Oncogenic role of Epstein-Barr virus-encoded RNAs in Burkitt's lymphoma cell line Akata

Our previous reports indicated that Epstein-Barr virus (EBV) contributes to the malignant phenotype and resistance to apoptosis in Burkitt's lymphoma (BL) cell line Akata (N. Shimizu, A. Tanabe-Tochikura, Y. Kuroiwa, and K. Takada, J. Virol. 68:6069-6073, 1994; J. Komano, M. Sugiura, and K. Takada, J. Virol. 72:9150-9156, 1998). Here we report that the EBV-encoded small RNAs (EBERs) are responsible for these phenotypes. Transfection of the EBER genes into EBV-negative Akata clones restored the capacity for growth in soft agar, tumorigenicity in SCID mice, resistance to apoptotic inducers, and upregulated expression of bcl-2 oncoprotein that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. This is the first report which provides evidence that virus-encoded RNAs (EBERs) have oncogenic functions in BL cells.     

Activation of latent EBV via anti-IgG-triggered, second messenger pathways in the Burkitt's lymphoma cell line Akata

Anti-IgG treatment activated latent EBV genomes in 50 to 70% of the cells of the Burkitt's lymphoma cell line Akata. The EBV-activating role of intracellular Ca2+, as potentiated by diacylglycerol (DAG) and suppressed by cAMP, was analyzed in the cells through effects of agonists and antagonists of these second messenger pathways. Early Ag (EA) was induced in 10% of cells with the calcium ionophore A23187 (A23187). EA induction with anti-IgG or A23187 was blocked by a calmodulin antagonist, trifluoperazine. The DAG pathway had a potentiating but not direct effect on EBV activation because: 1) the DAG analog, dioctanoylglycerol (diC8), an agonist for protein kinase C, alone induced only 2% EA-positive cells, 2) diC8 synergized with A23187 for EA induction, and 3) the protein kinase C antagonist, staurosporine, almost completely inhibited EA induction by anti-IgG. When cells were reincubated in medium with fresh diC8 and A23187 at 3, 6, 9, and 12 h, EA induction at 24 h reached the levels seen with anti-IgG stimulation. A cAMP-mediated pathway suppressed EBV activation because dibutyryl cAMP or 8-bromo-cAMP, plus blockage of phosphodiesterase by theophylline, or use of forskolin, inhibited EA induction with anti-IgG. Although the principal stimulatory role in EBV activation of a Ca2(+)-mediated, second messenger pathway, as synergized by DAG and inhibited by cAMP, was established, we did not explain the significant lag in EA induction by A23187 and diC8 as compared with anti-IgG induction of EA. We conclude that EBV genome activation with anti-IgG is mediated by Ca2+/calmodulin and DAG pathways in Akata cells, that the cAMP pathway suppresses EA induction by anti-IgG, and that a mechanism regulating the speed of EA induction remains unexplained.     

Involvement of caspase activation and mitochondrial stress in trichostatin A-induced apoptosis of Burkitt's lymphoma cell line, Akata

Epstein-Barr virus (EBV) infects more than 90% of the human population and has a potential oncogenic nature. Trichostatin A (TSA) has potent antitumor activity, but its exact mechanism on EBV-infected cells is unclear. This study examined the effects of TSA on proliferation and apoptosis of the Burkitt's lymphoma cell line, Akata. TSA treatment inhibited cell growth and induced cytotoxicity in both the EBV-negative and -positive Akata cells. TSA sensitively induced apoptosis in both cells, as demonstrated by the increased number of positively stained cells in the TUNEL assay, the migration of many cells to sub-G1 phase by flow cytometric analysis, and the formation of DNA ladders. This suggests that EBV has no effect on the sensitivity to TSA. Western blot analysis showed that the cleavage of PARP and Bid and the activation of caspases are closely related to the TSA-induced apoptosis of the cells. The reduction in mitochondrial transition potential and the release of apoptosis-inducing factor from mitochondria to cytosol was also observed after the TSA treatment, but was suppressed by treating the cells with a cathepsin B inhibitor. Overall, these findings suggest that besides the caspase-dependent pathway, mitochondrial events are also associated with the TSA-induced apoptosis of Akata cells.     

Isolation of Epstein-Barr virus (EBV)-negative cell clones from the EBV-positive Burkitt's lymphoma (BL) line Akata: malignant phenotypes of BL cells are dependent on EBV.

During cultivation of the Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) line Akata, it was noted that EBV DNA is lost from some of the cells. Isolation of EBV-positive and EBV-negative clones with the same origin made it possible to examine the effects of EBV in BL cells. The results indicate that malignant phenotypes of BL, such as growth in low serum, anchorage-independent growth in soft agar, and tumorigenicity in nude mice, are dependent on the presence of EBV genomes and underline the oncogenic function of EBV in human cancer.     

Epstein-barr virus (EBV) nuclear protein 2-induced disruption of EBV latency in the Burkitt's lymphoma cell line Akata: analysis by tetracycline-regulated expression

The Burkitt's lymphoma (BL) cell line Akata retains the latency I program of Epstein-Barr virus (EBV) gene expression and cross-linking of its surface immunoglobulin G (IgG) by antibodies results in activation of viral replication. When EBV nuclear antigen 2 (EBNA2) was artificially expressed by a constitutive expression vector, the Cp EBNA promoter remained inactive and accordingly the latency III program was not induced. In contrast, expression of LMP2A and activity of the Fp lytic promoter were activated. Consistent with this Fp activity, the rate of spontaneous activation of the EBV replicative cycle was increased significantly, suggesting the possibility that EBNA2 can induce EBV replication. The efficiency of anti-IgG-induced activation of the viral replication was reduced in Akata cells expressing EBNA2. To obtain more direct evidence for EBNA2-induced activation of the EBV replicative cycle, this protein was next expressed by a tetracycline-regulated expression system. EBNA2 was undetectable with low doses (<0.5 microgram/ml) of tetracycline, while its expression was rapidly induced after removal of the antibiotic. This induced expression of EBNA2 was immediately followed by expression of EBV replicative cycle proteins in up to 50% of the cells, as shown by indirect immunofluorescence and immunoblot analysis. These results suggest an unexpected potential of EBNA2 to disrupt EBV latency and to activate viral replication.     

Cellular and Epstein-Barr virus specific DNA polymerases in virus-producing Burkitt's lymphoma cell lines.

We have determined the levels of cellular DNA polymerases and Epstein-Barr virus specific DNA polymerase in three Burkitt's lymphoma cell lines producing varying amounts of EBV, one of which was induced by 12-0-tetra-decanoylphorbol-13-acetate (TPA). There was a proportional increase in the level of EBV-DNA polymerase with an increase in the percent of virus-producing cells. However, there was a reciprocal relationship between the levels of EBV-DNA polymerase and DNA polymerase alpha i.e., in cell line containing the highest level of EBV-DNA polymerase, activity of DNA polymerase alpha, but not of DNA polymerase beta, was reduced to an insignificantly low level. TPA does not have any direct effect on activities of either EBV-DNA polymerase or DNA polymerase alpha. EBV-DNA polymerases isolated from cells grown with or without TPA are indistinguishable in their properties such as elution position on phosphocellulose column, molecular weight, mono and divalent cation requirements, pH optimum, and other requirements for optimum activity. Addition of crude extracts of cells grown in presence of TPA to the purified DNA polymerase alpha did not inhibit its activity indicating that the observed loss was not due to any specific inhibitor present in TPA treated cells. Raji, a nonproducer cell line, did not contain EBV-DNA polymerase. There was no induction of EBV-DNA polymerase when Raji cells were grown in presence of TPA. The phenomenon of reduction in the levels of DNA polymerase alpha in cells induced to produce EBV may represent a mechanism by which the host DNA replication is shut off following virus infection.     

bcl-2高表达细胞株的构建

利用基因转染技术,将人的bcl-2基因cDNA克隆于逆转录病毒载体pLXSN,通过PA317细胞包装成病毒后感染HeLa细胞。经PCR及蛋白质印迹证明转染成功,得到bcl-2稳定高表达株Hbc17。用顺铂诱导凋亡,Hbc17比对照细胞株H1具有明显的抗凋亡能力。为进一步研究顺铂诱导肿瘤细胞凋亡的机理奠定基础。     

Apoptosis and restriction of G(1)/S cell cycle by fenretinide in Burkitt's lymphoma mutu I cell line accessed with bcl-6 down-regulation

Fenretinide (4-HPR) is a synthetic retinoid with cancer chemopreventative potential and clinically manageable side effects, compared to the prototype retinoid, all-trans retinoic acid (RA). 4-HPR has been shown to modulate cell proliferation and induce apoptosis in a variety of human tumor cell types, but its effects on B-cell non-Hodgkin's lymphomas (NHL-B) have not been explored. Treatment of Burkitt's lymphoma Mutu I cells with 3 microM 4-HPR is accompanied by growth arrest, induction of apoptosis, and restricted progression of the cell cycle at the G(1)/S checkpoint. We also observed that 4-HPR elicited a reduced expression of bcl-6 in these cells, which supports the proposed role of bcl-6 as an anti-apoptotic gene. While 4-HPR treatment had no effect on total Rb gene expression, it significantly reduced the state of hyperphosphorylation of Rb, resulting in the predominant existence of Rb in the underphosphorylated state.     

Epstein-Barr virus-positive Burkitt's lymphoma in a German woman during pregnancy

A fatal case of a Burkitt's lymphoma which occurred in a 34-year-old German woman during pregnancy is described. Nearly all organs showed either diffuse or nodular infiltration by tumor cells. Placenta and fetus were free of detectable tumor tissue. The patient had extremely high antibody titers (1 : 2056), both against Epstein-Barr virus capsid antigen (VCA) and the early antigen complex (EA). Within the tumor cells the Epstein-Barr virus-specific nuclear antigen EBNA and viral DNA was detected. A cell line established from a tumor biopsy displayed a translocation involving chromosomes 2 and 8. The role of Epstein-Barr virus in the development of Burkitt's lymphoma is discussed.     

Accumulation of Epstein-Barr virus (EBV) BMRF1 protein EA-D during latent EBV activation of Burkitt's lymphoma cell line Raji

As a new model to elucidate molecular mechanisms in Epstein-Barr virus (EBV) activation, we tested the tetracycline-inducible (Tet-On)/BZLF1-oriP plasmid system in Raji cells. Cells transfected with this Tet-On plasmid did not activate EBV by doxycycline and surprisingly EBV latency was disrupted with large amounts of BMRF1 protein (EA-D) being accumulated in the cells. Brilliant EA-D fluorescence was markedly condensed in small sized cells, intra-cellular vesicles, and extra-cellular particles. Scanning electron microscopy demonstrated the extra-cellular particles to be covered with a membrane. EA-D molecules of 58, 50, 48, and 44kDa were expressed in the cells. The high (58 and 50kDa) and low (48 and 44kDa) EA-D molecules appeared in the early and late stages, respectively. Low EA-D molecules were detected mostly in EA-D positive cells separated into the heaviest density layer of a discontinuous Percoll gradient. Such molecules could be created from high EA-D molecules by protein phosphatase treatment. The EA-D molecules that appeared similar were detected in EBV-activated P3HR-1 and Akata cells. Several hypotheses concerning the accumulation of EA-D molecules of various polymorphic forms and their phosphorylation/dephosphorylation in this model system are presented, with possible biological and clinical relevance.     

Epstein-Barr virus RNA confers resistance to interferon-alpha-induced apoptosis in Burkitt's lymphoma

We investigated whether Epstein--Barr virus (EBV) infection could counteract the antitumor effect of interferon (IFN)-alpha. EBV-negative subclones isolated from EBV-positive Burkitt's lymphoma (BL) cell lines Akata, Daudi and Mutu were found to fall into apoptosis after IFN-alpha treatment. On the other hand, EBV-positive counterparts exhibited striking resistance against IFN-alpha-induced apoptosis. Transfection of an individual EBV latent gene into EBV-negative BL cells revealed that EBV-encoded poly(A)(-) RNAs (EBERs) were responsible for IFN resistance. EBERs bound double-stranded (ds) RNA-activated protein kinase (PKR), a key mediator of the antiviral effect of IFN-alpha, and inhibited its phosphorylation. Transfection of dominant-negative PKR, which was catalytically inactive and could block phosphorylation of endogenous PKR, made EBV-negative BL cells resistant to IFN-alpha-induced apoptosis. Furthermore, EBERs did not bind mutant PKR, which was catalytically active but lacked dsRNA-binding activity, nor did they inhibit its phosphorylation. These results indicate that EBERs confer resistance to IFN-alpha-induced apoptosis via binding to PKR and inhibition of its phosphorylation. This is the first report that the virus counteracts IFN-induced apoptosis in virus-associated tumors.     

Identification of genes deregulated during serum-free medium adaptation of a Burkitt's lymphoma cell line

Abstract.  Objective : Serum is usually added to growth media when mammalian cells are cultured in vitro to supply the cells with growth factors, hormones, nutrients and trace elements. Defined proteins and metal ions, such as insulin, growth factors, transferrin and sodium selenite, are sometimes also included and can in some cases substitute serum components. How adaptation to serum free media influences cells has not been studied in detail. Materials and Methods : We have adapted the Burkitt's lymphoma line Ramos to a serum-free medium that supports long-term survival and studied gene expression changes that occurred during the adaptation process. Results and Conclusions : The adaptation process was characterized by initial cell population growth arrest, and after that extensive cell death, followed by proliferation and long-term survival of clonal cultures. Proliferation and cell cycle progression of the serum-free cultures closely mimicked that of serum-dependent cells. Affymetrix micro-array technology was used to identify gene expression alterations that had occurred during the adaptation. Most changes were subtle, but frequently the genes with altered expression were involved in basal cellular functions such as cell division, cell cycle regulation, apoptosis and cell signalling. Some alterations were restored when the cells were transferred back to serum-containing medium, indicating that expression of these genes was controlled by components in serum. Others were not, and may represent changes that were selected during the adaptation process. Among these were, for example, several genes within the Wnt signalling pathway.