Antigens In Raji Cells Research Articles

Phenylethylchromones with In Vitro Antitumor Promoting Activity from Aquilaria filaria

A new chromone, 2-(2-hydroxy-2-phenylethyl)chromone (1), was isolated together with ten known phenylethyl chromones from MeOH extracts of agarwood (Aquilaria filaria). The selected compounds were evaluated in an antiproliferative assay against five human tumor cell lines, including a multidrug-resistant cell line. They were also tested for antitumor promoting activity, as mediated by 12-O-tetradecanoylphorbol-13-acetate-induced activation of the Epstein-Barr virus early antigen in Raji cells. Among all compounds, 4',7-dimethyoxy-6-hydroxychromone (2) displayed broad spectrum antiproliferative activity against all tumor cell lines tested with IC50 values of 25-38 µM, while 8 was selectively inhibitory against multidrug-resistant cells. All tested compounds suppressed tumor promotion at noncytotoxic concentrations. 4',6-Dihydroxyphenylethylchromone (7) exhibited the most potent effect with an IC50 value of 319 mol ratio relative to 12-O-tetradecanoylphorbol-13-acetate. This study is the first to report the antitumor promoting activity of 2-(2-phenylethyl)chromone derivatives, as well as the selective antiproliferative activity of 8 against a multidrug-resistant tumor cell line.

CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins.

CD9, CD63, CD81, and CD82 are glycoproteins of unknown function which belong to the tetraspan superfamily. These molecules have short cytoplasmic sequences, four transmembrane domains and two unequal extracellular regions. Here, we show that these molecules are associated with each other on cell surface and with other glycoproteins such as very late antigen (VLA) integrins and HLA-DR antigens. Moreover, the VLA integrins and HLA-DR antigens were also found to be associated. The interactions of these molecules were analyzed by transfection experiments. It is demonstrated that overexpression of CD9 antigen in Raji cells leads to a lower efficiency of precipitation of CD81 and CD82, suggesting a direct interaction between these molecules. In these cells, the co-precipitation of CD81 and CD82 was not modified, suggesting that these tetraspans did not compete for association. However, in COS-7 cells, transfection of both CD81 and CD82 led to a marked reduction of the number of CD9/CD81 or CD9/CD82 complexes compared to single-transfected cells, and this was associated with the appearance of CD81/CD82 complexes. Therefore, in this cellular system, CD9 competes with CD81 and CD82 for association with the other tetraspan proteins. Finally, the tetraspans do not compete for the association with integrins or HLA-DR. Indeed, when CD9 was expressed in Raji cells, it was incorporated into the pre-existing complexes of these molecules with CD81 and CD82. These data suggest the existence of a tetraspan network which, by connecting several molecules, may organize the positioning of cell surface proteins and play a role in signal transduction, cell adhesion, and motility.

Tumor-promoting activity of staurosporine, a protein kinase inhibitor on mouse skin.

Staurosporine, which is a potent inhibitor of protein kinases, such as protein kinase C, inhibited both inductions of adhesion of human promyelocytic leukemia cells (50% effective dose = 9.0 nM) and Epstein-Barr virus early antigen in Raji cells (50% effective dose = 3.4 nM) by teleocidin. However, staurosporine induced irritation on mouse ear and histidine decarboxylase activity in mouse skin. It did not induce ornithine decarboxylase activity in mouse epidermis. The two-stage carcinogenesis experiments of staurosporine were carried out at two different doses. Experiment 1 revealed that the group treatment with a single application of 100 micrograms of 7,12-dimethylbenz(a)anthracene, followed by repeated applications of 50 micrograms of staurosporine, resulted in 85.7% of tumor-bearing mice at Wk 30, whereas group treatment with staurosporine alone or 7,12-dimethylbenz(a)anthracene alone gave 6.7% and 0%, respectively. Experiment 2 showed that group treatment with 7,12-dimethylbenz(a)anthracene followed by applications of 10 micrograms of staurosporine resulted in 33% of tumor-bearing mice at Wk 30. In addition, staurosporine treatment reduced the percentages of tumor-bearing mice treated with teleocidin from 100% to 67% in Wk 15. These results demonstrated that staurosporine is a weak tumor promoter of mouse skin compared with teleocidin, but staurosporine has some potency to inhibit tumor promotion by teleocidin.

Epstein‐barr virus internalization and infectivity are blocked by selective protein kinase C inhibitors

Selective protein kinase C inhibitors can either block or significantly reduce Epstein-Barr virus infectivity: inhibition of transformation and decreased 3H-thymidine (3H-TdR) incorporation in human B lymphocytes infected with B95-8 EBV, as well as a significant reduction in the induction of early antigens in Raji cells superinfected by P3HRI EBV was achieved by pre-treating the cells with the inhibitors. The inhibitors do not act by blocking binding of the virus to its cellular receptor CR 2, but rather are effective in the viral internalization process. Our results suggest that protein kinase C may be involved in the process of viral entry into cells.

Sites of Epstein-Barr Virus Replication in Acute and Chronic Active Epstein-Barr Virus Infections

We examined Epstein-Barr virus (EBV) in saliva samples and peripheral blood mononuclear cells (PBMC) from 9 patients with acute infectious mononucleosis (IM) and 4 patients with chronic active EBV infection (CEBV). All saliva samples from patients with acute IM were positive for EBV by both Southern blot hybridization assay and the cord transformation assay. In contrast, EBV DNA could not be detected in saliva samples from patients with CEBV. Only one of the saliva samples from 4 patients with CEBV could transform cord blood lymphocytes with extreme difficulty. No saliva samples from patients with CEBV were capable of inducing early antigens in Raji cells. On the other hand, EBV DNA was detected in PBMC from 3 of the 4 patients with CEBV but not in those from patients with acute IM by Southern blot hybridization. Spontaneous lymphoblastoid cell lines could be easily established from PBMC of patients with acute IM but not from PBMC of patients with CEBV, despite several attempts. Viral capsid antigen and early antigens were not observed in cultured cells from patients with CEBV. These results suggest that the main site of EBV replication in CEBV might not be oropharyngeal epithelial cells.

Transformation of C3H/10T [formula omitted] cells and induction of EBV-early antigen in Raji cells by altertoxins I and III

Moulds of the genus Alternaria are common contaminants of some food crops. Some isolates have been shown to produce mutagenic compounds called altertoxins. Altertoxin I (ATX-I) and altertoxin III (ATX-III) were examined for activity in the Raji cell Epstein-Barr virus early antigen (EBV-EA) induction system and in the C3H/10T 1 2 murine fibroblast cell transformation system. Exposure of Raji cells to ATX-I or ATX-III activated EBV-EA expression by 8- and 9.5-fold, respectively. A single exposure of C3H/10T 1 2 cultures to ATX-I or ATX-III resulted in significant increases in cell transformation, and the response to ATX-I was stronger. Both altertoxins enhanced the transformation of C3H/10T 1 2 cells, and chronic exposure of non-initiated C3H/10T 1 2 cells to ATX-I and ATX-III, starting 6 days after cells were plated, resulted in cell transformation in 8 59 and 12 37 dishes, respectively, compared with transformation in only 2 63 control dishes. Since activation of EBV-EA in Raji cells has been positively correlated with tumour promoters, these data together indicate that ATX-I and ATX-III are not just mutagens but have a potential role in cell transformation.

Inhibition of 12- O-tetradecanoylphorbol-13-acetate-induced induction of Epstein-Barr virus early antigen in Raji cells by some inhibitors of tumor promotion

The effects of some compounds, which have been reported to inhibit tumor promotion in vivo, on the induction of the early antigen (EA) of Epstein-Barr virus (EBV) by 12- O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells were examined. The inhibitors of the cascade process involving arachidonic acid, indomethacin, nordihydroguaiaretic acid, phenidone and p-bromophenacyl bromide, effectively inhibited EBV-EA induction by TPA. Two flavonoids, morin and kaempferol also inhibited EA induction. Among antioxidants, butylated hydroxytoluene effectively inhibited EA induction, though α-tocopherol did not show any inhibition of EA induction at concentrations of up to 150 μg/ml. N-(6-Aminohexyl)-5-chloro-1-naphthalenesul-fonamide, a calmodulin antagonist, and esculetin showed inhibitory effects on EA induction, though slight cytotoxicity was observed. l-1- p-Tosylamino-2-phenylethyl chloromethyl ketone, a protease inhibitor, showed cytotoxicity and no specific inhibition of EA induction. Five kinds of steroids, cortisone, hydrocortisone, prednisolone, dexamethasone and fluocinolone acetonide showed no inhibitory effect on EA induction at concentrations of up to 100 μg/ml. In addition, the relationship between the inhibition of EBV-EA induction and that of tumor promotion is discussed.

P3HR-1 Epstein-Barr virus with heterogeneous DNA is an independent replicon maintained by cell-to-cell spread

We present results of biological experiments which indicate that the subpopulation of Epstein-Barr virus strain P3HR-1 with heterogeneous (het) DNA consists of self-contained replicons which multiply alongside, but independently of, Epstein-Barr virus strain HR-1 containing standard DNA. When a population of HR-1 virions containing het DNA was introduced into X50-7 cells, the input heterogeneous DNA increased in abundance, as did the DNA of the endogenous virus of X50-7 cells. The input standard HR-1 viral DNA, however, was not amplified. When parental HR-1 cells or a cellular subclone containing het DNA were grown for several weeks in the presence of human serum with neutralizing antibody, the het DNA was lost from the culture; standard HR-1 DNA, however, was not affected by antiserum. Furthermore, virions containing het DNA could be serially propagated through cellular subclones of HR-1 cells which lack het DNA. After each serial passage, cells which acquired het DNA released virions with the ability to induce early antigens in Raji cells. These experiments define a novel in vitro life cycle of an Epstein-Barr virus variant which is maintained, not vertically by partitioning to daughter cells in cell division, but horizontally by cell-to-cell spread.

Effect of Heat Shock on Epstein-Barr Virus and Cytomegalovirus Expression

The effect of heat shock was investigated on lymphoblastoid cell lines Raji and P3HR1 harbouring Epstein-Barr virus (EBV) genomes, and on Vero cells abortively infected with human cytomegalovirus (HCMV). A heat shock at 44 degrees C for 10 min induced the appearance of EBV early antigens in Raji cells and increased the percentage of cells expressing EBV viral capsid antigens in P3HR1 cells. Heat shock performed on Vero cells just before HCMV infection resulted in an approximately fourfold increase in the number of cells exhibiting early nuclear antigens, and in the appearance of HCMV-induced Fc receptors.

Epstein-Barr virus with heterogeneous DNA disrupts latency.

By cloning the HR-1 Burkitt lymphoma line, we previously uncovered two distinct biological variants of nontransforming Epstein-Barr virus (EBV). The most commonly cloned variant has a low rate of spontaneous viral synthesis and is unable to induce early antigen in Raji cells (EAI-). A rare variant spontaneously releases virus which is capable of inducing early antigen in Raji cells (EAI+). Since EAI- virus lacks heterogeneous DNA (het-) and EAI+ virus contains heterogeneous DNA (het+), we suggested that spontaneous viral synthesis and induction of early antigen are biological properties which correlate with the presence of het sequences. The present experiments provide three new lines of experimental evidence in favor of this hypothesis. (i) Revertant subclones of the EAI+ het+ variant which have lost the het DNA concomitantly lost EAI ability. Thus, het DNA is not stably associated with the cells as are the episomes. (ii) het DNA was acquired by two het- subclones of the HR-1 line after superinfection with EAI+ virus. After superinfection, these clones synthesized EAI+ het+ virus. Thus, het DNA may be maintained in the HR-1 line by cell-to-cell spread. (iii) Virus with het DNA activated full expression of endogenous latent EBV of the transforming phenotype in a line of immortalized neonatal lymphocytes designated X50-7. By use of restriction endonuclease polymorphisms unique to both the superinfecting and endogenous genomes, we show that the genome of the activated virus resembles that of the virus which was endogenous to X50-7 cells. This result suggests that het sequences result in transactivation of the latent EBV. het DNA had homology with EBV sequences which are not normally contiguous on the physical map of the genome. het DNA was always accompanied by the presence of DNA of nonheterogenous HR-1. Thus, het DNA is a form of "defective" EBV DNA. However, the biological effect of this defective DNA is to enhance rather than to interfere with EBV replication. This is a novel property of defective virus.

Modulation by the polyoxotungstate HPA-23 of Epstein-Barr virus early antigen expression in Raji cells treated with iododeoxyuridine.

The polyoxotungstate HPA-23 was found to exert a differential effect on the induction of Epstein-Barr virus early antigen in Raji cells induced with 5-iodo-2'-deoxyuridine (IUdR). Thus, treatment of Raji cells concomitantly with IUdR and HPA-23 inhibited early antigen expression, and the extent of inhibition was proportional to the duration of treatment with HPA-23. In contrast, pretreatment of Raji cells with HPA-23 prior to induction with IUdR stimulated early antigen expression in exponentially multiplying but not in stationary-phase cells. HPA-23 alone had no effect on early antigen expression in Raji cells. Activation of the latent Epstein-Barr virus genome by IUdR is dependent upon incorporation of the thymidine analogue into cellular DNA during the S-phase of the cell cycle. Synthesis of Epstein-Barr virus DNA also takes place during S-phase, suggesting a possible participation in this process of cellular DNA polymerase alpha which is thought to be responsible for cellular DNA replication and the activity of which increases several-fold during S-phase. Treatment of Raji cells with HPA-23 caused a marked decrease in DNA polymerase alpha activity, which could result in an inhibition of IUdR incorporation leading to the observed reduction of early antigen expression in cells treated concomitantly with IUdR and HPA-23.

A new strain of Epstein-Barr virus derived from nasopharyngeal carcinoma hybrid cells.

A new strain of Epstein-Barr virus was isolated from an epithelioid hybrid cell line derived from nasopharyngeal carcinoma which had been cultured for 14 days at 32 degrees. This virus can transform human cord blood lymphocytes and induce early antigens in Raji cells.

Multiplicity-dependent induction of viral capsid antigen in raji cells superinfected with epstein-barr virus

A quantitative analysis of Epstein-Barr virus (EBV)-induced early antigen (EA) and viral capsid antigen (VCA) syntheses was carried out in Raji cells superinfected with purified, concentrated P3HR-1 EBV. When the cells were exposed to the virus and assessed by immunofluorescence and immunoprecipitation, EA induction occurred significantly (17%) but not VCA (<l% ), at a low-input multiplicity of infection (MOI) of 10 EBV DNA copies/cell. In contrast, at a high MOI of 500 EBV DNA copies/cell, the majority of cells were positive for both EA (82%) and VCA (61%). The latter VCA synthesis was accompanied by the replication of EBV DNA. Kinetic studies showed that EA induction was directly proportional to the dilution of the infecting virus, while VCA was made following three-hit kinetics. The implications of these results are discussed in relation to the heterogeneous nature of P3HR-1 EBV and a possible role of EA in VCA synthesis.

Inhibition of 12- O-tetradecanoylphorbol-13-acetate-induced induction in Epstein-Barr virus early antigen in Raji cells

Retinol, 5 flavonoids, 3 steroids and 7 sweetening agents were studied for their effects on 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced early antigen (EA) of Epstein-Barr virus (EBV) in Raji cells. Concomitant treatment of Raji cells with TPA and retinol showed inhibition of EA induction. Among flavonoids, quercetin resulted in effective inhibition of EA induction by TPA and α-naphthoflavone showed the weakly inhibitory effect. None of the other flavonoids such as rutin, catechin and β-naphthoflavone affected the induction of EBV-EA by TPA. β-Estradiol obviously inhibited EBV-EA induction by TPA, but hydrocortisone did not show any inhibitory effect on it. Glycyrrhetinic acid, steviol, phyllodulcin and perrillartine also showed the remarkable inhibition of EBV-EA induction. On the other hand, glycyrrhizin and stevioside, glycosides of glycyrrhetinic acid and steviol, did not inhibit the induction of EBV-EA by TPA. Some of the inhibitors reported here may be effective on the inhibition of the in vivo tumor promotion by TPA.

Epstein‐Barr virus induction by a serum factor: IV. Ubiquitous occurrence of the factor within vertebrates and its interaction with defined lymphoid cell lines

We have recently reported the existence of a serum factor that induces Epstein-Barr virus antigens in Raji cells (Bauer et al., 1982b, c). Here we demonstrate the ubiquitous presence of this factor in sera of vertebrates of all five classes. Furthermore, we tested 48 EBV-genome-positive lymphoid cell lines of different origins for responsiveness to the serum factor. Only cells from lines of two groups, i.e. Burkitt's lymphoma and EBV-transformed marmoset lines, responded to the factor, whereas human lymphoblastoid cell lines were not induced by the serum factor.

Steroids inhibit tumor promoting agent induced Epstein-Barr virus early antigens in Raji cells.

Four steroids and one protease inhibitor were evaluated for their effects on 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced and Epstein-Barr virus-induced early antigens (EBV-EA) in Raji cells. Continuous treatment with dexamethasone, prednisolone, hydrocortisone and cortisone inhibited TPA-induced EBV-EA to varying degrees, but the protease inhibitor N-alpha-p-tosyl L-lysine chloromethyl ketone-HCl (TLCK) had no significant effect. None of the agents tested inhibited EBV- induced EA. In addition, the effect of the steroids was reversible since the removal of these agents resulted in recovery of the percentage of EBV-EA-positive cells in TPA-treated cultures. These results were in agreement with the in vivo experiments of other investigators, who demonstrated inhibition of tumor promotion with steroids. Since TLCK failed to inhibit TPA-induced EA, it is unlikely that induction of EA by TPA is the result of production of proteases.

The spontaneous and induced synthesis of Epstein-Barr virus antigens in Raji cells immobilized on surface coated with anti-lymphocyte globulin.

Immobilization of Raji cells on surface coated with anti-lymphocyte globulin (ALG) at low cell densities lead to the synthesis of Epstein-Barr virus (EBV) early antigen (EA) in up to 5% of the cells. At higher cell densities the percentage of antigen-positive cells decreased and at confluency no antigen synthesis was observed. Addition of iododeoxyuridine (IdUrd) to low density cultures increased the expression of EA to 20%, whereas in confluent cultures the cells could not be induced to synthesize EA. Treatment of cells in suspension with ALG failed to induced EA synthesis and did not potentiate the effect of IdUrd. Immobilized Raji cells proved to be suitable targets for superinfection with EBV derived from P3HR1 cultures.

Expression of Epstein-Barr-virus-associated membrane antigen in Raji cells superinfected with two different virus strains

Abstract Papain treatment of Epstein-Barr virus (EBV; (P3HR-1 strain)-superinfected Raji cells removed the initially adsorbed membrane antigen (MA) positive material from the cell membranes. MA positive cells appeared again after 20 hr in culture, reaching a maximum level at about 30 hr. Puromycin, cycloheximide, and actinomycin D prevented the appearance of MA, whereas cytosine arabinoside had no effect. These results suggest that MA is synthesized de novo by P3HR-1 virus-infected cells and can be detected after 20 hr. The effects of the different metabolic inhibitors are in line with the concept that MA synthesis is an early function of the viral genome. Parallel EA induction tests showed that papain treatment had no effect on the frequency of EA positive cells compared to buffer-treated cells. A comparison between P3HR-1 and B95-8 virus strains with equal EBNA-inducing capacity showed that B95-8 virus was deficient with regard to its ability to induce MA in Raji cells, as judged by direct immunofluorescence.