肝细胞生长因子/c-Met系统在鼻咽癌中的表达及意义

目的　探讨肝细胞生长因子(HGF)及其受体c- Met蛋白在鼻咽癌组织中的表达水平,研究HGF/c- Met系统对鼻咽癌细胞侵袭转移的影响。方法　收集1999—2003年期间45例确诊的鼻咽原发癌活检组织标本,采用免疫组织化学(LSAB)法检测鼻咽癌组织中HGF α亚单位和c- Met的表达,并与患者的病理及临床资料相联系。采用流式细胞术检测鼻咽癌细胞株CNE- 2在HGF刺激前后c- Met阳性癌细胞百分率的改变;蛋白印迹法和逆转录PCR法分别用于检测癌细胞株c -Met蛋白表达和mRNA表达的变化。结果　在45例鼻咽原发癌组织中,癌细胞c- Met的阳性表达率为91. 1% (41 /45),但仅有1例鼻咽癌细胞表达HGF( 2 .2%, 1 /45 )。HGF主要在鼻咽癌间质中的淋巴细胞表达。癌细胞c Met的表达水平与淋巴结转移有关(P=0 .024 ),且与淋巴细胞表达HGF呈正相关(rs=0 .450,P=0 .002)。癌细胞c Met表达量在间质淋巴细胞高表达HGF的病例中明显高于淋巴细胞低表达HGF的癌组织(P=0 .009)。但癌细胞c Met的表达与患者性别、年龄、病理组织学分型以及临床分期均未显示相关性。鼻咽癌细胞株CNE 2在HGF诱导24h后,c Met阳性的癌细胞比例即有明显增加,由(46 .6±9 .02)%增加至(85. 8±6. 05)% (P=0 .003 ),癌细胞c Met蛋白表达相对强度和mRNA表达水平均有显著提高,     

Expression of chemokine receptor CXCR4 in nasopharyngeal carcinoma: pattern of expression and correlation with clinical outcome

Nasopharyngeal carcinoma (NPC) is a tumor derived from epithelial cells and Epstein-Barr virus infection has been reported to be a cause of this disease. Chemokine receptor CXCR4 was found to be involved in HIV infection and was highly expressed in human malignant breast tumors and the ligand for CXCR4, CXCL12 (SDF-1), exhibited high expression in organs in which breast cancer metastases are often found. The metastatic pattern of NPC is quite similar to that of malignant breast tumors. In this study, we investigated the expression of CXCR4 in nasopharyngeal carcinoma (NPC) tissues by immunohistostaining. We found different staining patterns, which included localization in the nucleus, membrane, cytoplasm or a combination of them. The staining intensity was also variable among samples. The metastatic rates in patients with high compared to low or absent expression was 38.6% versus 19.8%, respectively (P = 0.004). High expression of CXCR4 was associated with poor overall survival (OS = 67.05% versus 82.08%, P = 0.0225). These results suggest that CXCR4 may be involved in the progression of NPC and that a high level of CXCR4 expression could be used as a prognostic factor.     

Epstein-Barr virus (EBV) latent membrane protein-1 down-regulates tumor necrosis factor-alpha (TNF-alpha) receptor-1 and confers resistance to TNF-alpha-induced apoptosis in T cells: implication for the progression to T-cell lymphoma in EBV-associated hemophagocytic syndrome

The infection of T cells by Epstein-Barr virus (EBV) may result in hemophagocytic syndrome (HPS) through enhanced cytokine secretion, particularly tumor necrosis factor-alpha (TNF-alpha), by EBV latent membrane protein-1 (LMP-1). One bewildering observation of HPS patients is relapsing disease or progression to T-cell lymphoma. This finding raises the question whether EBV LMP-1-expressing T cells may survive and proliferate in the cytokine milieu of HPS. To explore this possibility, we tested the sensitivity of LMP-1-expressing T cells to apoptosis in the presence of TNF-alpha. LMP-1 up-regulated TNF-alpha through TRAF2,5 and nuclear factor-kappaB pathway in T cells. The LMP-1-expressing T cells then became resistant to TNF-alpha-induced apoptosis. Interestingly, the expression of TNFR1 was remarkably down-regulated by LMP-1 in T cells. Furthermore, the TNF-alpha/TNFR1 downstream death signal TNFR1-associated death domain protein was constitutively recruited by LMP-1, and the activities of apoptotic caspases 3, 8, and 9 were suppressed. Reconstitution of TNFR1 successfully reversed the TNF-alpha-induced apoptotic cascades. Therefore, EBV LMP-1 not only activates T cells to proliferate but also confers resistance to TNF-alpha-mediated apoptosis via down-regulation of TNFR1 in the cytokine milieu of HPS. This finding provides a potential mechanism to explain the disease persistence or progression to T-cell lymphoma in HPS patients.     

鼻咽癌组织中潜伏膜蛋白1的表达对癌细胞增殖和凋亡的影响

目的 探讨治疗前鼻咽癌组织中潜伏膜蛋白１（ＬＭＰ－１）的表达对癌细胞增殖和凋亡的影响。方法 收集未经治疗的鼻咽癌活检组织标本５５例,采用原位分子杂交和免疫组化检测ＥＢＵ病毒编码的小ＲＮＡｓ和ＬＭＰ－１以及癌细胞增殖细胞核抗原指数和癌组织中凋亡要关基因产物ｂｃｌ－２的表达,以末端标记原位细胞死亡指数（ＴＵＮＥＬ ｉｎｄｅｘ ＴＩ）表示癌细胞的凋亡程度。结果（１）５５例末治疗鼻咽癌组织中２７例呈ＬＭＰ     

Prevalence and relevance of EBV latency in nasopharyngeal carcinoma in Israel

BACKGROUND: Nasopharyngeal carcinoma (NPC) is frequently associated with Epstein-Barr virus (EBV). The incidence of NPC in Western countries is lower than in the Far East, and EBV latency in NPC is less prevalent. Israel, as a part of the Mediterranean area, is one of the countries with an intermediate risk for NPC. METHODS: Immunohistochemistry (IHC) for latent membrane protein 1 (LMP-1) and in situ hybridisation (ISH) for EBV encoded RNA (EBER) were used to evaluate the prevalence and possible prognostic value of EBV latency among Israeli patients with NPC. Forty five patients with different NPC histologies were studied. RESULTS: LMP-1 IHC was positive in six samples only, all with undifferentiated histology. EBER ISH was positive in 40 of the 45 samples. According to histological type, three of five patients with squamous cell carcinoma were EBV positive and 37 of 40 non-keratinising and undifferentiated carcinoma cases were positive. Although EBV was more prevalent in patients with non-squamous carcinoma, the difference was not significant, probably because of the small number of patients with keratinising carcinoma. With regard to the clinical categories and survival, no significant difference could be detected between patients who were positive or negative for EBER ISH. No association was found between EBV latency and patient sex, age, origin, stage, or survival. CONCLUSIONS: NPC in Israel is highly associated with EBV latency as detected by EBER ISH. LMP-1 IHC is considerably less sensitive in detecting EBV latency in NPC among the same patient group.     

Encoded latent membrane protein 1 of Epstein-Barr virus on follicular dendritic cells in residual germinal centres in Hodgkin's disease.

AIMS--To determine if there is an association between Epstein-Barr virus (EBV) infection and Hodgkin''s disease. METHODS--Fifty cases of Hodgkin''s disease and 25 reactive lymph nodes were screened for the presence of EBV-RNA (EBER) using in situ hybridisation, and for the expression of EBV encoded latent membrane protein 1 (LMP-1) by immunohistochemistry. RESULTS--In 42% of the cases of Hodgkin''s disease, EBER was detected in the nuclei of the malignant cells, and in LMP-1 expression was found 36%. Both EBER and LMP-1 positivity were seen in 34% of the cases. An additional finding was the presence of LMP-1 on follicular dendritic cells in residual germinal centres in two cases of Hodgkin''s disease. EBER was not detected in these germinal centres. In reactive lymph nodes only occasional EBER positive, small, lymphoid cells were found, without LMP-1 expression. CONCLUSIONS--These results show a strong correlation between the presence of EBER and the LMP-1 expression in the Reed-Sternberg cells. They corroborate a role for EBV in at least some cases of Hodgkin''s disease. LMP-1 is probably presented as an immune complex in the germinal centres, as part of an immune response against EBV.     

Construction and characterization of monoclonal antibodies specific to Epstein-Barr virus latent membrane protein 1

Epstein-Barr virus (EBV) has been implicated in the development of many human neoplasias including B lymphomas and nasopharyngeal carcinoma (NPC). The EBV latent membrane protein 1 (LMP-1) has been found to participate in diverse cellular signaling pathways and is essential for virus-induced B-cell immortalization. In order to determine quantitatively the amount of LMP-1 in cells, five monoclonal antibodies (Mabs) specific to LMP-1 were generated. The epitopes recognized by these Mabs were found to cluster within the repeat region between the CTAR1 and CTAR2 domains, corresponding to amino acid positions 254-319 of LMP-1. These Mabs were capable of recognizing LMP-1 proteins of both lymphoid and epithelial origin as revealed by immunoblot, enzyme-linked immunosorbent assay (ELISA) and immunocytofluorescence analysis. A sandwich ELISA for the quantification of LMP-1 has been established using these Mabs. Taken together, our results indicate that the Mabs generated in this study are suitable for the detection of LMP-1 in biomedical research.     

Coexpression of hepatocyte growth factor and its receptor c-Met correlates with high MIB-1 proliferative index in malignant fibrous histiocytoma

Hepatocyte growth factor (HGF) is a multifunctional cytokine that variably affects cell motility, proliferation, and morphogenesis. Little information is currently available on the HGF and its receptor c-Met expression in malignant fibrous histiocytoma (MFH). We immunohistochemically investigated the HGF and c-Met expression in 43 MFH tissue specimens. Furthermore, the correlation of the HGF and c-Met expression with tumor proliferative activity assessed by MIB-1 indices was analyzed. Our results showed that positive cytoplasmic immunoreactivity for HGF and c-Met was identified in tumor cells in 36 (84%) and 20 (47%) of the 43 MFH cases analyzed, respectively. Coexpression of HGF and c-Met was observed in 20 (47%) of the 43 MFHs, and was correlated with high MIB-1 proliferative indices (p = 0.0446). These findings strongly indicate that the HGF/c-Met signaling system plays an important role in promoting cell proliferation of human MFHs via an autocrine loop.     

转录调节因子Ets-1在大鼠血管平滑肌细胞增殖与凋亡中的作用

目的： 研究转录调节因子Ets-1对大鼠血管平滑肌细胞增殖与凋亡的影响及其可能的机制。 方法： 用定量细胞DNA片段的方法观察大鼠血管平滑肌细胞增殖与凋亡。用Western印迹法检测磷酸化视网膜母细胞瘤（RB-P）蛋白表达。 结果： Ets-1抑制大鼠血管平滑肌细胞凋亡。反义P21WAF1/CIP1能够阻断Ets-1的抗凋亡作用，并抑制Ets-1诱导的平滑肌细胞增殖。Ets-1能够上调RB-P蛋白表达，反义P21WAF1/CIP1可以阻断Ets-1诱导的RB-P蛋白表达。 结论： 在大鼠血管平滑肌细胞中，Ets-1通过P21WAF1/CIP1旁路发挥抑制凋亡和促进增殖作用。     

EB病毒编码的RNA及EB病毒潜在膜蛋白在中线T淋巴瘤中的表达

应用免疫组化和原位杂交技术及抗ＥＢ病毒潜在膜蛋白（ＬＭＰ－１）单克隆抗体和ＥＢ病毒编码的ＲＮＡ（ＥＢＥＲ－１）探针对９例中线Ｔ淋巴瘤（ＭＴＬ）进行了ＥＢ病毒（ＥＢＶ）检测。结果显示：８例肿瘤细胞核ＥＢＥＲ－１阳性；７例肿瘤细胞膜和胞浆ＬＭＰ－１阳性。结果表明：（１）ＥＢＶ与我国的ＭＴＬ存在密切关系，很可能在其发病中起着重要作用；（２）ＥＢＶ在ＭＴＬ的检出率高于全身其它部位和其它类型的周围型Ｔ淋巴瘤；（３）ＥＢＥＲ－１原位杂交和ＬＭＰ－１免疫组化在检测ＭＴＬ中ＥＢＶ方面都很敏感、可靠，而后者更经济简便。     

Epstein-Barr virus LMP1 modulates the malignant potential of gastric carcinoma cells involving apoptosis.

About 10% of gastric carcinomas including lymphoepithelioma-like carcinoma and adenocarcinoma are associated with Epstein-Barr virus (EBV) infection. In EBV-associated gastric carcinomas, the tumor cells express Epstein-Barr nuclear antigen 1 (EBNA-1) but not EBNA-2, -3A, -3B, or -3C, leader protein, or latent membrane proteins (LMPs) because of gene methylation. Only a few exceptional cases have LMP1 expression in tumor cells as demonstrated by immunohistochemical studies. To elucidate the biological effects of LMP1 and the significance of its restricted expression in EBV-associated gastric carcinomas, the LMP1 gene was transferred into EBV-negative gastric carcinoma cell lines (SCM1 and TMC1) and into EBV-negative nasopharyngeal carcinoma (NPC) cells (HONE-1) as a control. The biological effects of LMP1 in gastric carcinoma cells were monitored in vitro and in vivo. These results showed that the consequence of LMP1 expression is a growth enhancement in NPC cells, but it is a growth suppression in gastric carcinoma cells. The LMP1-expressing gastric carcinoma cells had a reduced growth rate, colony-forming efficiency, mean colony size, and tumorigenicity and a lower malignant cytological grade. The reduced growth rate, colony-forming efficiency, and mean colony size were partially reversible in vitro with treatment with LMP1 antisense oligonucleotide. In addition, enhanced apoptosis was found in the LMP1-expressing gastric carcinoma cells. This suggests that LMP1 may negatively modulate the malignant potential of gastric carcinoma cells via an enhancement of apoptosis. We concluded that the restriction of LMP1 expression in EBV-associated gastric carcinomas may lead to a growth advantage for tumor cells by avoiding LMP1 apoptotic effects and immunologically mediated elimination.     

上皮型钙黏素基因启动子甲基化及其mRNA和蛋白表达在鼻咽癌组织中的意义

目的　探讨鼻咽癌癌细胞上皮型钙黏素基因启动子甲基化的程度及其mRNA和蛋白表达水平在鼻咽癌早期浸润和转移中的作用。方法　采用甲基化特异性聚合酶链反应 (methylation specificPCR ,MSP)、蛋白印迹、免疫组织化学和逆转录聚合酶链反应 (RT PCR)等方法检测 2 1例鼻咽癌患者鼻咽原发癌和淋巴结转移癌配对组织中的上皮型钙黏素基因启动子甲基化程度和上皮型钙黏素、β 链接素在不同组织中mRNA和蛋白水平的表达。 结果　 ( 1)在 2 1例鼻咽原发癌组织中 ,11例( 5 2 4% )可以检测到上皮型钙黏素基因启动子的甲基化 ,而在配对的 2 1例淋巴结转移癌组织中 17例 ( 80 9% )则可检测到 ,二者差异具有显著性 (P <0 0 5 )。 ( 2 )在鼻咽原发癌组织中 ,80 %的癌细胞均表达上皮型钙黏素蛋白 ,在淋巴结转移癌组织中只有平均 5 0 %的癌细胞表达 ,两者亦差异有显著性 (P =0 0 0 4) ;但β 链接素蛋白在原发癌和淋巴结转移癌组织中均有较高的表达量 (均为 85 % )。 ( 3 )蛋白印迹检测表明 ,上皮型钙黏素在鼻咽原发癌组织中表达的平均相对强度为 2 0 6 7± 3 2 7,明显高于在转移癌组织中的蛋白平均相对强度 65 0± 15 9;而 β 链接素在不同组织中的蛋白表达相对强度则差异无显著性 (P =0 75 4)。 ( 4)上皮型钙黏     

Down-regulation of Epstein-Barr virus nuclear antigen 1 in Reed-Sternberg cells of Hodgkin's disease.

AIMS--To demonstrate Epstein-Barr virus (EBV) encoded nuclear antigen 1 (EBNA-1) gene expression in EBV associated disorders using a new monoclonal antibody (1H4-1) on routinely processed tissues. METHODS--The pressure cooker antigen retrieval method was used for the immunohistochemical demonstration of EBNA-1 gene expression in formalin fixed, EBV positive tissues from Hodgkin's disease, infectious mononucleosis, HIV associated non-Hodgkin's lymphomas, post-transplant lymphomas, and undifferentiated nasopharyngeal carcinoma (NPC). EBV encoded EBNA-2, latent membrane protein 1 (LMP-1) and BZLF-1 gene expression was also examined using commercially available monoclonal antibodies. RESULTS--Of the 34 EBER in situ hybridisation positive cases of Hodgkin's disease examined, none expressed EBNA-1 in the Reed-Sternberg cells. These cells were nevertheless strongly LMP-1 positive in all cases. Strong EBNA-1 staining was seen in all cases of EBER positive HIV associated non-Hodgkin's lymphoma (five of five), nasopharyngeal carcinoma (five of five), infectious mononucleosis (three of three), and post-transplant lymphoma (one of one). These cases also expressed LMP-1, EBNA-2 and BZLF-1, but at differing levels. CONCLUSION--The pressure cooker antigen retrieval procedure is a sensitive and reliable adjunct to immunohistochemistry, especially with antibodies which are otherwise ineffective on routinely processed tissues. The EBNA-1 gene is not expressed at detectable levels in the malignant cells of Hodgkin's disease, but is consistently expressed in other EBV associated disorders. This finding has important implications for the role of EBNA-1 in the biology of EBV.     

Expression of c-Met and heparan-sulfate proteoglycan forms of CD44 in colorectal cancer

In colorectal cancer patients, prognosis is not determined by the primary tumor but by the formation of distant metastases. Molecules that have been implicated in the metastatic process are the proto-oncogene product c-Met and CD44 glycoproteins. Recently, we obtained evidence for functional collaboration between these two molecules: CD44 isoforms decorated with heparan sulfate chains (CD44-HS) can bind the c-Met ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF). This interaction strongly promotes signaling through the receptor tyrosine kinase c-Met. In the present study, we explored the expression of CD44-HS, c-Met, and HGF/SF in the normal human colon mucosa, and in colorectal adenomas and carcinomas, as well as their interaction in colorectal cancer cell lines. Compared to the normal colon, CD44v3 isoforms, which contain a site for HS attachment, and c-Met, were both overexpressed on the neoplastic epithelium of colorectal adenomas and on most carcinomas. Likewise, HGF/SF was expressed at increased levels in tumor tissue. On all tested colorectal cancer cell lines CD44v3 and c-Met were co-expressed. As was shown by immunoprecipitation and Western blotting, CD44 on these cells lines was decorated with HS. Interaction with HS moieties on colorectal carcinoma (HT29) cells promoted HGF/SF-induced activation of c-Met and of the Ras-MAP kinase pathway. Interestingly, survival analysis showed that CD44-HS expression predicts unfavorable prognosis in patients with invasive colorectal carcinomas. Taken together, our findings indicate that CD44-HS, c-Met, and HGF/SF are simultaneously overexpressed in colorectal cancer and that HS moieties promote c-Met signaling in colon carcinoma cells. These observations suggest that collaboration between CD44-HS and the c-Met signaling pathway may play an important role in colorectal tumorigenesis.