Immunolocalization of urokinase-type plasminogen activator in adenomas and carcinomas of the colorectum

Carcinogenesis in the human colon is associated with a marked increase in the tissue content of the urokinase-type plasminogen activator (u-PA). This study was performed to determine the type of cells responsible for the u-PA increase in carcinomas of the colon and in their precursor lesions, the adenomas, by immunohistological evaluation applying monoclonal antibody 3689 directed to the beta-chain of u-PA. Normal intestinal mucosa (n = 17) showed hardly any staining of u-PA, but some lamina propria cells were faintly positive. Carcinomas (n = 17) and adenomas (n = 16) showed a considerable and comparable staining intensity of u-PA in neoplastic columnar epithelial cells, and this staining was found to be diffuse and cytoplasmic. In a majority of the neoplastic tissues the u-PA staining was found to be patchy and not related to known risk markers of malignancy such as dysplasia in the adenomas, or to prognostic determinants such as Dukes' classification or differentiation in the carcinomas. The observation of strong u-PA positive lamina propria cells in adenomas but infrequently observed in normal mucosa and carcinomas was noteworthy. u-PA staining intensity of the tissue sections was found to correlate well with the u-PA antigen level in the tissue extracts determined by ELISA (r = 0.52, P = 0.0001) but poorly with the u-PA activity determined enzymatically (r = 0.28, P = 0.05). In conclusion, the u-PA increase in neoplasia of the human colon can be attributed to an increased diffuse cytoplasmic content of u-PA in neoplastic columnar epithelial cells.     

A morphometrical analysis of minute depressed adenomas in familial polyposis coli

In a morphometrical study of minute adenomas in familial polyposis coli (FPC), ranging from 0.8 mm to 1.5 mm in diameter, five parameters of depressed adenomas were compared with those of ordinary polypoid adenomas. The density of glands (DG), an index of structural atypia, and the nuclear-cytoplasmic (N/C) ratio, an index of cellular atypia, were higher in depressed adenomas than in polypoid adenomas (74.8% vs 66.8% and 37.8% vs 32.7%, respectively). The cell area (C-area) of depressed adenomas was less than that of polypoid adenomas (107.3(μm2 vs 121.1 μm2), but there was no significant difference in values for the nuclear area (N-area) in the two types (40.0 μm2 vs 39.2 μm2). The higher N/C ratio of depressed adenomas was, therefore, not caused by enlargement of the nuclei but by reduction of the cell size. In addition, values of the sphericity of nuclei (SN) (3.14 vs 3.65) indicated rounder nuclei in depressed adenomas than in ordinary ones. The higher DG and N/C ratio of the depressed adenomas indicates their higher grade of atypia than that of ordinary polypoid adenomas.     

APC in the regulation of intestinal crypt fission

The functional effects of APC (adenomatous polyposis coli gene) germ-line mutations on crypt fission and cell proliferation were investigated in the normal intestine of human familial adenomatous polyposis (FAP) and multiple intestinal neoplasia (MIN) mice. Compared with controls, there was a 19-fold increase in the proportion of crypts in fission in FAP colon [95 per cent confidence interval (CI):11–32, P<0·0001], and a 75 and 61 per cent increase in MIN colon (95 per cent CI:1·08–2·82, P<0·02) and small bowel, respectively (95 per cent CI:1·31–1·99, P<0·001). In marked contrast, no significant differences in intra-cryptal epithelial cell proliferation or mitotic distribution were seen. Furthermore, 10·9 per cent of crypts in FAP were in asymmetrical fission as opposed to only 1 per cent in controls (P=0·001). The largest relative increases in MIN crypt fission were in the colon (proximal and distal colon:190 per cent, P=0·02 and 83 per cent, P=0·01), suggesting that Apc mutations exert their maximal influence site-specifically. However, sites with the highest relative increases were also those with the largest eventual tumour sizes, but not the highest polyp counts. Three-dimensional serial section reconstruction analysis corroborated that FAP adenomas enlarge by crypt fission, which was frequently both asymmetrical and atypical. It is proposed that the absence of an increase in intestinal cell division infers that APC regulates intestinal crypt differentiation, specifically through the crypt cycle. This role appears analogous to the control of axis re-duplication in embryonic development, when downstream targets of APC are over-expressed. It is concluded that in vivo, the major defect in pre-neoplastic intestine harbouring APC mutations is elevated rates of crypt fission, and that this is also the mode by which micro-adenomas enlarge. © 1998 John Wiley & Sons, Ltd.     

P-选择素对ApcMin/+小鼠肠道肿瘤的作用

目的: 研究P-选择素(P-selectin)在Apc^Min/+小鼠肠道肿瘤中的作用。方法: 采用P-selectin 基因缺失的基因工程小鼠和肠道肿瘤模型Apc^Min/+小鼠杂交,计数Apc^Min/+小鼠与Apc^Min/+ P-selectin ^-/-杂交小鼠小肠及大肠肿瘤的数目,并测量其肿瘤体积,研究P-selectin对Apc^Min/+小鼠肠道肿瘤的作用。结果: 与Apc^Min/+小鼠相比,Apc^Min/+P-selectin^-/-杂交小鼠在9周龄时肠道肿瘤数目与总负荷明显减少。结论: P-selectin 缺失能够显著抑制Apc^Min/+小鼠肠道肿瘤的生长。     

Electron microscopic studies of endocrine hyperplasia in duodenal adenomas in familial adenomatous polyposis

Summary Electron microscopical studies on endocrine cell hyperplasia of duodenal adenomas from five patients with familial adenomatous polyposis were performed. All the endocrine cell types normally found in the duodenal mucosa were identified. A constant feature was proliferation of duodenal-enterochromaffin cells but an increase in the number of all other endocrine cell types apart from pyloricgastrin cells and somatostatin cells, was also observed. Certain types of intestinal endocrine cells (the intestinal enterochromaffin cell and the glicentin cell) are rare cells in the normal duodenal mucosa. The finding of these cells may indicate increased biological aggressivity.     

Pkhd1基因的缺失可促进ApcMin/+小鼠肠道肿瘤的发展演进

目的 确定人类常染色体隐性遗传性多囊肾病(ARPKD)致病基因Pkhd1在ApcMin/小鼠肠道肿瘤发生中的作用.方法 Pkhd1基因敲除小鼠模型(Pkhd1-/-)与肠道肿瘤模型ApcMin/+小鼠杂交后,比较不同月龄(1、3、6月)单一ApcMin/+小鼠与缺失Pkhd1的ApcMin/+小鼠(Pkhd1-/-;ApcMin)肠道肿瘤的数目及大小,并观察其肠道肿瘤组织病理的变化.结果 与单一的ApcMin/+小鼠相比,Pkhd1-/-;ApcMin/+小鼠在1月龄时,肠道肿瘤数目及大小均无显著差异.但该小鼠在3月龄和6月龄时肠道肿瘤数目及大小均显著增加(P=0.017、P=0.022).此外,在6月龄时,Pkhd1-/-;ApcMin/+小鼠病理表型有向恶性转化的明显趋势.结论 具有Pkhd1缺失的Apc Min/+小鼠与单一的ApcMin/小鼠相比能够显著促进肠道肿瘤的发生和发展,并具有促进其肿瘤恶性转化的趋势.     

Mutations and nuclear accumulation of beta-catenin correlate with intestinal phenotypic expression in human gastric cancer

AIMS: Abnormal localization of beta-catenin is frequently observed in human gastric cancers. The aim of the present study was to evaluate relationships among gastrointestinal differentiation phenotypes, beta-catenin localization and mutations of Wnt signalling genes. METHODS AND RESULTS: Seventy-seven regions in 39 gastric adenocarcinomas were classified according to beta-catenin localization and gastric and intestinal phenotypes. Cases with membranous beta-catenin localization showed a gradual decrease from gastric (G) (55% = 6/11) and gastric-and-intestinal-mixed (GI) (17% = 5/29) to intestinal (I) (0% = 0/21) phenotypes, while those with nuclear localization showed a concomitant increase: 18% (2/11), 41% (12/29), 95% (20/21) and 63% (10/16) for G, GI, I and null type (N), respectively (P < 0.001, membranous versus nuclear localization in G, GI through I). Mutations in exon 3 of the beta-catenin gene were found in G (50% = 1/2), GI (67% = 8/12), I (45% = 9/20) and N (0% = 0/10) regions with nuclear beta-catenin localization (GI versus N, P < 0.01; I versus N, P < 0.05). Adenomatous polyposis coli (APC) gene mutations were demonstrated only in GI, I and N types, irrespective of beta-catenin localization. Molecular analysis of these genes revealed 10 tumours to be heterogeneous out of 16 informative cases (62.5%). CONCLUSION: Intestinal phenotypic expression is accompanied by a shift from membranous to cytoplasmic/nuclear accumulation of beta-catenin. In contrast, N-type regions may progress along a different pathway.     

遗传性非腺瘤病性结直肠癌结肠腺瘤病基因突变研究

目的 分析遗传性非腺瘤病性结直肠癌(hereditary non-polyposis colorectal cancers，HNPCC)结肠腺瘤病(adenomatous polyposis coli，APC)基因突变的特点及错配修复缺陷对其影响。方法 采用体外蛋白合成试验和序列分析确定19例HNPCC病例APC体细胞突变。结果 19例病例中有11例(13个突变点)发生APC突变，发生率为58％(11／19)，其中移码突变9个，无义突变4个，移码突变占多数(69％)。所有移码突变表现为1—2个碱基的缺失或插入，大多(7／9)发生在简单核苷酸重复序列，特别是单腺苷酸重复序列(A)n(5／9)。检出的由单个碱基替换而导致的无义突变都发生在CpG岛，表现为C向T的转换。结论 多于半数的HNPCC发生APC突变，其突变多发生在编码区单核苷酸重复序列(移码突变)或CpG岛(点突变)上，提示APC基因失活在HNPCC为常见的分子事件；错配修复缺陷所致的微卫星DNA不稳定性等内源性机理可能对APC突变产生影响。     

应用变性高效液相色谱检测31例家族性腺瘤性息肉病家系结肠腺瘤性息肉病基因突变

目的 应用变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)技术检测我国家族性腺瘤性息肉病(familial adenomatons polyposis,FAP)家系的结肠腺瘤性息肉病(adenoinatous pelyposis coli,APC)基因变异特征,研究其病因机制.方法 采集31个家系的先证者、患者和家系成员的外周血淋巴细胞,抽提DNA并以降落式PCR扩增APC基因各外显子和启动子.基因突变检测先由DHPLC进行筛选,发现异常峰者进行测序鉴定并TA克隆鉴定,结果与网络数据进行比对.结果 31个家系中共有15个家系检出了12种不同的突变类型,FAP家系APC基因的突变检出率为48.39%.发现了4种新的突变及3例不同的内含子突变.4个新的突变分别位于255、677、1192、1403密码子,均为移码突变.证明了DHPLC能检出APC基因的突变.在APC基因的突变中,移码突变占86.67%,无义突变占13.33%,说明移码突变是中国人APC基因突变的主要方式.在突变位点上,第15外显子突变最常见,约占86.67%.结论 FAP家系APC基因的突变检出率为48.39%,发现了4种新的导致蛋白编码改变的突变.证实中国人FAP家系中APC基因突变位点以第15外显子最常见,类型以移码突变为主.     

The role of PSGL-1 in the growth of intestinal tumor in ApcMin/+ mice

目的 研究P-选凝素糖蛋白配体1(P-selectin glycoprotein ligand 1,PSGL-1)在ApcMin/ +小鼠肠道肿瘤中的作用.方法 PSGL-1基因缺失的基因工程小鼠和肠道肿瘤模型ApcMin/ +小鼠杂交后,统计ApcMin/ +小鼠与(ApcMin/ +;PSGL-1-/-)杂交小鼠小肠及大肠肿瘤的数目和总负荷,观测其肠道肿瘤的发生变化.结果 相比ApcMin/ +小鼠,(ApcMin/ +;PSGL-1-/-)杂交小鼠在18周龄时肠道肿瘤数目与总负荷有明显增加.结论 PSGL-1的缺失促进了ApcMin/ +小鼠肠道肿瘤的发生,PSGL-1在人类肠道肿瘤中可能发挥着抑制肿瘤形成的作用.     

家族性腺瘤性息肉病一家系调查及APC基因胚系突变分析

目的 探讨家族性腺瘤性息肉病(familial adenomatous polyposis,FAP)家系调查及高危亲属基因筛查的意义,报道云南省一FAP家系发病相关基因APC基因的胚系突变结果.方法 查阅对2001年昆明医学院第一附属医院1例FAP患者病例,电话联系及登门随访进行其家系调查,绘制家系图谱.抽取该家系成员外周静脉血提取DNA,利用PCR方法扩增APC基因,应用DNA自动测序仪进行测序.结果 该家系三代共计9人,成员Ⅰ1、Ⅱ1、Ⅱ2、Ⅱ3、Ⅱ4、Ⅲ2、Ⅲ3、Ⅲ48人检出APC基因胚系突变c.3587C>A(S1196X),其中Ⅱ2、Ⅱ2、Ⅱ4、Ⅲ2、Ⅲ3经肠镜检查证实有结直肠多发息肉,Ⅲ4未检出息肉,为基因突变携带者.结论 通过家系调查对高危亲属进行基因筛查可以发现早期患者,尤其是无临床表现的FAP基因突变携带者,以早期进行医学干预及预防性手术治疗,降低FAP的癌变率、病死率;APC基因c.3587C>A(S1196X)胚系突变是引起该家系FAP患者发病的原因.     

家族性腺瘤样息肉病中APC基因的胚系突变分析

目的 探索有效的突变检测技术，系统分析家族性腺瘤样息肉病(familial adenomatous polyposis，FAP)相关基因结肠腺瘤病(adenomatous polyposis coli，APC)基因的胚系突变，及其与疾病表型的关系。方法 从22例临床确诊的FAP患者，外周静脉血中提取基因组DNA。变性高效液相色谱、蛋白截短检测、测序技术结合应用进行全基因分析。根据患者临床资料，进行基因型-表型分析。结果 22例FAP患者中13例检出APC基因胚系突变，均为无义或移码突变。基因型-表型关系的初步分析表明，在基因5′端或3′端发生突变的患者临床症状较轻，在基因中段发生突变的患者临床症状典型或严重。结论 本研究中所采用的技术体系可敏感、高效地检出APC基因突变，APC基因的突变型与FAP患者的临床表型存在关联，所采用的技术体系适用于FAP症状出现前的基因诊断。     

中国人家族性腺瘤性患肉病患者结肠腺瘤性患肉病基因的胚系突变

目的 探讨中国人家族性腺瘤性息肉病(familial adenomatous polyposis,FAr)患者的结肠腺瘤性息肉病(adenomatous polyposis coli,APC)基因的胚系突变类型.方法 对9个FAP家系18名成员进行多重连接依赖性探针扩增(multiplex ligation-dependent probe amplification,MLPA)检测APC基因有无大片段缺失.再应用PCR扩增APC基因的15个外显子区域,经变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)对每个扩增片段进行筛查,流出峰异常的片段,经DNA测序验证小片段的改变.结果 9个家系中有3个家系发现有APC基因的胚系突变:家系2为c.3184-3187 del CAhA,家系4为c.5432C＞T,家系9为c.3925-3929 del AAAAG.3种突变中c.5432C＞T在数据库中未见报道.结论 中国人不同的APC基因的胚系突变可引起FAP;无APC胚系突变的FAP患者的发病可能存在其他的机制.     

Germline mutations in APC and MUTYH are responsible for the majority of families with attenuated familial adenomatous polyposis

A small fraction of families with familial adenomatous polyposis (FAP) display an attenuated form of FAP (AFAP). We aimed to assess the presence of germline mutations in the MUTYH and adenomatous polyposis coli (APC) genes in AFAP families and to compare the clinical features between the two causative genes. Families with clinical AFAP were selected from the Dutch Polyposis Registry according to the following criteria: (a) at least two patients with 10-99 adenomas diagnosed at age >30 years or (b) one patient with 10-99 adenomas at age >30 years and a first-degree relative with colorectal cancer (CRC) with a few adenomas, and, applying for both criteria, no family members with more than 100 polyps before the age of 30 years. All probands were screened for germline mutations in the APC and MUTYH genes. Twenty-five of 315 Dutch families with FAP (8%) met our criteria for AFAP. These families included 146 patients with adenomas and/or CRC. Germline APC mutations were identified in nine families and biallelic MUTYH mutations in another nine families. CRC was identified at a mean age of 54 years (range 24-83 years) in families with APC and at 50 years (range 39-70 years) in families with MUTYH (p = 0.29). APC and biallelic MUTYH mutations are responsible for the majority of AFAP families. Based on our results and those reported in the literature, we recommend colonoscopy once every 2 years in AFAP families, starting surveillance from the late teens in APC mutation carriers and from age 20-25 years in biallelic MUTYH mutation carriers.     

Hypermethylation of APC promoter 1A is associated with moderate activation of Wnt signalling pathway in a subset of colorectal serrated adenomas

Aims:  The role of Wnt signalling pathway in serrated adenomas (SAs) remains to be identified. The aim of this study was to determine whether Wnt signalling plays a role in the pathogenesis of SAs, and to clarify the mechanism of Wnt signalling activation in SAs.
Methods and results:  This study investigated immunoreactivities of adenomatous polyposis coli (APC) and β-catenin, mutations of APC and β-catenin genes, methylation status of APC promoter 1A in 12 SAs, and compared the findings with normal colorectal mucosa, hyperplastic polyps, traditional adenomas (TAs) and colorectal cancers (CRCs). APC expression was moderately decreased in SAs. Cytoplasmic accumulation of β-catenin was demonstrated in 41.7% (5/12) of SAs, but membranous immunoreactivity of β-catenin was lost in only 8.3% (1/12) of SAs. No β-catenin mutation was detected in any of 12 SAs, and only one SA was found to be positive for APC gene mutation. Complete methylation of APC promoter 1A was found in 41.7% (5/12) of SAs, but in no TAs or CRCs.
Conclusions:  Hypermethylation of APC promoter 1A, instead of mutations involving APC and β-catenin , contributes to moderate activation of Wnt signalling in a subset of SAs.     

家族性腺瘤性息肉病患者APC基因启动子区异常甲基化及大片段结构异常

目的 研究3个家族性腺瘤性息肉病(familial adenomatous polyposis,FAP)家系的腺瘤样息肉病(adenomatus polyposis coli)基因(APC)启动子1A区异常甲基化及DNA大片段结构异常.方法 对3个FAP家系成员的肿瘤组织标本和正常组织标本DNA进行化学修饰,应用甲基化特异PCR(methylation-speeif-ic PCR,MsP)和DNA序列分析方法筛查APC基因启动子1A区甲基化情况.采用多重连接依赖性探针扩增(multiplex ligation-dependent probe amplification,MIPA)分析系统检测5例FAP患者肿瘤组织标本和正常标本的APC基因的15个外显子及启动子区DNA大片段结构异常.结果 在1个家系中发现2例患者存在APC基因启动子1A区异常甲基化.同一个家系中另1例患者存在APC基因全基因杂合性缺失.结论 APC基因启动子1A区异常甲基化可影响APC功能,可能是结直肠癌进展过程中的早期事件;大片段缺失可能是导致典型FAP的一个因素.     

Downregulation of the urokinase-type plasminogen activator receptor through inhibition of translation by antisense oligonucleotide suppresses invasion of human glioblastoma cells

We previously showed that downregulation of the urokinase-type plasminogen activator receptor (uPAR) in the SNB19 human glioblastoma cell line by the stable transfection of a plasmid expressing a 300 bp antisense sequence to the 5′ end of the uPAR gene produced a decrease in the amount of target mRNA. In a more recent study, we found that adenovirus-mediated transduction (Ad-uPAR) of the same uPAR antisense gene construct in SNB19 cells also downregulated uPAR protein levels. We report here that Ad-uPAR-transfected SNB19 cells produced the same amounts of target uPAR mRNA but significantly less protein by in vitro translation and by in situ [³⁵S] labeling compared to Ad-CMV vector-transfected and mock-transfected cells. This antisense construct also inhibited glioblastoma cell invasion confirming previous results. We conclude that downregulation of uPAR by this antisense gene construct results from inhibition of protein translation. This revised version was published online in July 2006 with corrections to the Cover Date.     

APC基因甲基化定量检测芯片的建立

目的 建立抑癌基因APC（adenomatous polyposis coli，APC）启动子1A的甲基化定量芯片检测方法。方法选取一段420bp的APC基因启动子1A CpG密集序列作为靶序列，针对M0、M1、M2、M3、M4 5个CpG靶位点，设计一套检测甲基化与非甲基化的探针。采用脐带血DNA克隆体作为阴性、阳性质控品。结果甲基化阳性、阴性质控的芯片结果与测序吻合。每组探针中荧光强度由强至弱依次为，阳性质控（甲基化）：探针1〉2、3〉4；阴性质控（非甲基化）：探针3〉4、1〉2。5个位点的5条荧光强度标准曲线，尺。范围是0．93～0．99。M0、M1、M2、M3、M4 5个位点甲基化杂合型的检测范围分别为50．0％±3．6％、50．0％±6．9％、50．0％±3．5％、50．0％±8．5％、50．0％±7．3％。结论建立了APC基因启动子5个COG位点的甲基化定量检测芯片。     

Immunohistochemical detection of the receptor for urokinase plasminogen activator in human colon cancer

Paraffin-wax embedded specimens from 30 cases of colonic adenocarcinoma were investigated for immunoreactivity for the receptor of urokinase-type plasminogen activator (uPAR). In all cases there was a strong signal, predominantly at the invasive foci. The positive cells were mainly tumour-infiltrating macrophages but neutrophils and eosinophils were also strongly stained. The neoplastic cells were positive in 19 of the samples with staining of occasional or a moderate number of cells. In uninvolved, normal-appearing mucosa adjacent to the malignant infiltrates, immunostaining of both macrophages and neutrophils was seen, but the labelling was less intense than that seen in the malignant lesions. Weak to moderate staining of normal intestinal epithelium was also seen at the luminal surface. Comparison between immunoreactivity and in situ hybridization showed a similar distribution of protein and mRNA with two exceptions: first, neutrophils (strongly immunoreactive for uPAR) were negative or only weakly positive for uPAR mRNA; and second, many cancer cells at invasive foci showed prominent hybridization signals but no detectable uPAR immunoreactivity. Together with previous findings of urokinase plasminogen activator (uPA) protein and mRNA being expressed in tumour-infiltrating fibroblast-Iike cells at the invasive foci, these results support the view that the uPA pathway of plasminogen activation is involved in tissue degradation in colon cancer. The results also extend and consolidate an emerging picture of non-neoplastic tumour stromal cells producing molecules involved in the generation and regulation of extracellular proteolysis in cancer.