Hydrocortisone Enhancement of Both EBV Replication and Transformation of Human Cord Lymphocytes

In vitro evaluation of antileukemic activity of 17 commonly used fruits and vegetables in Taiwan

PHOSPHONOACETIC acid (PAA) is an inhibitor of herpes simplex virus and of several other herpes viruses. It acts as a specific inhibitor of two viral DNA polymerases, those of herpes simplex and of Marek's disease (a herpes-induced leukaemia in chickens)1,2. We have used PAA as a probe to investigate the possible existence of a virus-directed DNA polymerase and its role in replication of viral genomes during the transformation and proliferation of normal human peripheral lymphocytes induced by Epstein–Barr virus (EBV), -which is a herpes virus closely associated with the development of several neoplasms in man.

Epstein-Barr virus DNA synthesized in superinfected raji cells

We report the use of monoclonal antibody against the early antigen diffuse component (anti-EA-D) of Epstein-Barr virus (EBV) to analyze, both qualitatively and quantitatively, the expression of EA-D in various human lymphoblastoid cell lines activated by chemical inducers. The kinetics of synthesis of EA-D in P3HR-1, B95-8, and Ramos/AW cells were similar in that they all reached the peak of synthesis on day 5 after induction. Surprisingly, no expression of EA-D was found in induced BJAB/GC, an EBV-genome-containing cell line. EBV-negative cell lines, BJAB and Ramos, were negative for EA-D. Raji cells had no detectable EA-D but responded rapidly to induction, reaching a peak on day 3. Superinfection of Raji cells also resulted in marked induction of EA-D, which reached a plateau between 8 to 12 h postinfection. Western blotting coupled with the enzyme-linked immunosorbent assay was employed to identify polypeptides representing EA-D. A family of four polypeptides with molecular weights of 46,000 (46K protein), 49,000, 52,000, and 55,000 were identified to be reactive with monoclonal anti-EA-D antiserum. The pattern of EA-D polypeptides expressed in each cell line was different. Of particular interest was the expression of a large quantity of 46K protein both in induced Raji and P3HR-1 cells, but not in superinfected Raji cells. A 49K doublet was expressed in activated p3HR-1, B95-8, and Ramos/AW cells and in superinfected Raji cells. In addition, two distinct 52K and 55K polypeptides were expressed in induced Ramos/AW and superinfected Raji cells. However, none of these EA-D polypeptides was detectable in BJAB/GC, BJAB, Ramos, and mock-infected Raji cells. To approximate relative concentrations of EA-D in cell extracts, we employed the enzyme-linked immunosorbent assay and immunoblot dot methods by using one of the purified EA-D components to construct a standard curve. Depending upon the cell lines, it was estimated that ca. 1 to 3% (determined by the enzyme-linked immunosorbent assay) and 0.8 to 1.6% (determined by immunoblot dot) of total proteins from maximally induced cells were EA-D. These results suggest that differential expression of EA-D polypeptides could be of importance in the diagnosis of state of EBV infection.

Phosphonoformate inhibits synthesis of epstein-barr virus (EBV) capsid antigen and transformation of human cord blood lymphocytes by EBV

Transfer RNA's and human lymphocyte transformation.

The effect of interferon (IFN) on protein synthesis was studied in the Burkitt's lymphoma cell line P3HR-1 by [35S]methionine labelling of the cells, followed by two-dimensional gel electrophoresis of cell extracts. De novo synthesis of three proteins (mol. wts. 33 000, 62 000, and 98 000, respectively) and alterations in the rate of synthesis for a small number of additional proteins were observed during the first 12 h of treatment, while the rate of overall protein synthesis was unaffected. Treatment of P3HR-1 cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or hydrocortisone (HC), which induce similar changes in cell cycle distribution as does IFN, did not induce comparable changes in the rates of protein synthesis. Thus, the effects were specific for IFN and not induced by the change in cell cycle distribution per se, i.e., accumulation in G0. Treatment of cells with 2'-5' pA core did not mimic the effect of IFN at the translational level. A substrain of P3HR-1 cells, selected for resistance to the anti-proliferative effect of IFN, lacked six proteins found in the wild-type. The 62 000 mol. wt. protein was induced in this substrain as well as in native P3HR-1 cells on addition of IFN. The resistant substrain still developed an anti-viral effect in response to IFN. Thus, it seems as if the anti-proliferative and anti-viral effects of IFN, at least in some cells are mediated by different intracellular molecular mechanisms.

The experiments show that 30 virus-induced or virus-specified proteins were synthesized in Raji cells after superinfection with Epstein-Barr virus (EBV) derived from P3HR1 cells. Using a combination of pulse labelling, application of cycloheximide blocks at different times post-infection, treatment with amino acid analogues and inhibition of DNA synthesis it was shown that three groups of proteins appear in Raji cells after superinfection; the synthesis of the proteins in any one group appears to be coordinately regulated. Amongst the six virus-induced proteins which were synthesized immediately after release from an early cycloheximide block one would expect to find those proteins essential for the transition from EBNA to EA synthesis. Using human sera with differing specificities for the various antigen groups 11 proteins were identified as being specifically precipitated by sera having high titres against the EBV-induced early antigen complex.

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.

Migration of peripheral leukocytes in samples from sensitized [Epstein-Barr virus (EBV) antibody-positive] humans was greatly inhibited when challenged by antigen prepared from EBV-producing P3HR-1 cells but not by antigen prepared from EBV-nonproducing RAJI cells, EBV-negative human fibroblasts, or epithelial cells. Such inhibition was not observed when peripheral leuocytes from subjects or neonates not sensitized to EBV were challenged. Similar results were obtained in a two-stage test when the same leukocyte samples were challenged in vitro by antigen prepared from P3HR-1 cells and the cell-free supernatant was assayed for migration inhibition factor (MIF) in the guinea pig macrophage migration inhibition test; migration of guinea pig peritoneal exudate cells was greatly inhibited by the supernatant filtrates of leukocyte cultures only from subjects positive for EBV-antibody. Furthermore, this inhibitory effect was not observed if supernatant filtrates from leukocyte cultures challenged by antigens prepared from RAJI cells, fibroblasts, or epithelial cells were used. The EBV antigen transformed peripheral leukocytes and induced early antigen production in RAJI cells; however, a "killed" preparation (by UV irradiation) was sufficient for eliciting MIF production.

Effect of colchicine, vinblastine and cytochalasin B on human lymphocyte transformation by phytohemagglutinin

Mitogen-induced lymphocyte transformation (LT) has been used extensively to describe proliferation abnormalities of lymphocytes in various diseases such as lymphoproliferative syndromes (1). Since LT results from the cooperation of different cell types (2) we further analyzed this phenomenon in order to define partial cellular defects underlying secondary immunodeficiency syndromes such as in Hodgkin’s disease.KeywordsMitogenic SignalLymphocyte TransformationStimulative CapacityNylon WoolHuman Blood LymphocyteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Epstein-Barr virus (EBV) is unusually adapted for transformation of human B lymphocytes (bone marrow-derived lymphocyte). In this chapter, presently available information on the events leading to transformation will be reviewed. For reasons which will become apparent, we will assume that some event occurs at about 3 days after infection of human B lymphocytes with EBV in vitro, following which the cells are transformed into permanently established lines. It is assumed that this event is the integration of one or more genomes or fractional genomes of EBV into cellular DNA. In other systems which may be regarded as models for the transformation of lymphocytes by EBV, integration has been established. The integration of viral genomes or fractional genomes into the host genome, occurs, for example, in the transformation of appropriate cells by SV40 (Sambrook et al., 1975; Botchan et al., 1976), by adenovirus (Sambrook et al., 1975; Green et al., 1976; van der Eb and Houweling, 1977), by ultra violet-inactivated or temperature-sensitive mutants of herpes simplex viruses 1 and 2 (HSV) (Macnab, 1974; Rapp and Li, 1975; Galloway et al., in preparation), which are related to EBV, and by RNA tumor viruses in which a form of the DNA provirus is the intermediate for integration (Guntaka etal., 1975; Weinberg, 1977). A more general model for this kind of process is the lysogenization of Escherichia coli by phage lambda or phage mu.

Acyclic nucleoside phosphonates (ANPs), such as (S)-1-[(3-hydroxy-2-phosphonomethoxy)propyl)]cytosine (HPMPC), are an important group of broad-spectrum antiviral agents with activity against DNA viruses. In this report, we present the in vitro potencies of novel ANPs against gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus (EBV), and three animal gammaherpesviruses. 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC), (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine (3-deaza-HPMPA), and their cyclic derivatives have emerged as highly potent antigammaherpesvirus agents. Interestingly, cyclic prodrugs of ANPs exhibited reduced activities against EBV strain P3HR-1, but not against EBV strain Akata. Cell culture metabolism studies with HPMPC and cyclic HPMPC revealed that these differences were attributable to an altered drug metabolism in P3HR-1 cells after EBV reactivation and, more specifically, to a reduced hydrolysis of cyclic HPMPC by cyclic CMP phosphodiesterase. We did not correlate this effect with phosphodiesterase downregulation, or to functional mutations. Instead, altered cyclic AMP levels in P3HR-1 cells indicated a competitive inhibition of the phosphodiesterase by this cyclic nucleotide. Finally, both HPMPC and HPMP-5-azaC emerged as highly effective inhibitors in vivo through significant inhibition of murine gammaherpesvirus replication and dissemination. With the current need for potent antigammaherpesvirus agents, our findings underline the requirement of appropriate surrogate viruses for antiviral susceptibility testing and highlight HPMP-5-azaC as a promising compound for future clinical development.

A functional role of class II antigen in the binding of Epstein-Barr virus (EBV) was deduced from the study of membrane proteins on Jijoye, an EBV receptor (EBVR)-positive B cell line, and its mutant, EBVR-negative daughter cell line, P3HR-1. From gel electrophoresis of radiolabeled microsomal membrane proteins and immunoprecipitates, we identified class II antigen on Jijoye but not on P3HR-1 cells and the presence of Ii on both cell lines. The role of these molecules in EBVR function was tested by antibody blocking of virus adsorption. Anti-p23,30 serum (to class II antigen) was found to block binding of EBV to B lymphoblasts under conditions in which normal rabbit serum, rabbit antiserum to butyrate-treated P3HR-1 cells (with ample anti-Ii antibodies), and rabbit anti-p44,12 (to class I antigen and beta 2-microglobulin) serum did not block virus binding. Only one of four commercial monoclonal antibodies (MoAb) to framework epitopes on class II antigens blocked binding of EBV, whereas all four MoAb demonstrated immunofluorescent reactivity with the EBVR+ Raji cells. In previous studies of binding of EBV to hairy leukemic cells, a substantial subpopulation of HLA-DR+, EBVR- cells was identified, in addition to HLA-DR+, EBVR+ cells. These findings were consistent with the view that the HLA-DR complex has a role in the binding of EBV but that other components are also needed for the expression of EBVR function.