ENHANCED MALIGNANT PROGRESSION OF NASOPHARYNGEAL CARCINOMA CELLS MEDIATED BY THE EXPRESSION OF EPSTEIN–BARR NUCLEAR ANTIGEN 1 IN VIVO

Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein–Barr virus (EBV) and mostly classified as poorly differentiated squamous cell carcinoma or undifferentiated carcinoma with early metastasis and a rapidly progressive clinical course. The EBV-encoded latent proteins, Epstein–Barr nuclear antigen 1 (EBNA 1) and latent membrane proteins (LMPs), may be expressed in NPC, but their biological effects are poorly understood. EBNA 1 may predispose B lymphocytes to lymphomagenesis in transgenic mice, but its biological effects in NPC are still unknown. This study investigated the biological effects of EBNA 1 by expressing it in an EBV-negative NPC cell line (HONE-1), which was then inoculated into both nude and severe combined immunodeficiency mice. The EBNA 1 caused HONE-1 cells to grow in a less differentiated pattern and to progress more rapidly, as well as increasing their tumourigenicity and metastatic capability. These data suggest that EBNA 1 may play a critical role in the progressive evolution of NPC.     

A20 RNA expression is associated with undifferentiated nasopharyngeal carcinoma and poorly differentiated head and neck squamous cell carcinoma

A20 is an anti-apoptotic gene that can be induced in human epithelial cell lines in response to expression of the Epstein–Barr virus (EBV) gene product latent membrane protein 1 (LMP1). EBV is a ubiquitous, persistent human herpesvirus that is consistently associated with undifferentiated nasopharyngeal carcinoma (NPC), in which antigen expression includes LMP1. Consistent with a potential role in the development of NPC, LMP1 has profound effects on epithelial cell growth. A20 may be a key downstream effector of LMP1 in NPC, as LMP1-induced A20 blocks p53-mediated apoptosis in H1299 epithelial cells and most NPCs have wild-type p53. Moreover, the potential role of A20 in the development of epithelial malignancies may extend to tumours not associated with EBV. The purpose of this study was to develop an in situ hybridization assay to assess expression of A20 RNA in undifferentiated NPC and in non-EBV-associated poorly differentiated head and neck squamous cell carcinomas (SCCs) and well-differentiated SCCs of the skin. A20 RNA expression was also examined in normal samples of oral mucosa and skin. Expression of A20 was demonstrated in 76 per cent of undifferentiated NPCs and in 80 per cent of poorly differentiated head and neck SCCs, suggesting a role for A20 in the pathogenesis of these epithelial malignancies. By contrast, A20 RNA was not detected in well-differentiated SCCs of the skin, or in any normal samples of squamous epithelial tissue. The pathway leading to A20 expression in non-EBV-associated poorly differentiated head and neck SCCs is clearly LMP1-independent. LMP1 expression was demonstrated in 29 per cent of NPC biopsies, suggesting an LMP1-independent pathway to A20 induction in undifferentiated NPC. Copyright © 1999 John Wiley & Sons, Ltd.     

Detection of latent Epstein-Barr virus (EBV) DNA in paraffin sections of nasopharyngeal carcinomas expressing no EBV-encoded small RNAs using in situ PCR

Summary.  In situ hybridization (ISH) with EBER 1 (Epstein-Barrvirus (EBV)-encoded small RNA1) probes is widely used for in situ detection of EBV-infected cells. ISH with an EBER1 probe showed that 10 of 40 NPC cases were negative for EBER1 expression. For in situ detection of EBV DNA, we used in situ PCR method which can detect one copy of EBV DNA per cell. Of the 10 EBER1-negative cases, three cases including one each of well- and poorly differentiated carcinomas and undifferentiated carcinoma were EBV DNA-positive by in situ PCR. The remaining seven were truly negative for the presence of EBV DNA. All the EBV genome-negative NPC cases examined here were histologically classified as poorly differentiated or undifferentiated carcinomas which are known to be closely associated with EBV, indicating the existence of EBV DNA-negative NPC cases, regardless of histological type or differentiation. These results indicate that there are EBV genome-positive NPC cases expressing no EBER1 and that in situ PCR can be suitable for in situ detection of EBV-infected cells, especially those expressing no EBER1 in paraffin sections. Received April 14, 1997 Accepted June 5, 1997     

三株人鼻咽癌Scid小鼠移植瘤的建立及特性研究

目的 建立鼻咽癌鼠移植瘤,为鼻咽癌的特异性免疫治疗研究提供实验模型。方法 从26例未治的鼻咽癌病人鼻咽部铰取肿瘤组织,分别皮下接种BALB／c裸小鼠（14只）和Scid小鼠（12只）;成功建立移植瘤株后观察其生长特性、形态、染色体和LMP的表达情况。结果 成功建立鼻咽癌Scid小鼠移植瘤株CSNET－1、CSNET－2和CSNET－3,已分别传至第11代（23个月）、第14代（17个月）和第9代（16个),其光镜和电镜下形态符合低分化磷癌的特征,电镜下在CSNET－1中可观察到Epstein-Barr病毒样颗粒,CSNET－1的染色体全部为人体细胞来源的染色体,免疫组化检测结果,3个瘤株均可见EB病毒晚期膜蛋白LMP－1和LMP－2阳性细胞。结论 CSNET－1、CSNET－2和CSNET－3是人类来源的鼠移植瘤株,是鼻咽癌免疫治疗研究的理想动物模型。     

The mechanism of Epstein-Barr virus infection in nasopharyngeal carcinoma cells.

To investigate the relationship between Epstein-Barr virus (EBV) and nasopharyngeal carcinoma (NPC) cells, we examined the pathway of EBV infection in NPC cell lines. We used immunolocalization to investigate the EBV receptor (C3d-R) and polymeric immunoglobulin receptor [secretory component (SC) protein]. We incubated IgA anti-EBV and EBV particles with NPC cells and observed the EBV DNA signal by in situ polymerase chain reaction hybridization and polymerase chain reaction plus Southern blotting. We also colocalized SC protein and EBV RNA in NPC biopsy specimens. Results showed that: 1) NPC cells did not express the EBV receptor but did express SC protein in each line; 2) SC protein was also expressed in some tumor cells but not in untransformed squamous metaplastic epithelia in NPC biopsy specimens; 3) EBV could infect NPC cells through an EBV-IgA and SC complex and retained an EBV viral genome in their nuclei; SC expression could be down-regulated by EBV proteins; and 4) in biopsy specimens, a fraction of tumor cells showed SC protein expression; only a portion of tumor cells contained EBV, and of these cells only a few expressed SC protein. These findings indicate that EBV cannot infect untransformed nasopharyngeal squamous metaplastic epithelia but can enter NPC cells through IgA-mediated endocytosis.     

稳定表达鼻咽癌来源潜伏膜蛋白1基因的鼻咽癌细胞系的建立

目的:建立稳定表达鼻咽癌(NPC)来源潜伏膜蛋白1(LMP1)的鼻咽癌细胞系。方法:利用基因重组技术构建NPC来源LMP1的一般性真核表达载体及上皮特异性表达载体,并将其转染鼻咽癌细胞系CNE-2,用PCR、RT-PCR及蛋白印迹等检测N-LMP1在CNE-2中的整合和表达。结果:①成功构建了N-LMP1的一般性和上皮特异性表达载体。②N-LMP1基因在CNE-2细胞中获得了正确表达。结论:成功建立了稳定表达NPC来源LMP1的鼻咽癌细胞系,为进一步研究LMP1在鼻咽癌细胞中的作用机制奠定基础。     

Antitumor activity of the pachymic acid in nasopharyngeal carcinoma cells

Objective: To investigate the antitumour efficacy of pachymic acid (PA), which is a fungal extract component, on nasopharyngeal carcinoma (NPC) cells CNE-1, CNE-2. Methods: We have chosen NPC cell line CNE-2 for the study, and the cells were treated with PA before the detection. CCK-8 assay was used to detect the proliferative ability, and Annexin V-PI double staining was used for the detection of apoptosis rate; and the nucleus damage was detected by transmission electron microscope, the protein expression of the DNA damage pathway were detected by Western blot. Results: PA can significantly inhibited proliferation of CNE-1, CNE-2 cells. The proportion of apoptotic cells of all cell lines gradually increased in a dose-dependent manner induced by PA, P < 0.05. Meanwhile, the nucleus could be caused morphological changes and the expression of DNA damage-related proteins was upregulated by PA in CNE-2. Conclusions: PA can significantly inhibit cell proliferation and increase the apoptosis rates and may induce the apoptosis of the human NPC cells.     

Detection of Epstein-Barr virus DNA sequences in nasopharyngeal carcinoma cells by enzymatic DNA amplification.

The presence of Epstein-Barr virus (EBV) DNA sequences was examined by the polymerase chain reaction in 50 nasopharyngeal carcinoma (NPC) biopsy specimens and in two primary epithelial tumor cell cultures derived from patients with NPC. The detection limit was a single EBV genome equivalent by agarose gel electrophoresis followed by Southern blot analysis of the amplified products. EBV DNA sequences were detected in all 41 undifferentiated NPC cell specimens, in 2 of 4 moderately differentiated NPC cell specimens, and in 3 of 5 keratinized NPC cell specimens. Undifferentiated NPC cells were also found to contain higher copy numbers of EBV than cells of the other two types of NPC. Our data suggest that EBV replication may be closely associated with the differentiation of NPC tumor cells. The results also demonstrated a sensitive and specific method for the detection of EBV DNA sequences in NPC tumor cells.     

Epstein-Barr virus infection to Epstein-Barr virus-negative nasopharyngeal carcinoma cell line TW03 enhances its tumorigenicity

Almost all nasopharyngeal carcinomas (NPCs) are infected by Epstein-Barr virus (EBV), but most ex vivo NPC cells lose EBV genomes during passages. In this study, an EBV-negative NPC cell line, TW03, established from EBV-carrying NPC was reinfected with EBV by cocultivation with irradiated Akata cells carrying recombinant EBV containing a neomycin-resistant gene. The reinfected EBV (+) TW03 cells expressed EBERs and EBNA1, but not EBNA2, lytic proteins (ZEBRA and EA-D), or LMP1. They had an epithelial appearance similar to that of EBV (-) TW03 cells. The doubling times of EBV (+) and EBV (-) TW03 cells were almost identical. However, the EBV (+) TW03 cells formed larger colonies with ragged contours in anchorage-independent cultures. An in vitro invasion assay showed that EBV (+) TW03 cells had a higher invasive activity than EBV (-) TW03 cells (p < 0.01). Both EBV (-) and EBV (+) TW03 cells formed poorly differentiated squamous cell carcinomas in SCID and nude mice. EBV (+) TW03 cells showed a higher tumorigenicity to nude mice (12 of 13) than EBV (-) TW03 cells (1 of 9) (p < 0.001). In the severe combined immunodeficiency (SCID) tumors of EBV (+) TW03 cells, not all of the tumor cells were EBER-1 positive. EBER-1 was more frequently detected in the peripheral regions and daughter nodules of the tumors than in the central areas. The microdissection polymerase chain reaction showed that the EBER-1-negative TW03 cells in the EBV (+) TW03 SCID tumors lost EBV genomes. EBER-1-negative cells showed as high a rate of Ki-67 positivity as EBER-1-positive cells, indicating that the former were proliferating rather than dead or dying. In horny pearls, keratinizing cells were ZEBRA-positive and EBER-negative. Loss of EBV genomes was not associated with squamous differentiation. These data indicated that reinfection of EBV promotes the tumorigenicity of EBV (-) TW03 cells by enhancing the invading activity.     

Incidence of latent infection of Epstein–Barr virus in lung cancers—an analysis of EBER1 expression in lung cancers by in situ hybridization

To evaluate the presence of Epstein–Barr virus (EBV) in lung cancers of Japanese patients, 81 lung cancers were examined using a highly sensitive in situ hybridization (ISH) method, employing an antisense oligonucleotide probe for EBV-encoded small nuclear RNA-1 (EBER). EBER1 expression was demonstrated in one poorly differentiated squamous cell carcinoma associated with marked lymphoid stroma (PDSCC-LS), two well differentiated adenocarcinomas, and two moderately differentiated squamous cell carcinomas, but was not detectable in other lung cancers, including small cell carcinomas. Unlike lymphoepithelioma-like undifferentiated carcinoma (LELC) of the lung, the PDSCC-LS consisted of poorly differentiated cells with distinct cell borders and nuclei with a coarse chromatin pattern and some prominent nucleoli. Most of the cancer cells expressed intense EBER1 signals. Although small to moderate numbers of cells positive for EBER1 were present in two adenocarcinomas and two squamous cell carcinomas, EBER1 signals varied in intensity and number in these four cases. Although polymerase chain reaction (PCR) and Southern blot hybridization with a ³²P-labelled probe internal to the primers were conducted to detect the EBV genome in 24 lung cancers, including five EBER1-positive cases, the genome was found to be positive in the five cases with EBER1-positive staining, including the PDSCC-LS, two adenocarcinomas and two squamous cell carcinomas, but not in the other cases. This study indicates that the morphological features of EBV-associated lung cancers are not restricted to the typical LELC type.     

Growth and metastatic behavior of human tumor cells implanted into nude and beige nude mice

The growth and metastatic behavior of several human tumor lines grown in adult nude mice, nude mice pretreated with antiserum against asialo GM1 glycoprotein, and beige nude mice were studied. The cell lines were all injected s.c. and i.v. A human colon carcinoma line was also injected into the spleen, and two human renal carcinoma lines were injected into renal subcapsule. All the tumor lines grew as fast or faster in adult nude mice compared with beige nude mice. There were no discernible differences in the production of experimental lung metastasis among the three groups of mice, but human colorectal carcinoma cells and human renal carcinoma cells produced more metastases in nude mice than in beige nude mice after intrasplenic or renal subcapsule injection, respectively. In vitro cytotoxicity assays confirmed that adult nude mice had high levels of natural killer (NK) cell activity whereas nude mice pretreated with anti-asialo GM1 serum and beige nude mice did not. The in vitro NK cell activity of nude mice was demonstrable against mouse lymphoma cells but not against human leukemia cells which were sensitive to lysis by human NK cells. These results suggest that the implantation of human tumor cells into beige nude mice, which are deficient in NK cell activity does not provide an advantageous model for the study of growth and metastasis of human neoplasms.Abbreviations NK natural killer - HCC human colon carcinoma - HRCC human renal cell carcinoma - anti-asGM1 anti-asialo GM1 - i.s. intrasplenic - RSC renal subcapsule - HBSS Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution     

巨噬细胞移动抑制因子提高鼻咽癌细胞体外侵袭能力

目的　研究巨噬细胞移动抑制因子 (MIF)体外提高鼻咽癌细胞侵袭能力的原因 ,探讨鼻咽癌细胞早期发生侵袭和转移的机制。方法　采用微孔迁移分析法 (micron migrationassay)检测鼻咽癌细胞株CNE 1、CNE 2在细胞因子MIF诱导下通过 8μm直径微孔的细胞数量变化 ,采用Western蛋白印迹法、流式细胞术和酶联免疫吸附法检测侵袭转移相关因子基质金属蛋白酶 2、9(MMP2 ,MMP9)及白介素 8(IL 8)在此过程中的相应改变。结果　 (1)经MIF诱导后 ,CNE 1通过 8μm微孔的细胞数由原来的 2 3 7± 11 0 2增加至 113 7± 2 0 9,CNE 2则由原来的 3 4 7± 7 41增加至 3 11 3± 48 9,差异均呈显著性 (P =0 0 0 5,P =0 0 0 1)。 (2 )经MIF诱导后 ,癌细胞表达MMP9蛋白的细胞比例均明显增加 ,CNE 1细胞由 2 8 5%± 2 45%增加至 82 4%± 3 49% (P =0 0 0 1) ,而CNE 2细胞则由刺激前的 3 2 8%± 3 48%增加至 86 1%± 1 62 % (P =0 0 0 2 )。同时癌细胞MMP9蛋白表达强度也显著增强 ,CNE 1和CNE 2细胞MMP9蛋白的平均相对强度均比诱导前提高 3倍以上 (P <0 0 5)。但MIF刺激前后 ,MMP2蛋白的细胞表达数量和表达强度在两株细胞中却未见明显变化 (P值均大于 0 0 5)。 (3 )MIF诱导后 ,CNE 2细胞培养上清液中的IL 8浓度为 12     

The ATM tumour suppressor gene is down‐regulated in EBV‐associated nasopharyngeal carcinoma

A micro‐array analysis using biopsies from patients with EBV‐positive undifferentiated nasopharyngeal carcinoma (NPC) and from cancer‐free controls revealed down‐regulation of tumour suppressor genes (TSG) not previously associated with this disease; one such gene was the ataxia telangiectasia mutated (ATM) gene. Q‐PCR confirmed down‐regulation of ATM mRNA and ATM protein expression in tumour cells was weak or absent in almost all cases. In NPC cell lines, however, ATM was down‐regulated only in the EBV‐positive line, C666.1, and in none of five EBV‐negative lines. In vitro infection of EBV‐negative NPC cell lines with a recombinant EBV was followed by the down‐regulation of ATM mRNA and protein, and only EBV‐positive cells showed a defective DNA damage response following γ‐irradiation. Our data suggest that loss of ATM function could be an important step in the pathogenesis of NPC, and may have implications for the treatment of this disease. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Desialylation of metastatic human colorectal carcinoma cells facilitates binding to Kupffer cells

Cell surface hypersialylation of human colorectal carcinoma (HCRC) cells correlates with increased metastatic potential after intrasplenic injection, while desialylation with various agents has been shown to inhibit hepatic metastases. In this study we examined the effects of desialylation of HCRC cell lines with a novel intracellular inhibitor of the CMP-sialic acid transport protein (KI-8110). HCRC cells, which are poorly differentiated and poorly metastatic in nude mice (Clone A and MIP-101) were compared to well-differentiated, highly metastatic cells (CX-1 and CCL-235). KI-8110 treatment has previously been shown to reduce sialic acid levels in each of these cell lines and to reduce hepatic metastases in CX-1 and CCL-235 cell lines. This study attempts to identify a mechanism by which desialylation inhibits hepatic metastases. After KI-8110 treatment,in vitro adhesion assays were performed with each cell line to examine binding to Kupffer cells and the extracellular matrix protein fibronectin. Binding of Clone A, CX-1, and CCL-235 to Kupffer cells was significantly increased after KI-8110 treatment. Desialylation had no significant effect on binding of HCRC cell lines to fibronectin. While the metastatic cascade involves many complex interactions, the cytotoxic effects of Kupffer cells in the hepatic sinusoid are known to be an important mechanism of host defense against tumor cells. Cell surface sialic acids may well mask Kupffer cell binding to HCRC cells, preventing their cytotoxic effects and enhancing the metastatic potential of circulating tumor cells.     

Epstein–Barr virus infection in non-carcinomatous gastric epithelium

Gastric tissue specimens from 20 patients with chronic atrophic gastritis, one of whom also had an early gastric carcinoma, were studied for evidence of Epstein–Barr virus (EBV) infection by Southern blot analysis, DNA and RNA in situ hybridization, and immunohistochemistry for the presence of the EBV-determined nuclear antigen 1 (EBNA-1) and the latent membrane protein 1 (LMP-1). EBV DNA was detected in two cases with chronic atrophic gastritis and in the case with early gastric carcinoma by Southern blot hybridization. DNA in situ hybridization showed EBV genomes in the epithelial cells of two other cases with chronic atrophic gastritis and in non-carcinomatous and carcinomatous epithelial cells of the early gastric carcinoma case. EBNA-1 was detected in all cases. LMP-1 was detected in areas of intestinal metaplasia in eight patients with chronic atrophic gastritis. EBV-encoded small RNA 1 (EBER-1) expression was limited to carcinoma cells. These results show that gastric epithelium is frequently infected with EBV and suggest that prolonged EBV persistence may contribute to the development of gastric carcinoma. © 1997 John Wiley & Sons, Ltd.     

临床PET-CT在裸鼠鼻咽癌移植瘤动物模型中的应用

目的探讨临床PET-CT在裸鼠鼻咽癌移植瘤放疗模型中的应用。方法①18只裸鼠鼻咽癌移植瘤动物模型分别于放疗前测量瘤体大小并行18F-FDGPET-CT显像。②按瘤体大小分别将裸鼠分成3组,显像结果与组织病理学结果进行比较;③取其中3只荷瘤鼠,分别按每床位3min、5min、8min时间采集,图像处理后由两位以上有经验的核医学科医师判断图像质量。结果①放疗前裸鼠鼻咽癌移植瘤18F-FDGPET-CT显像瘤体处均为阳性。②组1,瘤体平均T/N=1.203±0.189;组2,瘤体平均T/N=2.086±0.456;组3,瘤体平均T/N=1.535±0.160。③不同采集时间对图像质量影响不大。结论①临床PET-CT可用于裸鼠鼻咽癌移植瘤动物模型的研究,对于裸鼠鼻咽癌移植瘤动物模型,3min/床位采集是可行的。②瘤体直径在1.0～1.4cm进行PET-CT显像比较适宜。     

Epstein-Barr virus-associated lymphoepithelioma-like carcinoma of the esophagus

Epstein-Barr virus (EBV)-associated lymphoepithelioma-like carcinoma (LELC) rarely occurs in the esophagus. We report a case of such tumor arising in the esophagus of a 64-year-old Taiwanese woman. No tumors were detected outside the esophagus including nasopharynx by thorough clinical studies. She underwent subtotal esophagectomy. Light microscopy disclosed a poorly differentiated carcinoma morphologically reminiscent of nasopharyngeal undifferentiated carcinoma (lymphoepithelioma). Immunohistochemical stain for latent membrane protein-1 showed positivity on the tumor cells. The infiltrating lymphocytes were chiefly composed of CD8-positive cytotoxic T cells. EBV DNA was demonstrated by both nested polymerase chain reaction (PCR) in the main tumor and metastatic lymph node, and localization in the tumor cells by in situ PCR in situ hybridization (ISH). However, the result of EBV-encoded small RNA-1 ISH was negative. Our case suggests that LELC of the esophagus may be associated with EBV in the endemic area. Due to its distinct histological features, the association with EBV, and possible prognostic implication, LELC of the esophagus should be precisely diagnosed and discerned from the usual poorly differentiated carcinoma.