Reduced gene expression of E-cadherin and associated catenins in human cervical carcinoma cell lines

Cell–cell adhesion mediated by E-cadherin is often lost or disturbed in human carcinomas. For regular adhesive function, E-cadherin has to form complexes with peripheral cytoplasmic catenins which are multifunctional proteins that are also involved in signal transduction and growth regulation. We have analyzed the expression levels of the genes encoding -catenin, β-catenin and plakoglobin in correlation to the E-cadherin expression levels in cell lines derived from human cervical carcinomas. Reduced mRNA and protein levels were detected for plakoglobin, whereas - and β-catenin showed only reduced protein (but not mRNA) levels. The alterations in catenin gene expression were often associated with absent or reduced E-cadherin. The findings indicate that a reduction of catenin gene expression may contribute to the development of cervical carcinomas.     

5-杂氮-2'-脱氧胞苷对人结肠癌细胞E-cadherin基因表达及甲基化的影响

背景与目的:结肠癌是最常见的恶性肿瘤之一,其发病机制仍未完全明了。目前认为DNA甲基化导致转录抑制是恶性肿瘤发生的重要机制之一。E-cadherin能抑制肿瘤的浸润和转移,被公认为是浸润、转移抑制基因。结肠癌常表现有E-cadherin基因失活。本研究通过检测人结肠癌细胞株HT-29中E-cadherin基因表达及甲基化状态,初步探讨结肠癌的发病机制。方法:光镜观察5-杂氮-2'-脱氧胞苷(5-Aza-CdR)干预前后细胞形态学变化;免疫细胞化学及RT-PCR检测不同浓度5-Aza-CdR干预对HT-29细胞中E-cadherin蛋白及mRNA表达的影响;甲基化特异性PCR(MSP)分析细胞中E-cadherin基因甲基化状态。结果:5-Aza-CdR干预能使其甲基化的E-cadherin基因重新表达;免疫细胞化学染色检测1μmol/L5-Aza-CdR干预24h后细胞E-cadherin阳性率由(21±7)%提高到5μmol/L5-Aza-CdR干预时的(39±13)%;E-cadherin基因在HT-29细胞中有甲基化修饰。结论:E-cadherin基因启动子区异常甲基化是结肠癌发生、发展的重要机制之一,...     

Tet-off系统调控人IFNγ基因在小鼠骨髓基质细胞中的表达

背景与目的:Tet-off系统是一个高效、低毒、具有严密开关功能的基因调控系统,本研究构建质粒pTre-IFNγ,初步探讨Tet-off系统调控人干扰素γ(interferon-gamma,IFNγ)基因在小鼠骨髓基质细胞中的表达。方法:SacⅡ、XbaⅠ双酶切载体pTre及目的基因,T4连接酶连接,重组体经双酶切、测序鉴定;脂质体介导pTet-off及pTre-IFNγ共转染小鼠骨髓基质细胞;RT-PCR法检测IFNγmRNA,ELISA法检测分泌到培养液中的IFNγ蛋白,分析共转染后IFNγ的分泌规律、共转染后48h中不同浓度盐酸四环素对IFNγ蛋白分泌的影响及在共转染后第48h加入盐酸四环素在不同时间阶段对IFNγ蛋白分泌的影响。结果:双酶切及测序证实质粒pTre-IFNγ构建成功;共转染后48hRT-PCR检测到IFNγmRNA表达;ELISA法示IFNγ蛋白分泌持续6天,第3天的分泌高峰为每1×106个细胞(720.09±241.51)pg;共转染后48h中蛋白分泌量随四环素浓度增加逐渐减少,10～100ng/ml四环素存在时蛋白分泌接近零;第48h时添加盐酸四环素后8h即能明显抑制IFNγ蛋白表达,延长作用时间,抑制效应加重。结论:Tet-off系统能迅速而高效地下调人IFNγ基因在小鼠骨髓基质细胞中的表达。     

E-cadherin expression in human epithelial ovarian cancer and normal ovary

The ovarian surface epithelium (OSE) is the origin of the majority of human ovarian cancers. These adenocarcinomas are characterized by initial local growth followed by spreading into the peritoneal cavity at later stages of tumor progression. The cell-adhesion molecule E-cadherin (E-cad) plays an important role in maintaining tissue integrity. Disappearance or impaired function of E-cad have often been associated with tumor formation and invasion in vivo and in vitro. The cell-specific expression of E-cad was investigated in normal human ovaries (n = 12), in benign (n = 5) and borderline (n = 4) ovarian epithelial tumors and in adenocarcinomas of different stages and histological grades (n = 18), by immunohistochemistry and immunoblotting. An ovarian cancer cell line (NIH-OVCAR3) was used as a reference. The epithelial origin of the cells was confirmed with cytokeratin (AE1/AE3) staining. In normal ovaries, the expression of E-cad was limited to inclusion cysts or deep clefts lined with OSE, whereas no staining of the OSE could be demonstrated at the surface of the ovary. In contrast, benign and borderline tumors uniformly expressed E-cad. This was observed in malignant tumors of all stages despite their degree of differentiation. E-cad was also present in metastasis from such tumors. The cell-specific expression of E-cad in inclusion cysts of normal ovaries and in epithelial layers of borderline tumors indicates a role for E-cad in the early events of the progression to a malignant phenotype. E-cad was not downregulated in later stages of ovarian cancer progression. Int. J. Cancer 74:275-280, 1997. © 1997 Wiley-Liss, Inc.     

The association of E-cadherin expression and the methylation status of the E-cadherin gene in nasopharyngeal carcinoma cells

Loss of E-cadherin (E-cad) has been associated with progression and poor survival in nasopharyngeal carcinoma (NPC). In this study, we investigated the role of methylation on E-cad inactivation in NPC cell lines, as well as in NPC tissue samples. Using 6 NPC cell lines, we found that methylation of the E-cad 5' CpG island promoter region was correlated with the loss of both mRNA and E-cad protein expression in these cell lines. In addition, using 29 NPC and 10 non-malignant nasopharyngeal samples, we also observed 5' CpG methylation of the E-cad gene in 52% (15 out of 29) NPC samples, but in only 10% (1 out of 10) of the non-malignant nasopharyngeal tissues. Our findings indicate that 5' CpG island methylation of the E-cad gene may play an important part in the inactivation of E-cad in NPC. Our results also suggest that reducing the methylation of the E-cad gene may be a potential therapeutic strategy for NPC.     

Altered expression of E-cadherin in breast cancer. patterns, mechanisms and clinical significance

Reduced cell adhesion brought about by altered surface expression of E-cadherin has been implicated in invasive and metastatic malignant growth. We investigated the patterns of immunohistochemical E-cadherin expression in 120 breast carcinomas. Furthermore, we analysed DNA from the same samples for loss of heterozygosity (LOH) using three separate microsatellite markers on chromosome 16q22.1. Finally, the clinical outcome was ascertained for 108 patients. 19% (18/97) of infiltrating ductal carcinomas showed complete loss of E-cadherin expression compared with 64% (9/14) of infiltrating lobular carcinomas. LOH was detected in 46% (24/52) of infiltrating ductal carcinomas and 89% (8/9) of infiltrating lobular carcinomas. In the infiltrating lobular carcinomas, LOH was associated with complete loss of cell membrane expression of E-cadherin, although a cytoplasmic expression pattern was evident. In contrast, this association was not seen in the infiltrating ductal carcinomas. In a multivariate analysis, loss of E-cadherin expression was shown to be a significant independent risk factor for a poorer disease-free survival (P=0.019), in particular in the node-negative subset of patients (P=0.029). Significance was also approached for breast cancer corrected survival (P=0.056). We conclude that different mechanisms are involved in the altered E-cadherin expression seen in different subtypes of breast carcinomas. Furthermore, we implicate loss of E-cadherin, regardless of the genetic causes, as an independent prognostic marker for disease recurrence, especially in node-negative breast cancer patients, irrespective of the histological type.     

Chromosome alterations and E-cadherin gene mutations in human lobular breast cancer

We have studied a set of 40 human lobular breast cancers for loss of heterozygosity (LOH) at various chromosome locations and for mutations in the coding region plus flanking intron sequences of the E-cadherin gene. We found a high frequency of LOH (100%, 31/31) at 16q21-q22.1. A significantly higher level of LOH was detected in ductal breast tumours at chromosome arms 1p, 3p, 9p, 11q, 13q and 18q compared to lobular breast tumours. Furthermore, we found a significant association between LOH at 16q containing the E-cadherin locus and lobular histological type. Six different somatic mutations were detected in the E-cadherin gene, of which three were insertions, two deletions and one splice site mutation. Mutations were found in combination with LOH of the wild type E-cadherin locus and loss of or reduced E-cadherin expression detected by immunohistochemistry. The mutations described here have not previously been reported. We compared LOH at different chromosome regions with E-cadherin gene mutations and found a significant association between LOH at 13q and E-cadherin gene mutations. A significant association was also detected between LOH at 13q and LOH at 7q and 11q. Moreover, we found a significant association between LOH at 3p and high S phase, LOH at 9p and low ER and PgR content, LOH at 17p and aneuploidy. We conclude that LOH at 16q is the most frequent chromosome alteration and E-cadherin is a typical tumour suppressor gene in lobular breast cancer.     

Kallikrein 10的调控机制及其肿瘤临床应用的价值

人组织激肽释放酶10（Kallikrein10,KLK10）,是Kallikrein家族(KLK 1-15)的成员之一。KLK10编码激肽释放酶10（hK10）,hK10是一种分泌型丝氨酸蛋白酶,生理功能不明。KLK10与多种恶性肿瘤的发生、发展密切相关,KLK10异常表达受甲基化、激素受体等多种因素的调节,miRNA对KLK10的调控也成为新的热点。KLK10与KLK家族其他成员在部分肿瘤中平行表达,KLK10与其他KLKs可能存在共同的激素和miRNA调节通路。KLK10作为一种新的肿瘤生物学标志物,在卵巢癌、乳腺癌和胃结直肠癌等恶性肿瘤的早期诊断和预后评估及靶向治疗中的意义也日益显现。     

Immunohistochemical expression of E-cadherin and beta-catenin in the normal and malignant human endometrium: an inverse correlation between E-cadherin and nuclear beta-catenin expression

BACKGROUND: The E-cadherin/beta-catenin complex plays a crucial role in epithelial cell-cell adhesion and in the maintenance of tissue architecture. We previously reported aberrant expression of beta-catenin in endometrial carcinomas. However, the expression and correlation of E-cadherin and beta-catenin in normal and malignant endometrial tissues are not fully understood. MATERIALS AND METHODS: Immunohistochemical expression of E-cadherin and beta-catenin was detected in 30 cases of normal endometrium and 73 cases of endometrial carcinoma. RESULTS: In the normal endometrium, the expression of E-cadherin and cytoplasmic beta-catenin in glandular cells was predominantly observed in the proliferative phase, and decreased in the secretory phase. In endometrial carcinomas, the expression of E-cadherin and cytoplasmic beta-catenin decreased compared to that in the normal proliferative endometrial glands. The expression of E-cadherin and cytoplasmic beta-catenin tended to be reduced in histologically high-grade tumors compared to low-grade tumors. Nuclear expression of beta-catenin was observed in the glandular cells in the late proliferative and early secretory phases, as well as in high-grade endometrial carcinomas. Interestingly, nuclear beta-catenin expression was associated with the loss of E-cadherin expression in normal and carcinoma cells, indicating an inverse correlation. CONCLUSION: The cyclic expression of E-cadherin and beta-catenin in the normal endometrium suggests that the adhesion complex may act to maintain the endometrial architectures. In addition, nuclear beta-catenin expression associated with loss of E-cadherin expression may be involved in the acquisition of aggressive biological behavior, especially in high-grade tumors.     

The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas

Our study was designed to clarify the significance of silencing the E-cadherin gene, which is located on 16q22.1, due to CpG methylation during hepatocarcinogenesis. The CpG methylation status of primary hepatocellular carcinomas (HCCs) and corresponding liver tissues showing chronic hepatitis or cirrhosis, which are widely considered to be precancerous conditions, were assessed by digesting DNA with methylation-sensitive and non-sensitive restriction enzymes. CpG methylation around the promoter region of the E-cadherin gene was detected in 46% of liver tissues showing chronic hepatitis or cirrhosis and 67% of HCCs examined. Immunohistochemical examination revealed reduced E-cadherin expression in 59% of HCCs examined. CpG methylation around the promoter region correlated significantly with reduced E-cadherin expression in HCCs (p < 0.05). CpG methylation around the promoter region, which increases during the progression from a precancerous condition to HCC, may participate in hepatocarcinogenesis through reduction of E-cadherin expression, resulting in loss of intercellular adhesiveness and destruction of tissue morphology. Int. J. Cancer 71:355-359, 1997. © 1997 Wiley-Liss Inc.     

E-cadherin expression in upper-urinary-tract carcinoma

Carcinoma of the upper urinary tract is a relatively rare neoplasm, and few studies have dealt with clinicopathological findings and prognosis in a large number of cases. The purpose of our investigation was to look for a possible relation between E-cadherin (E-CD) immunoreactivity and clinicopathologic findings or clinical outcome in transitional-cell carcinoma of the upper urinary tract (TCC-UUT). To this end, we investigated E-CD immunoreactivity in 154 cases of TCC-UUT. E-CD immunoreactivity was recognized as being of “normal” pattern in 29.2% of samples. The relationship between E-CD immunoreactivity and clinicopathologic findings was significant for stage, grade and pattern of growth. The 5-year disease-free rate for 147 cases and 5-year overall survival rate for 154 cases were 55.7% and 71.5%, respectively. A univariate analysis of survival revealed that stage, grade, pattern of growth and E-CD immunoreactivity all had a significant effect on disease-free and overall survival rates. In the final models of multivariate analysis, however, we found that, for disease-free survival and for overall survival, only stage was a factor for progression or prognosis. Detection of E-CD immunoreactivity appears to be of limited value in deciding the prognosis of TCC-UUT. Int. J. Cancer 74:446–449, 1997. © 1997 Wiley-Liss, Inc.     

E-钙黏附素及α-链接素的表达与胃癌生物学行为及淋巴结转移规律的关系

目的 探讨上皮细胞黏附分子E－钙黏附素（E－CD）及链接素－α（α-CA）在胃癌的表达及其与胃癌生物学行为的关系。方法 采用免疫组化技术（ABC法）对70例手术切除新鲜胃癌组织进行了E－CD及α-CA表达情况的检测,每例均行腹腔脱落癌细胞和系统病理学检查。结果 胃癌组织E－CD及α-CA表达阳性率分别为38．6％和28．6％。在侵袭型、未分化型、弥漫状生长、侵透浆膜（T3／T4）、淋巴结转移阳性及Ⅲ、Ⅳ期胃癌中,E－CD及α-CA表达显著减弱（P＜0．025－0．005）。在淋巴结多数转移（＞5个）,转移度＞50％及转移至Ⅱ站以远的胃癌中,E－CD及α-CA表达亦明显减弱,且α-CA比E－CD降低更明显（P＜0．05－0．005）。E－CD（＋）α-CA（-）及E－CD（－）／α-CA（-）组淋巴结转移率和脱落癌细胞阳性率均高于E－CD（＋）／α-CA( )组（P＜0．05－0．005）,但E－CD（＋）／α-CA（-）联合检测对评估胃癌侵袭深度、淋巴结转移程度、肝转移、TNM分期及腹腔癌细胞脱落状态有较大意义,其中α-CA比E－CD表达更敏感。     

Clinical outcomes of downregulation of E-cadherin gene expression in non-small cell lung cancer

Objective: To investigate the promoter methylation status of the E-cadherin gene in non-small cell lung cancer(NSCLC) and its association with clinical pathological parameters, and to explore the relationship betweendownregulation of E-cadherin gene expression and the methylation status of its promoter region. Methods:Nested methylation-specific PCR was performed to examine CpG methylation within the 5’ CpG island of theE-cadherin gene in lung cancer and para-cancerous tissue from 37 patients with primary non-small cell lungcancer. Quantitative real-time PCR was performed to measure the level of E-cadherin mRNA. Results: Ofthirty-seven cases, 12 (32.4%) samples showed aberrant CpG methylation in tumor tissues compared with thecorresponding normal tissues. In addition, a reduction in E-cadherin mRNA levels was observed in 11 of the12 (91.7%) tumor tissues carrying a methylated E-cadherin gene. However, only 10 (43.5%) cases displayedreduced mRNA levels in tumor tissues from the remaining 23 cases (excluding 2 samples from which mRNA wasunavailable) without methylation events. Downregulation of E-cadherin gene expression significantly correlatedwith the promoter methylation status of this gene. Conclusion: These results provide strong evidence that themethylation status of E-cadherin gene contributes to a reduction in the expression of E-cadherin mRNA, andmay play a role in the development and progression of NSCLC.     

Multiple ways of silencing E-cadherin gene expression in lobular carcinoma of the breast

The cell-cell adhesion receptor gene E-cadherin (CDH1) is expressed by epithelial cells, in which it mediates adhesion and morphogenesis. Invasive lobular carcinoma (ILC) characteristically infiltrates diffusely as single cells; by immunohistochemistry, many of these tumours lack E-cadherin expression. In the present study we investigated various ways in which loss of function of the E-cadherin gene could occur in ILCs, namely, promoter methylation, mutation and allelic loss. We analysed 22 ILCs and found 12 (55%) E-cadherin-negative samples by immunohistochemical analysis. Methylation-specific polymerase chain reaction (PCR) showed that 17/22 (77%) of these tumours had methylation of the CDH1 promoter, including 11/12 (91%) of the E-cadherin-negative tumours. All 16 exons of E-cadherin (including intron-exon boundaries) were amplified from chromosomal DNA and screened for mutations by conformation-sensitive gel electrophoresis (CSGE). Bands with altered mobility were analysed by direct sequencing. We identified five frameshift mutations, which resulted in downstream stop codons and one splice site mutation in six different tumours (29%). Loss of heterozygosity (LOH) was assessed using microsatellite markers, and 9/18 (50%) informative tumours showed LOH. We conclude that most ILCs show genetic or epigenetic changes affecting the E-cadherin gene and that many of these tumours lack E-cadherin expression. In all cases in which there was loss of expression, this was consistent with biallelic inactivation of CDH1 by promoter methylation, mutation or allelic loss in any combination.     

MeCP2 and promoter methylation cooperatively regulate E-cadherin gene expression in colorectal carcinoma

Reduced expression of E-cadherin (E-cad) owing to aberrant 5'CpG island hypermethylation has been regarded as one of the main molecular events involved in the dysfunction of the cell-cell adhesion system. The molecular mechanisms providing diversity and heterogeneity of E-cad expression in colorectal carcinoma were explored. In 29 cases of colorectal carcinoma in Indonesia, the expression of E-cad was analyzed by immunohistochemical staining, the methylation status of the E-cad promoter was determined by methylation-specific PCR, and the expression of methyl-CpG-binding protein (MeCP) 2 was studied by in situ hybridization. E-cad expression was strong, and no methylation was observed in normal colon mucosa and most of the well-differentiated adenocarcinoma. In contrast, both signet-ring cell carcinoma and mucinous adenocarcinoma showed fully methylated patterns and strong MeCP2 expression. In moderately- and poorly-differentiated adenocarcinomas, however, E-cad expression was rather heterogeneous, especially at the front of invasion and in the dissociated areas, where loss of MeCP2 expression correlated with E-cad reexpression even in the presence of E-cad promoter methylation. We conclude that both CpG methylation of the E-cad promoter and significant MeCP2 expression cooperatively and epigenetically regulate E-cad expression in colorectal cancer. (Cancer Sci 2003; 94: 442–447)