NPC-KT Cells Research Articles

Oncogenic role of Epstein-Barr virus-encoded small RNAs (EBERs) in nasopharyngeal carcinoma

Objective Epstein-Barr virus (EBV) is a causative agent of nasopharyngeal carcinoma (NPC), and EBV gene expression is considered to be closely associated with the pathogenesis of NPC. Among EBV genes expressed in NPC, EBV-encoded non-polyadenylated RNAs, termed EBERs, are the most abundant transcripts of EBV in NPC. However, the role of EBERs still remains unclear. This study was designed to investigate the relevance of EBERs to the oncogenesis of NPC. Methods Two types of EBERs expression vectors (EBERs-high-expression vector and EBERs-low-expression vector) were constructed and transfected into EBV-negative cells, MDCK or EBV-negative clones of NPC-KT cells. Then, malignant transformation, represented by anchor independent growth, was evaluated between the EBERs-transfected cells and EBERs-negative cells using a soft agar colony formation assay. Apoptosis was induced by serum deprivation (0.1% concentration of fetal bovine serum) and interferon-α (IFN-α) (500 U/ml) treatment. Cell viability was evaluated with a trypan blue exclusion test. The activation of cellular transcriptional factor NF-κB was studied with the IL-8 promoter sequence using a luciferase reporter assay. Results EBERs-high-expression vector-transfected MDCK cells showed enhanced growth ability in soft agar compared with either EBERs-low-expression vector-transfected MDCK cells or EBERs-untransfected MDCK cells. However, they did not show the acquisition of any anti-apoptotic potential against either IFN-α or serum deprivation. Introduction of EBERs-low-expression vector into MDCK cells did not show anchor independent growth characteristics. Neither EBV-negative NPC-KT cells nor MDCK cells transfected with EBERs-high-expression vector showed any difference from EBERs-untransfected EBV-negative NPC-KT cells. Introduction of EBERs into MDCK cells did not transactivate the IL-8 promoter, indicating that neither NF-κB nor AP-1 was activated by EBERs. Conclusion EBERs are believed to induce the initial transformation of epithelial cells, thus contributing to the oncogenesis of NPC. Expression of abundant EBERs is considered to be critical for this transforming property of EBERs.

Ribonucleotide reductase inhibitors enhance cidofovir‐induced apoptosis in EBV‐positive nasopharyngeal carcinoma xenografts

In nasopharyngeal carcinoma (NPC), Epstein-Barr virus (EBV) infection is mainly latent, and the tumor cells contain episomal viral DNA. We have shown that the acyclic nucleoside phosphonate analog, cidofovir [(S)-1-(3-hydroxy-2-(phosphonylmethoxypropyl))cytosine] (HPMPC), inhibits growth of NPC xenografts in nude mice by causing apoptosis. The ribonucleotide reductase (RR) inhibitors, hydroxyurea and didox (3,4-dihydroxybenzohydroxamic acid), have been demonstrated to inhibit neoplastic growth and are used as antiviral and anticancer agents. Here we show that RR inhibitors enhance the antitumor effect of cidofovir in EBV-transformed epithelial cells. MTT assays indicate that hydroxyurea and didox enhance cidofovir-induced cell toxicity in NPC-KT cells, an EBV-positive epithelial cell line derived from NPC. The effect is due to enhancement of apoptosis through the caspase cascade as shown by pronounced cleavage of poly(ADP-ribose) polymerase. Finally, hydroxyurea strikingly enhanced the cidofovir-induced growth-inhibitory effect on NPC grown in athymic mice. The results suggest that RR inhibitors should enhance the antitumor effect of acyclic nucleoside phosphonate analogs on NPC.

Epstein‐Barr Virus (EBV) Latent Membrane Protein 1 Induces Interleukin‐8 through the Nuclear Factor‐κB Signaling Pathway in EBV‐Infected Nasopharyngeal Carcinoma Cell Line

Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic malignant tumor and is associated with Epstein-Barr virus (EBV) infection that exhibits type II latency. Angiogenesis is essential for tumor growth, invasion, and metastasis. Our previous studies have indicated that interleukin (IL)-8 was over-expressed in many NPC tissues and was found to be significantly correlated with angiogenesis by immunohistochemistry. In vitro design. The influence of the EBV genome for IL-8 gene expression was studied using the EBV-genome-positive and -negative epithelial/NPC hybrid cell line NPC-KT. The EBV-positive and -negative clones were selected by polymerase chain reaction and in situ hybridization. EBV-positive clones expressed abundant IL-8 mRNA compared with EBV-negative clones. This result indicated that over-expression of IL-8 depended on the presence of EBV genomes in NPC-KT cells. Two encoded genes, latent membrane protein (LMP)1 and EBV-encoded small RNAs (EBERs), expressed in NPC were transfected in EBV-negative NPC-KT cells. LMP1 transactivated the IL-8 promoter, whereas EBERs did not. Moreover, the nuclear factor (NF)-kappa B binding site in the IL-8 promoter was essential for the response to LMP1, and the activator protein (AP)-1 binding site played only a partial role. LMP1 induces IL-8 mainly through the activation of NF-kappa B and partly through AP-1 in NPC model cell lines, NPC-KT, and this suggests that LMP1 plays an important role in the angiogenesis of NPC.

Epstein-Barr virus lytic cycle spreads via cell fusion in a nasopharyngeal carcinoma hybrid cell line.

NPC-KT cl.S61, a subclone derived from an epithelial-nasopharyngeal carcinoma hybrid cell line NPC-KT, showed extensive Epstein-Barr virus (EBV) production and cell fusion when the EBV replicative cycle was induced by 5-iodo-2'-deoxyuridine. On the contrary, parental NPC-KT cells produced virus at a lower level and did not show cell fusion. Cell fusion in cl.S61 cells was blocked by 2-deoxyglucose and acyclovir, inhibitors of glycosylation and EBV DNA polymerase, respectively, with a concomitant decrease in the number of cells expressing EBV growth-associated antigens. However, the frequency of virus antigen expression in parental NPC-KT cells was not significantly affected by these drugs. This result suggests that efficient production of EBV from cl.S61 cells is due to the spreading of viral replicative cycle via cell fusion. It was also demonstrated by in situ autoradiography that cl.S61 cells producing virus fused to not only EBV receptor/CR2 positive Raji and BJAB cells, but also to receptor-negative Jurkat cells. The possible mechanism of EBV entry into cells devoid of virus receptor by cell fusion is discussed.

Effects of d-nicotine on contraction and relaxation induced by I-nicotine in the tracheal and main bronchial preparations of guinea pig and rabbit

An Epstein-Barr virus (EBV) genome-positive epithelial hybrid cell line, NPC-KT, derived from the fusion of primary nasopharyngeal carcinoma cells with a human epithelial cell line of adenoid origin has previously been described (Takimoto, Kamide, and Umeda, 1984a; Takimoto, Ogura, Sato, and Hatano, 1984b; Takimoto, Ogura, Sato, Umeda, and Hatano, 1985). The NPC-KT cells produce virus (NPC virus) with both transforming and lytic properties. An EBV genome-negative human lymphoblastoid cell line, Ramos, was infected with NPC virus. Three months after infection, more than 90 % of the cell population expressed EBV-associated nuclear antigen (EBNA). Approximately 40% of the cells exhibited a brilliant pattern, whereas the remaining 50% of the cells showed a faint granular pattern. The result suggests that there may be heterogeneity in NPC virus.

Epstein-Barr Virus (EBV) Receptors on Epithelial Hybrid Cells Derived from Nasopharyngeal Carcinoma

We previously established an Epstein-Barr virus (EBV) genome-positive epithelial hybrid cell line, designated NPC-KT, which was prepared by fusing primary epithelial cells derived from nasopharyngeal carcinoma (NPC) with an epithelial Ad-AH cell line. In this study, we measured the presence of the EBV receptors using radiolabelled EBV. It was determined that similar amounts of Burkitt's lymphoma (BL)-derived P3HR-1 or NPC-KT-derived EBV can attach BL-derived Raji cells, but that only P3HR-1 virus can attach to NPC-KT cells. In addition, the superinfection of NPC-KT cells with P3HR-1 virus could not be inhibited by pretreatment with a monoclonal antibody against C3d (OKB7). Raji cells adsorbed with OKB7 became much less susceptible to superinfection with P3HR-1 or NPC virus. The data suggest that EBV receptors unrelated to C3d receptor exist.

Antinuclear antibodies in the sera of patients with nasopharyngeal carcinoma

We studied the production of heterophile antinuclear antibodies (ANAs) in the sera of 50 patients, 20 with nasopharyngeal carcinoma (NPC) and 30 with other head and neck cancers (laryngeal cancer and maxillary cancer), before and after radiation therapy. A higher incidence of ANAs was found in the sera of patients with NPC and ANA production in these patients was higher after radiation therapy. We therefore performed in vitro experiments to explore the mechanisms of ANA production in the serum of postirradiated NPC patients. X-ray-irradiated NPC-derived cells (NPC-KT) produced a large amount of Epstein-Barr virus (NPC EBV) compared with non-irradiated NPC-KT cells. Nasopharyngeal carcinoma EBV-infected lymphocytes produced high levels of ANAs. These data suggest that lymphocytes infected by EBV from NPC cells may produce ANAs in the sera of NPC patients.

Differences in the ability of cells to fuse are mediated by strains of epstein‐barr virus

Epstein-Barr virus (EBV) preparations from both NPC-KT cells (NPC-EBV) and P3HR-1 cells (HR-1-EBV) can induce cell fusion between EBV receptor (EBVR)-positive Raji cells and EBVR-negative cells, but other strains of EBV cannot induce cell fusion. The effect of these two EBV isolates on ability of cells to fuse has been studied to determine if there are differences in the biological properties of the different EBV isolates, particularly the isolates obtained from nasopharyngeal carcinoma such as NPC-EBV. The frequency of cell fusion between NPC-EBV-superinfected Raji cells and EBVR-negative epithelial cells (Ad-AH) was increased more than 30-fold in the presence of medium containing 1% dimethylsulfoxide (DMSO). However, the frequency of cell fusion between HR-1-EBV-superinfected Raji cells and Ad-AH cells was unaffected under the same conditions. The data show that differences in the ability of cells to fuse are induced by variants of EBV in response to DMSO. These differences may be important in elucidating the different biological properties of EBV isolates and might have implications for the pathophysiology of EBV-associated illness.

Epstein-Barr Virus with Transforming and Early Antigen-inducing Ability Originating from Nasopharyngeal Carcinoma: Mapping of the Viral Genome

Epstein-Barr virus (EBV) from a human hybrid epithelial cell line (NPC-KT), derived from the fusion of human adenoidal cells and EBV genome-containing primary nasopharyngeal carcinoma cells (NPC) has both transforming and early antigen (EA)-inducing abilities. EBV DNA from NPC-KT cells was partially digested with TaqI and ligated to the cloning vector pJB8 at the ClaI site. This cosmid library encompassed the whole genomic DNA of the virus except for several kb of the terminal fragments. The identification and location of each of the cloned DNA fragments have been defined by hybridization to blots prepared with B95-8 and NPC-KT virion DNA. Defective heterogeneous restriction enzyme fragments of the viral DNA were not identified in any of the cosmid clones nor detected in hybridizations to virion DNA, which indicates that a single virus population derived from the NPC tissue has both transforming and EA-inducing activities.

Gene Mapping of an Epstein-Barr Virus Isolate Obtained from a Nasopharyngeal Carcinoma

Gene mapping of an Epstein-Barr virus (EBV) isolate derived from a nasopharyngeal carcinoma (NPC), designated NPC-EBV, has been performed. This isolate was rescued from an NPC epithelial hybrid cell line (NPC-KT), and used to transform cotton-top tamarin lymphocytes which, along with the epithelial NPC-KT cells, were used in the mapping studies. Using the BamHI and EcoRI restriction enzymes, we found that the NPC-EBV isolate did not contain the deletions observed in the genomes of the prototype HR-1 and B95-8 isolates, and may represent a 'more complete' virus genome. Polymorphism was observed in the BamHI-X, T, H, and L regions as compared to the HR-1 and B95-8 isolates. The NPC-EBV DNA in the epithelial NPC-KT cells was compared to the NPC-EBV genome in three different cotton-top tamarin lymphoblastoid cell lines transformed with the isolate. The restriction patterns were the same with one exception; there were differences in the size of the BamHI N-J het regions. This finding is consistent with the idea that the BamHI N-J het region may be the switch point of EBV DNA from the linear to the episomal form. The NPC-EBV is the first NPC-derived isolate to be obtained from epithelial cells in vivo and maintained in epithelial cells in vitro, and will be useful for studying biological variability of EBV.

Analysis of the DNAs of Epstein-Barr virus (EBV) derived from two EBV genome-positive epithelial/hybrid cell lines (NPC-KT and A2L/AH)

The authors recently reported the establishment of two Epstein-Barr virus (EBV)-producing epithelial/hybrid cell lines (A2L/AH and NPC-KT), A2L/AH cells were derived from the fusion of epithelial cell line (Ad-AH) with non-producer EBV genome-positive lymphoblastoid cells transformed with the B95-8 strain of EBV. NPC-KT cells were derived from the fusion of Ad-AH cells with primary nasopharyngeal carcinoma cells. In this study, viral DNAs derived from A2L/AH and NPC-KT cells were analyzed in comparison with viral DNA derived from B95-8 cells by use of the restriction endonuclease EcoRI and Hind III and separation of fragments in 0.4% agarose. The digestion fragments of these two viral DNAs were obviously similar, but could be distiguished each other. The molecular weight of DNA of virus from NPC-KT cells was approximately 181K base pairs (Kb), which was 12Kb larger than that of B95-8 virus DNA. Key words: NPC-KT, A2L/AH, Epstein-Barr virus, DNA A90-1177-51360

Marmoset lymphoblastoid cells transformed by NPC-KT-derived Epstein-Barr virus which possesses transforming and superinfecting characteristics.

New world monkey (cotton-top marmoset; CTM) lymphocytes were transformed by two different strains of Epstein-Barr virus (EBV) derived from human nasopharyngeal epithelial/hybrid cells (NPC-KT and A2L/AH). M-KT cells were a CTM lymphoblastoid cell line which was transformed by EBV derived from NPC-KT cells (NPC-KT EBV). M-BA2L cells were a CTM lymphoblastoid cell line which was transformed by EBV derived from A2L/AH cells (A2L/AH EBV). EBV obtained from M-KT cells, like parental NPC-KT EBV, could transform human cord blood lymphocytes (HCBLs) and superinfect Raji cells. EBV obtained from M-BA2L cells, like parental A2L/AH EBV, could transform HCBLs but could not superinfect Raji cells. We have compared the EBV-DNA associated with M-KT cells to NPC-KT EBV-DNA. The results obtained using a DNA restriction enzyme (Hind III) showed that virus DNA prepared from NPC-KT cells is different from EBV-DNA prepared from CTM lymphocytes transformed by EBV derived from infectious mononucleosis (B95-8), but that virus DNA prepared from M-KT cells is identical with parental NPC-KT EBV. These results indicate the possibility that a single NPC-KT EBV is associated with two biological activities.

Transfection of human nasopharyngeal epithelial cells with Epstein-Barr virus DNA derived from nasopharyngeal carcinoma

Transfection experiments using Epstein-Barr virus (EBV) DNA obtained from NPC-KT cells were carried out to examine whether the Ad-AH human adenoid epithelial cells could be transfected. We found that the Ad-AH cells were transfectable using NPC-KT virus DNA, as determined by the synthesis of EBV specific antigens. Concentrates of supernatant fluids obtained from transfected Ad-AH cells were capable of transforming human cord blood lymphocytes. Similar virus concentrates were unable to superinfect Raji cells.

Recover of transforming Epstein-Barr virus (EBV) from Raji cells superinfected with NPC-KT EBV

We previously established an epithelial/hybrid cell line, NPC-KT, by fusion 8-azaguanineresistant epithelial cells, Ad-AH, with a primary nasopharyngeal carcinoma cells. Virus preparations (NPC virus) from NPC-KT cells can transform human B-lymphocytes and superinfect Raji cells. In addition, the treatment of NPC virus-superinfected Raji cells with 1% dimethyl sufoxiside led to early antigen and virus DNA synthesis, and the formation of virus particles. The virus rescued from the superinfected Raji cells transforms human cord blood lymphocytes but is not able to induce early antigen in superinfected Raji cells.

Establishment of an Epstein-Barr Virus (EBV) Genome-Positive Subline of Ramos (Ramos/NPC) Following Infection of Ramos with Nasopharyngeal Carcinoma (NPC)-Derived EBV

An Epstein-Barr virus (EBV) genome-positive epithelial hybrid cell line, NPC-KT, derived from the fusion of primary nasopharyngeal carcinoma cells with a human epithelial cell line of adenoid origin has previously been described (Takimoto, Kamide, and Umeda, 1984a; Takimoto, Ogura, Sato, and Hatano, 1984b; Takimoto, Ogura, Sato, Umeda, and Hatano, 1985). The NPC-KT cells produce virus (NPC virus) with both transforming and lytic properties. An EBV genome-negative human lymphoblastoid cell line, Ramos, was infected with NPC virus. Three months after infection, more than 90% of the cell population expressed EBV-associated nuclear antigen (EBNA). Approximately 40% of the cells exhibited a brilliant pattern, whereas the remaining 50% of the cells showed a faint granular pattern. The result suggests that there may be heterogeneity in NPC virus.

Epstein-Barr virus (EBV) receptors on three EBV genome-positive epithelial hybrid cells

The authors have measured the relative number of Epstein-Barr virus (EBV) receptors on the surface of A2L/AH, NPC-KT, D98/HR-1, Raji, and P3HR-1 cells using an absorption index. It was determined that similar amounts of P3HR-1 or NPC-KT EBV can attach to D98/HR-1 cells, but that only P3HR-1 EBV can attach to NPC-KT cells. Both P3HR-1 and NPC-KT EBVs adsorb to Raji cells to a similar degree and to a greater level than either epithelial cells (D98/HR-1 and NPC-KT). Neither P3HR-1 or NPC-KT EBV adsorb to A2L/AH and P3HR-1 cells. The results suggest that there may be differences in the receptors for P3HR-1 and NPC-KT EBVs.

Effects of human interferon on cell proliferation and antigens induced by Epstein-Barr virus in epithelial hybrid cells derived from nasopharyngeal carcinoma.

The effects of human leukocyte interferon (HulFN-alpha) and fibroblast interferon (HulFN-beta) on cell growth and induction of antigens related to Epstein-Barr virus (EBV) in a nasopharyngeal carcinoma hybrid cell line (NPC-KT) were studied. HulFN-alpha showed little antiproliferative and anti-EBV effect on NPC-KT cells. On the other hand, HulFN-beta showed modest antiproliferative effect on NPC-KT cells; the effect was dependent on time and HulFN-beta concentration. HulFN-beta alone was ineffective in inhibiting EBV-related antigens, but NPC-KT cells treated for more than 1 week with HulFN-beta inhibited the action of the chemical inducer of EBV, n-butyric acid, and the appearance of EBV-induced early antigen and EB viral capsid antigen was inhibited more than 50% compared with NPC-KT cells treated with n-butyric acid alone.

Immunofluorescence and Electron Microscopic Investigations of Epithelial Hybrid Cells Derived From Nasopharyngeal Carcinoma (NPC-KT)

The present study was made to investigate some characteristics of the epithelial hybrid cells derived from nasopharyngeal carcinoma (NPC-KT cells) both in vivo and in vitro, using immunofluorescence and electron microscopic techniques. Immunofluorescence and electron microscopic studies have shown that the appearance of Epstein-Barr virus (EBV)-related early antigens, EB-viral capsid antigens and virus particles in nude-mouse-grown-tumour cells were rather repressed, in contrast to, in vitro culture of the NPC-KT cells. The tumours after transplantation of the NPC-KT cells to nude mice showed pathological pictures of poorly differentiated carcinoma with EBV-associated nuclear antigen and derived from the NPC-KT cells by means of cytogenetic studies. More importantly, we have detected EBV-related membrane antigens (MA) on the epithelial NPC-KT cells. To our knowledge, the presence of MA on the malignant epithelial cells of the nasopharynx have never been demonstrated. The results reported here show for the first time the presence of MA on nasopharyngeal carcinoma cells.

Isolation of Transforming and Early Antigen-Inducing Epstein-Barr Virus From Nasopharyngeal Carcinoma Hybrid Cells (NPC-KT)

The biologic activities of Epstein-Barr (EB) virus from an epithelioid-nasopharyngeal carcinoma hybrid cell line (NPC-KT) and three subclones from the NPC-KT cells were examined. Much infectious virus was released by treatment with 5-iodo-2'-deoxyuridine. All virus preparations from NPC-KT cells and the subclones were found to possess both transforming and EB virus-induced early antigen (EA)-inducing activities. The lymphoblastoid cell lines, which were established by infection of human cord-blood lymphocytes with the virus, were diploid with normal karyotypes. The cell lines were positive for EB virus-associated nuclear antigen but negative for EA and EB viral capsid antigen.

Epstein-Barr virus activation of human epithelial—Nasopharyngeal carcinoma hybrid cells

The authors used indirect immunofluorescence and electron microscopic techniques to study the effects of 5-iododeoxyuridine (IUDR) and the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) on the epithelial hybrid cell line (NPC-KT) derived from nasopharyngeal carcinoma. The hybrid cells contained Epstein-Barr virus (EBV)-induced early antigens at a very low level but not Epstein-Barr viral capsid antigens in spontaneous production; the hybrid cells responded significantly to both IUDR and TPA induction. The chemicals induced both early antigens and viral capsid antigens in the NPC-KT cells. Moreover, the virus particles were detected in the altered NPC-KT cells by IUDR treatment. The data suggest that the EBV-induced agents activate the EBV productive cycle in a proportion of nasopharyngeal carcinoma cells.