ki-67 did not. In the neoplastic lesions, the expression of p21^WAF1/CIP1 was detected in 78 per cent (112/144) of the adenomas and 76 per cent (262/343) of the adenocarcinomas. The incidence of p21^WAF1/CIP1 expression did not differ among histological types of gastric carcinoma. The strong expression of p21^WAF1/CIP1 was more frequently observed in carcinomas invading into submucosa or in cases of stages 2, 3, and 4 than in carcinomas limited to the mucosa or in stage 1 cases. The incidence of strongly positive cases was higher in carcinomas with lymph node metastasis than in those without metastasis. There was no apparent correlation between the expression of p21^WAF1/CIP1 and the abnormal accumulation of p53 or with proliferative activity measured by Ki-67 expression. These findings overall suggest that p21^WAF1/CIP1 might be associated with the senescence of non-neoplastic gastric epithelial cells; that a p53-independent pathway might be substantially involved in the induction of p21^WAF1/CIP1 in gastric neoplasia; and that the proliferative activity of gastric cancer might not be solely dependent on control of the cell cycle by p21^WAF1/CIP1.     

肌源神经营养因子(MDNF)对体外培养的大鼠脊髓运动神经元缺氧时p53表达的影响

本研究目的是观察缺氧时肌源神经营养因子(MDNF)对体外培养的大鼠脊髓运动神经元p53蛋白表达的影响。从成鼠和胎鼠骨骼肌中提取MDNF。在胎鼠脊髓运动神经元培养第7 d时将其分成对照组和实验组(包括成鼠MDNF组和胎鼠MDNF组),取100μl MDNF(0.1μg/ml)加入培养大鼠脊髓运动神经元的培养液中,对照组加入等量的PBS。然后用液体石蜡封闭液面造成神经元缺氧损伤,缺氧3 d后,应用Nissl染色、原位末端标记、免疫组化方法,对损伤的大鼠脊髓运动神经元进行检测。结果表明:培养的大鼠脊髓运动神经元缺氧3 d后,经MDNF孵育可使脊髓运动神经元TUNEL、p53表达均较对照组明显减弱,神经元损伤程度减轻,神经元存活数明显高于对照组,且胎鼠MDNF的效果更好。提示:大鼠脊髓运动神经元缺氧后可出现神经细胞凋亡,但胎鼠MDNF较成鼠MDNF更能减弱缺氧时脊髓运动神经元p53的表达,抑制缺氧后神经元的死亡。     

青蒿素调控p53蛋白诱导肝癌细胞凋亡的机制研究

目的 通过探讨青蒿素调控p53表达进而诱导肝癌细胞凋亡的情况研究青蒿素抗肿瘤的机理.方法 20只C57小鼠随机分为空白组、PBS组、青蒿素组及青蒿素+Pifithrin-α组,每组5只.以PBS组、青蒿素组及青蒿素+Pifithrin-α组小鼠联合应用DEN/CCM/乙醇构建肝癌模型.空白组小鼠正常饲养3周,PBS组腹腔注射PBS 3周,青蒿素组腹腔注射青蒿素(100mg/kg)3周,青蒿素+Pifithrin-α组应用Pifithrin-α (50 mg/kg)隔日作用1周后,再与青蒿素作用3周.处死各组小鼠,分离小鼠肿瘤组织,Western blot检测肿瘤组织中Caspase3及p53的表达.结果 成功构建小鼠肝癌模型,青蒿素组荷瘤小鼠经作用后肿瘤组织Caspase3及p53蛋白表达显著增加,青蒿素+Pifithrin-α组荷瘤小鼠经Pifithrin-α预处理后,青蒿素诱导的细胞凋亡蛋白Caspase3显著降低.结论 青蒿素通过调控p53的表达诱导肝癌细胞凋亡,进而发挥抗肿瘤作用.     

诱导型NOS与p53、Bax蛋白在胆囊良恶性病变中的表达及相关意义

目的　探讨诱导型一氧化氮合酶 (iNOS)与凋亡诱导基因p5 3、Bax蛋白在胆囊良恶性病变中的表达及相关意义。　方法　采用免疫组织化学SP法检测iNOS及p5 3、Bax蛋白在 16例慢性胆囊炎、11例慢性胆囊炎伴胆囊腺肌瘤增生及 2 4例胆囊腺癌中的表达。　结果　 1 在胆囊良恶性病变的胆囊壁中均有iNOS、Bax表达 ,在胆囊腺癌中iNOS、Bax表达下降 (P <0 0 5 ) ,p5 3仅在部分胆囊腺癌细胞核中有较强阳性表达 ;2 在胆囊良恶性病变中 ,iNOS与Bax表达呈正相关 (P <0 0 1) ,p5 3与Bax表达呈负相关 (P <0 0 1)。　结论　慢性胆囊炎尤其是慢性胆囊炎伴腺肌瘤增生是胆囊腺癌重要的危险因素 ,NO是重要中介分子 ,NO诱导胆囊良性病变恶变的作用与细胞凋亡有密切关系。     

p21/WAF1/CIP1 EXPRESSION IN NORMAL MUCOSA AND IN ADENOMAS AND ADENOCARCINOMAS OF THE COLON: ITS RELATIONSHIP WITH DIFFERENTIATION

The immunoreactivity of p21 was evaluated in normal mucosa and in adenomas and adenocarcinomas of the human large bowel. In normal mucosa, p21 immunoreactivity was seen in the superficial third of the crypts (maturation compartment) and in surface (terminally differentiated) epithelium, and was mutually exclusive with Ki67/MIB1 reactivity. These data show that p21 expression is inversely related to proliferation and directly related to terminal differentiation. In adenomas, p21-reactive cells were frequently clustered in the superficial areas and were non-reactive for MIB1. In adenocarcinomas, p21 staining was heterogeneous: high p21 expression (19 cases) was associated with lower stage and lack of p53 overexpression. p21-reactive cells were devoid of MIB1 immunoreactivity, but no relationship could be found between p21 and MIB1 labelling indices. p21 heterogeneity may be related to alterations in the p53-dependent induction pathway: high p21 expression was associated with low to absent p53 reactivity, with presumed normal p53 function; low p21 expression was associated with p53 overexpression, with presumed p53 alteration resulting in loss of function.     

人喉鳞状细胞癌p53蛋白和PCNA的表达

目的：探讨Ｐ５３蛋白和增殖细胞核抗原（ＰＣＮＡ）在人喉鳞状细胞癌的表达。方法：应用免疫组织化学Ｓ－Ｐ法对８０例喉鳞状细胞癌、１０例声带息肉、１０例喉乳头状瘤进行检测。结果：（１）Ｐ５３蛋白在喉鳞状细胞癌阳性率为４１％，阳性细胞指数为５４．５９±２２．８２，与临床Ｔ分期，分化程度以及淋巴结转移无关；（２）ＰＣＮＡ指数在声带息肉，喉乳头状瘤，喉鳞的逐渐提高，在有淋巴结转移的比无淋巴结转移的指数明显增高     

长期吸烟对大鼠肺组织p53、K-ras蛋白表达和基因突变的影响

目的 观察长期吸烟对Wistar大鼠肺组织p53、K-ras表达的影响,研究吸烟与肺组织p53、K-ras基因突变的关系.方法 建立Wistar大鼠被动吸烟模型.72只雄性Wistar大鼠分为实验组和对照组,实验组被动吸烟6个月,对照组正常呼吸条件下饲养,每个月末分别取6只大鼠肺组织,免疫组织化学观察p53和K-ras蛋白表达情况,聚合酶链式反应-单链构象多态性(PCR-SSCP)技术检测p53第5、6、7～8外显子和K-ras第1外显子的突变情况.结果 免疫组织化学结果显示,p53蛋白表达于细胞核,K-ras蛋白表达于胞浆,吸烟组p53、K-ras蛋白表达强度与正常组比较有显著性差异.随着吸烟时间的增加,p53、K-ras蛋白阳性表达率均随吸烟时间的延长而升高.PCR-SSCP显示,p53基因随吸烟时间的延长突变率增加;K-ras基因突变率随吸烟时间延长增加不显著.结论 吸烟可以导致大鼠肺组织p53、K-ras蛋白表达增强和基因突变率增加,随吸烟时间的延长呈上升趋势,为吸烟对肺的组织基因突变的判定及吸烟者肺癌的早期诊断提供理论依据,具有重要的理论和应用价值.     

M2型肿瘤相关巨噬细胞通过p53途径下调肝癌化疗敏感性的机制研究

目的 通过研究M2肿瘤相关巨噬细胞调控p53表达来下调肝癌化疗敏感性的机制,以此拓宽建立抗肿瘤耐药的措施.方法 构建小鼠肝癌模型及分离肝癌组织,研磨组织提取巨噬细胞,通过RT-PCR方法检测巨噬细胞表面CD206及CD163分子表达情况.培养人单核巨噬细胞(THP-1),PMA (100ng/mL)和IL-4 (100ng/mL)作用诱导分化成肿瘤相关巨噬细胞后,与肝癌细胞(HepG2、SMMC7721,Hep 3B)共培养,加入奥沙利铂(20μ g/mL)作用24小时,通过Western blot方法检测凋亡相关蛋白Caspase 3、抗凋亡蛋白Bcl-2及p53的表达,通过MTT方法检测化疗对肝癌细胞增殖情况.结果 成功构建了肝癌模型,分离出了肝癌组织巨噬细胞,RT-PCR检测CD206和CD163的表达显著升高;人单核细胞(THP-1)经加入PMA(100ng/mL)和IL-4 (100ng/mL)分化诱导48h后其细胞的表面CD206和CD163的表达明显高于THP1分化诱导的细胞表面表达量;肝癌细胞SMMC7721和HepG2与M2型肿瘤相关巨噬细胞共培养24h,经奥沙利铂作用后,肿瘤细胞Bcl-2表达明显升高,Caspase 3和p53的表达显著降低;肿瘤细胞的增殖抑制率明显降低,而Hep 3B细胞的凋亡则明显降低.结论 M2型肿瘤相关巨噬细胞调控肝癌细胞中p53的表达,进而抑制了肝癌化疗的敏感性.     

二乙基己烯雌酚诱发成年仓鼠睾丸生精异常及对生精细胞表达bcl-2、P53和cadherin的影响

目的探讨在二乙基己烯雌酚(DES)诱发成年动物生精异常过程中,原癌基因bcl-2、p53及黏附分子cadherin在生精细胞中表达的变化,旨在阐明DES诱发生精异常的作用机理。方法成年雄性仓鼠皮下注射DES连续7d后,取其睾丸,进行光镜及电镜的观察和原癌基因bcl-2、p53及黏附分子cadherin的免疫组织化学染色。结果DES处理组的成年仓鼠睾丸生精细胞发育异常,bcl-2和p53表达量均比对照组有显著增加,并以精母细胞和圆形精子细胞较为明显。生精上皮中cadherin的表达比对照组有明显减少。结论DES增加精母细胞和圆形精子细胞表达bcl-2和p53;同时抑制cadherin表达,是诱发成年仓鼠生精异常的原因之一。     

人脑胶质瘤中LN、FN及p53基因蛋白的免疫组织化学与其侵袭微生态系统的相关性探讨

目的探讨人脑胶质瘤中层粘连蛋白(LN)、纤维连接蛋白(FN)及突变型p53基因蛋白的免疫组织化学与肿瘤侵袭微生态系统(tumorous invasion microecosystem,TIMES)的相关性. 方法利用透射电镜观察TIMES中微血管特征和免疫组织化学SP法评价LN、FN、p53的表达情况,并作比较分析. 结果 1.脑胶质瘤微血管基底膜(base membrane,BM)连续,多数呈局限性或广泛性增厚,并与LN、FN染色结果相一致,也与p53染色与否有关,p53阳性染色者BM增厚较p53阴性者明显.2.LN、FN在所有胶质瘤微血管BM及内皮细胞上呈阳性染色,恶性程度越高,BM上LN、FN染色阳性越强,血管管壁越厚(P<0.01和P<0.05),而瘤细胞未见染色.LN在脑内转移瘤BM和内皮细胞上却未见染色,但可见散在瘤细胞浆膜染色;FN在脑内转移瘤上的染色则与脑胶质瘤相类似.3. 45例胶质瘤中p53阳性染色21例,其p53阳性染色与否也与BM上LN、FN染色结果存在密切正相关(P<0.01).p53阳性染色率在脑内转移瘤和恶性胶质瘤中无统计学差别(P>0.05). 结论脑微血管内皮细胞上LN、FN的过分表达可能是脑胶质瘤TIMES中BM形态学增厚的原因之一,p53对TIMES的影响也与微血管的内皮细胞功能状态有关.血管内皮细胞可能是TIMES的调控者之一.