Expression of Egr-1 correlates with the transformed phenotype and the type of viral latency in EBV genome positive lymphoid cell lines.

In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines, including both EBV genome negative and EBV carrying cell lines. Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines. Thus, constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines. In contrast, Egr-1 expression is abrogated in group I Burkitt tumor cells, irrespective of the EBV genome carrying status. Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression. Several forms of EGR-1 protein are found within the different groups of cell lines, and the binding activity to DNA consensus sequences was investigated. Finally, time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte.

Regulation of G protein-coupled receptor kinase subtypes by calcium sensor proteins.

G protein-coupled receptor homologous desensitization is intrinsically related to the function of a class of S/T kinases named G protein-coupled receptor kinases (GRK). The GRK family is composed of six cloned members, named GRK1 to 6. Studies from different laboratories have demonstrated that different calcium sensor proteins (CSP) can selectively regulate the activity of GRK subtypes. In the presence of calcium, rhodopsin kinase (GRK1) is inhibited by the photoreceptor-specific CSP recoverin through direct binding. Several other recoverin homologues (including NCS 1, VILIP 1 and hippocalcin) are also able to inhibit GRK1. The ubiquitous calcium-binding protein calmodulin (CaM) can inhibit GRK5 with a high affinity (IC(50)=40-50 nM). A direct interaction between GRK5 and Ca(2+)/CaM was documented and this binding does not influence the catalytic activity of the kinase, but rather reduced GRK5 binding to the membrane. These studies suggest that CSP act as functional analogues in mediating the regulation of different GRK subtypes by Ca(2+). This mechanism is, however, highly selective with respect to the GRK subtypes: while GRK1, but not GRK2 and GRK5, is regulated by recoverin and other NCS, GRK4, 5 and 6, that belong to the GRK4 subfamily, are potently inhibited by CaM, which had little or no effect on members of other GRK subfamilies.

Selective induction of allostimulatory capacity after 5-azaC treatment of EBV carrying but not EBV negative Burkitt lymphoma cell lines.

Epstein-Barr virus (EBV) negative and EBV carrying Burkitt lymphoma (BL) lines that remain phenotypically similar to the in vivo tumor cells (operationally defined group I BLs) express high levels of CD10 and CD77, and lack immunoblastic markers such as CD23 and CD39, and the cell adhesion molecules CD11a, CD18, CD54 and CD58. This cell phenotype is associated with poor stimulatory capacity in allogeneic mixed lymphocytes cultures (MLC) [Avila-Carino et al. Int. J. Cancer 40, 691-697 (1987)] EBV carrying BL lines tend to drift spontaneously towards an immunoblastic phenotype in parallel with up-regulation of six EBV-encoded nuclear antigens (EBNA-2 to -6) and two membrane proteins (LMP-1 and -2). These viral antigens are characteristically expressed in all EBV transformed lymphoblastoid cell lines (LCLs) of normal B cell origin and can be induced in group I BL lines by treatment with the DNA demethylating agent 5-azacytidine (5-azaC) [Masucci et al. J. Virol. 65, 1558-1567 (1989)]. We have now studied the effect of 5-azaC on the induction of allogenic T cell proliferation by three EBV negative (Ramos, BL28 and BL41) and four EBV carrying BL lines (Rael, Eli, Chep and Mutu) which stably express a group I phenotype. Pre-treatment with 4-15 microM 5-azaC had no effect on the EBV negative cells but increased the stimulatory capacity of all four EBV carrying lines. LMP-1 was the only viral antigen regularly induced suggesting that its expression may be required for the increase of allostimulation. This was corroborated by the observation that LMP-1 transfection increased 35-70-fold the stimulatory capacity of Rael cells. The cell adhesion molecule CD54 was the only cellular marker selectively up-regulated in all cell lines with increased stimulatory capacity.

Continuous subcutaneous infusion of apomorphine rescues nigro-striatal dopaminergic terminals following MPTP injection in mice.

Apomorphine has been introduced in the treatment of late-stage Parkinson's Disease (PD). The disadvantage of a short half-life of apomorphine is now overcome by the use of a continuous subcutaneous (s.c.) self-delivering system. We examined whether continuous s.c. infusion of apomorphine rescues nigro-striatal dopaminergic neurons from toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Apomorphine was continuously infused in mice by means of a s.c. minipump that delivered the drug at a rate of 0.5 or 3.15 mg/kg/day. MPTP induced a >80% reduction in striatal dopamine (DA) after one day. DA levels were still substantially reduced one month following MPTP injection, in spite of a partial recovery. Similarly, striatal immunoreactivity for tyrosine hydroxylase and dopamine transporter was markedly reduced at this time interval. Continuous s.c. infusion of apomorphine starting 40 h following MPTP injection rescued striatal dopaminergic terminals, as assessed by measurements of DA and its metabolites, as well as TH and DAT immunostaining after one month. The neurorescuing effect was more remarkable at a delivery rate of 3.15 mg/kg/day of apomorphine. In contrast, no rescue was observed when apomorphine was administered as a single daily s.c. bolus of 1 or 5mg/kg starting 40 h following MPTP. We conclude that apomorphine is able to rescue nigro-striatal dopaminergic neurons when continuously delivered at doses that are comparable to those delivered by minipumps in PD patients. These results suggest that continuous s.c. infusion of apomorphine not only relieves the symptoms, but also reduce the ongoing degeneration of nigro-striatal dopaminergic neurons in PD patients.

The E27 beta2-adrenergic receptor polymorphism reduces the risk of myocardial infarction in dyslipidemic young males.

In the present study we evaluated whether two polymorphisms of beta2-adrenergic receptors (beta2-AR) gene (R16G and Q27E) could modify the risk of myocardial infarction (MI). Using a case-control design, we analyzed the data from 125 male patients who had experienced a first episode of MI before the age of 45 years and 108 male controls matched for age. The allele frequencies for R16G and Q27E were: G16=0.56 and E27=0.36 in patients with MI and G16=0.61 and E27=0.42 in the control group. There was a trend (not statistically significant) of decreasing MI risk according to E27 or G16 alleles. Combined effect between E27 allele and history of dyslipidemia has been observed. Whereas dyslipidemia conferred a relative risk of MI of 4.8 (P<0.001) compared with normolipidemia in the entire study population, the relative risk increased to 9.0 (P<0.001) in Q27 homozygotes with dyslipidemia, and decreased to 1.8 (P=0.36) in E27 homozygotes. Our results show that the E27 allele of the beta2-adrenergic receptor has a significant protective effect on MI in dyslipidemic young male.

Human herpesvirus 6 variant A, but not variant B, infects EBV-positive B lymphoid cells, activating the latent EBV genome through a BZLF-1-dependent mechanism.

Human herpesvirus 6, a predominantly T lymphotropic virus, has been recently shown to infect some EBV-positive B cell lines, and to induce in them the activation of the EBV lytic cycle. Here we have confirmed and extended such observations, showing that (1) this phenomenon is restricted to the variant A of HHV-6: in fact two isolates belonging to the HHV-6 variant B (BA92 and Z29) were neither able to infect any B cell line, independently of the EBV status, nor to induce the EBV genome expression. The only exception is represented by the P3HR1 cells, in which, however, the infection by the variant B does not determine induction of EBV antigens; (2) the presence of the EBV genome contributes to the susceptibility of the B cell lines to HHV-6 infection, increasing the binding sites and the percentage of infectable cells, as detected by immunoelectron microscopy; and (3) HHV-6 infected T cells, transfected with plasmids bearing the promoter regions of the EBV early genes BZLF1 and BMRF1, show a strong transactivation of these promoters.

Expression of neurotrophin receptors in normal and malignant B lymphocytes.

In order to define a cellular model suitable for studying, in vitro, the molecular properties and functions of neurotrophin receptors in human lymphocytes, TrkA, TrkB, TrkC and p75(NTR) expression was investigated in a panel of EBV immortalized lymphoblastoid (LCL) and Burkitt lymphoma-derived cell lines (BLs) compared to primary B lymphocytes by RT-PCR and flow cytometric analysis. Our data show that trkA and trkB are transcribed in most B cell lines of normal and malignant origin. For several of them, we also gained first evidence of trkC expression in B cells. All cell lines and primary B cells lack p75(NTR) expression. These data suggest that neurotrophin receptors expression in the B cell lines correlates to some extent with the phenotypic maturation stage and endogenous viral activity levels. Our data suggest that TrkA and TrkB, once activated, provide a partial rescue from apoptosis, whereas TrkC stimulates the progression through the cell cycle without affecting cell survival. Finally, the identification of a number of cell lines showing single expression of one of the Trk receptors has disclosed the availability of a cellular tool for further studies on their function, and mechanisms of signal transduction in the B cell moiety in the absence of p75(NTR).

Stimulation with allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines generates HLA class I-specific CTLs with different target cell avidity.

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) are potent inducers of cytotoxic T-lymphocytes (CTL) in allogeneic mixed lymphocyte cultures (MLC). The contribution of EBV antigens to the induction of cytotoxic responses was investigated by comparing CTL clones derived from allogeneic MLCs of lymphocytes from one EBV seropositive and one seronegative donor for their capacity to lyse paired EBV positive and negative targets. The majority of the clones showed a conventional "HLA-specific" cytotoxicity and lysed equally well HLA-matched LCLs and mitogen-induced T- or B-blasts. A minority of the clones from both donors exhibited an "LCL-selective" killing potential as they lysed poorly T- and B-blasts. The LCL-selective clones did not recognize EBV antigens because they could not discriminate between EBV negative Burkitt lymphoma (BL) lines and their in vitro EBV-converted sublines. MAbs to CD3, CD8, and MHC class I antigens blocked the lysis of LCLs by HLA-specific and LCL-selective CTLs with comparable efficiency suggesting that the two effector types express T-cell receptors of similar affinity. T-blasts were unable to inhibit the lysis of LCLs in cross competition assays. This correlated with a significantly lower expression of the cell adhesion molecules ICAM-1 and LFA-3. The results suggest that stimulation with allogeneic LCLs activates HLA class I-specific CTLs with variable target cell avidity. Only CTLs that act independently of the enhancing effect of cell adhesion molecules are able to lyse mitogen-induced T- and B-blasts.

Expression of the Epstein-Barr virus (EBV)-encoded membrane protein LMP1 impairs the in vitro growth, clonability and tumorigenicity of an EBV-negative Burkitt lymphoma line.

In a previous study on several independently established Epstein-Barr virus (EBV)-converted sublines of the EBV-negative Burkitt lymphoma (BL) line BL41, we found that expression of the virally encoded membrane protein LMP1 was accompanied by reduced agarose clonability and tumorigenicity. In order to investigate whether LMP1 can induce these phenotypic changes by itself, we have now studied the growth in suspension culture, the clonability in agarose and the tumorigenicity in immunosuppressed and SCID mice of 4 LMP1-transfected sublines of BL41 that carry the gene under the control of the ZnSO4-inducible metallothionein promoter. Expression of LMP1 at levels comparable to those detected in EBV-transformed lymphoblastoid cell lines (LCL) correlated with impairment of growth in suspension and reduction of clonability and tumorigenicity. Only minor changes were observed in transfectants expressing low LMP1 levels. Up-regulation of LMP1 by ZnSO4 treatment of the low LMP1 clone MTLM5 was accompanied by a slowing down of proliferation, increased cell clumping and decreased clonability. The results suggest that expression of LMP1 at levels which are compatible with immortalization of normal B-cells antagonizes the ability of BL cells to grow in vitro and in vivo, and illustrate a possible mechanism by which down-regulation of this viral antigen may favor tumorigenicity in EBV-carrying BLs.

Differential regulation of Epstein-Barr virus (EBV) latent gene expression in Burkitt lymphoma cells infected with a recombinant EBV strain.

Epstein-Barr virus (EBV)-negative Burkitt lymphomas (BLs) can be infected in vitro with prototype EBV strains to study how the virus may affect the phenotype of tumor cells. Studies thus far have concentrated on the use of transforming B95-8 and nontransforming P3HR1 strains. Immunological and phenotypic differences between the sublines infected with these two strains were reported. The majority of these differences, if not all, can be attributed to the lack of EBNA-2 coding sequences in the P3HR1 strain. The recent development of a selectable Akata strain has opened up new possibilities for infecting epithelial and T cells as well. We infected five EBV-negative BL lines with the recombinant Akata virus. Our results indicate that the infected cell lines BL28, Ramos, and DG75 express EBNA-1, EBNA-2, and LMP1, the viral proteins associated with type III latency, and use both YUK and QUK splices. In contrast, two EBV-negative variants of Akata and Mutu when reinfected displayed restricted type I latency and expressed only EBNA-1. All clones of infected Mutu cells used the QUK splice exclusively. The usage of Qp was observed in a majority of Akata clones. Some Akata clones, however, were found to have double promoter usage (Qp and C/Wp) but at 4 months after infection did not express EBNA-2. The results demonstrate differential regulation of EBV latency in BLs with the same recombinant viral strain and suggest that the choice of latency type may be cell dependent. The restricted latency observed for infected Akata and Mutu cells indicates that a BL may opt for type I latency in the absence of immune pressure as well.

Expression of the Epstein-Barr virus (EBV)-encoded membrane antigen (LMP) increases the stimulatory capacity of EBV-negative B lymphoma lines in allogeneic mixed lymphocyte cultures.

Epstein-Barr virus (EBV)-negative Burkitt lymphoma (BL) lines are poor stimulators in allogeneic mixed lymphocyte cultures compared to EBV-transformed lymphoblastoid cell lines derived from the same individuals. We have previously shown that the stimulatory capacity of the tumor cells is increased after EBV conversion (Avila-Carino et al., Int. J. Cancer 1987. 40: 691). As a first step towards the identification of the viral gene product responsible for this change we have studied the influence of the EBV latent membrane protein (LMP) on the stimulatory capacity of the EBV-negative BL lines BL41 and DG75 and the B lymphoma line BJAB. Four LMP-transfected sublines of BL41, four DG75 LMP transfectants and one LMP-transfected subline of BJAB showed a significantly stronger stimulatory capacity than the original line. The effect was directly proportional to the amount of LMP detected in each transfectant but was not due to reactivation of LMP-specific memory cells since lymphocytes from EBV-seropositive and -seronegative individuals responded equally. In order to define the relation between LMP expression and induction of stimulatory capacity, DG75 was transfected with constructs containing the LMP gene under the control of an heat-shock promoter. The peak of LMP expression in heat shock-treated cells preceded the appearance of stimulatory capacity by 6-12 h suggesting that critical amounts of the protein may be required to induce the phenotypic change recognized by the T cells. LMP influenced in a dose-dependent manner the expression of the adhesion molecules LFA-1, LFA-3 and ICAM-1 and B cell activation markers CD23 and CD39 in transfected sublines of BL41, but did not affect the expression of these markers in the DG75 and BJAB cell line. All LMP-expressing transfectants showed an increased capacity to form conjugates with unprimed allogeneic lymphocytes.

Over-expression of C-myc increases the sensitivity of Epstein-Barr virus immortalized lymphoblastoid cells to non-MHC-restricted cytotoxicity.

Epstein-Barr virus (EBV)-carrying Burkitt lymphoma (BL) lines which maintain the phenotypic characteristics of the in vivo tumor cells are more sensitive to natural (NK), interferon-activated (IAK) and IL-2-activated (LAK) cytotoxicity than EBV-immortalized lymphoblastoid cell lines (LCL) of normal B-cell origin. All BL cells carry chromosomal translocations which lead to deregulated expression of the c-myc oncogene. LCLs transfected with constitutively active c-myc alleles display changes in growth properties and surface phenotype. In this study, we have examined the effect of c-myc deregulation on the sensitivity of LCLs to NK, IAK and LAK effectors. C-myc-transfected LCLs showed an increased sensitivity to lysis which correlated with the level of c-myc expression. Expression of HLA class I and sensitivity to allospecific and EBV-specific cytotoxic T-lymphocytes (CTL) remained unchanged. Transfection of a constitutively active v-H-ras gene, which also induces changes in growth properties and cell-surface phenotype, did not alter the sensitivity of LCLs to NK or LAK cytotoxicity.

Antigen processing and presentation by EBV-carrying cell lines: cell-phenotype dependence and influence of the EBV-encoded LMP1.

Burkitt's-lymphoma (BL) lines which have maintained in vitro the tumor-cell phenotype (group-I BLs) are poor antigen-presenting cells (APC), in spite of a relatively high surface expression of MHC class II. In order to investigate the mechanism of this deficiency, we have compared group-I BL lines, their sub-lines which have progressed in vitro towards an LCL-like phenotype (group-III BLs), and EBV-transformed lymphoblastoid cell lines (LCLs), for their ability to bind and process tetanus toxoid (TT). The uptake and internalization of 125I-labelled TT was equivalent in the 3 cell types. Only LCLs and group-III BL lines were able to process the TT, as shown by the identification of discrete proteolytic products after separation of whole-cell extracts in tricine-SDS-polyacrylamide gels, and by the recovery of TCA-soluble radioactivity in the culture supernatant. Processing of TT was induced by expression of the EBV-encoded membrane protein LMP 1 in transfected group-1 BLs. The present findings suggest that the inability of group-1 BLs to act as APC is due to their failure to process exogenous antigens. This function appears to be related to phenotypic properties that can be modulated by the expression of LMP1.

Integration of a short Epstein-Barr virus DNA fragment in a B95-8 virus converted Burkitt lymphoma line expressing Epstein-Barr nuclear antigens EBNA2 and EBNA5.

We have analysed the expression of transformation-associated viral antigens, the Epstein-Barr virus (EBV) DNA content and the phenotypic characteristics of two B95-8 virus-converted sublines of the EBV-negative Burkitt's lymphoma (BL) line BL28. The converted lines called E95A-BL28 and E95B-BL28, respectively, differed in their EBV gene expression. The E95B convertant expressed virus-encoded nuclear antigens EBNA1 to -6 and the membrane protein LMP1, but only EBNA2 and EBNA5 were detected by immunofluorescence and immunoblotting in the E95A convertant. Only the entire BamHI W, Y and H regions could be detected in the E95A convertant by hybridization of Southern blots with probes covering the BamHI C, W, Y, H, F, E, K and Nhet regions of the EBV genome. EBV episomes were found to be absent in the E95A convertant as seen by Gardella gels. The E95A convertant retained the phenotypic characteristics of the EBV-negative parental line, and remained highly clonable in agarose. In contrast, expression of EBNA1 to -6 and LMP1 was accompanied by a shift towards a more lymphoblastoid cell line-like phenotype and by loss of agarose clonability in the E95B convertant.

Augmentation of leukocyte infiltration in murine tumors expressing B-cell derived but not nasopharyngeal carcinoma derived EBV membrane protein LMP1.

The Epstein-Barr virus (EBV) encoded latent membrane protein of B cell origin, B-LMP1 (B95-8 prototype) and nasopharyngeal carcinoma (NPC) derived C-LMP1 (CAO prototype) were transfected individually in S6C adenocarcinoma cells of ACA (H-2f) origin. We have shown previously that inoculation of B-LMP1 expressing S6C cells led to tumor rejection in pre-immunized, immunocompetent syngeneic ACA mice, whereas the C-LMP1 transfectants were not immunogenic. Furthermore, B-LMP1 but not C-LMP1 expressing S6C cells grew with necrosis and extensive skin damage in non-immunized mice. A study was carried out to determine whether the in vivo growth pattern of S6C cells expressing two different LMP1 isolates could be correlated to any immunomodulatory mechanism. An increased infiltration of CD45+ leukocytes was found in B-LMP1 expressing S6C tumors originating in non-immunized, syngeneic ACA mice. The C-LMP1 expressors, vector transfectants and untransfected parental tumors had significantly lower number of infiltrating leukocytes. The immunoaccessory molecules ICAM-1, B7-1 and MHC Class I and II expression was unaltered in both B- and C-LMP1 transfectants. The data suggest that B-LMP1 but not C-LMP1 induce anti-tumor immune response.

Upregulation of Epstein-Barr virus-encoded latent membrane protein by human herpesvirus 6 superinfection of EBV-carrying Burkitt lymphoma cells.

The effect of HHV-6 strain A infection on the expression of Epstein-Barr virus- (EBV-) encoded growth transformation-associated genes in two EBV-positive Burkitt lymphoma cell lines, Akata and P3HR-3, was investigated. The results indicate that HHV-6A upregulates the expression of the latent membrane protein LMP-1 in both cell lines. Expression of EBNA-2 was also upregulated in Akata cells following HHV-6A infection. Transfection of reporter constructs carrying the LMP-1 regulatory sequences (LRS; -634/+40) or its 5' deleted derivatives in Akata and in a T-lymphoblastoid cell line, J-Jhan, confirmed the presence of positive and negative regulatory elements responsive to HHV-6A infection in LMP-1 regulatory sequence (LRS). The majority of LRS constructs were under the influence of dominant negative factors. HHV-6A was able to override the effect of such factors acting on reporter plasmids containing the -634/-54, -324/-54, -214/-54, and -106/-54 parts of LRS. The plasmid that carried only the -54/+40 LRS region was constitutively active in both Akata and J-Jhan cells; in Akata, its activity was influenced by HHV-6A. The finding that HHV-6A infection may activate LMP-1 and EBNA-2 expression, which is essential for the immortalization of B-lymphocytes by EBV, shows a novel aspect of the interaction between these two herpesviruses.

Human herpesvirus 6 infection in neoplastic and normal brain tissue.

The presence and variant distribution of human herpesvirus 6 (HHV-6) was investigated by a nested polymerase chain reaction (PCR) in 118 biopsies from patients affected by nervous tissue tumor (115 primary tumors and 3 metastasis) and in 31 autopsy samples from the brain of healthy individuals. HHV-6 DNA sequences were detected in normal and neoplastic nervous tissue at a frequency of 32% and 37%, respectively. In both tissues, variant A was three times more frequent than the variant B. Peripheral blood lymphocytes (PBLs) derived from seven tumor affected patients contained the same variant as their respective brain sample, as judged by PCR. The expression of HHV-6 encoded immediate early protein p41 was detected by immunohistochemistry in neoplastic but not in normal brain. This may reflect viral reactivation from latency in immunocompromised patients. The seroepidemiological data indicated a frequency distribution of anti-HHV-6 antibodies in patients with brain tumors similar to that found in healthy donors.

HHV-6 infection in Italy: characterization of an endemic isolate and seroepidemiologic analysis.

A biologic, immunologic and molecular characterization of an HHV-6 isolate (BA92) rescued by the peripheral blood mononuclear cells of a child affected by Exanthem subitum is reported. The comparison with the known HHV-6 prototype strains showed that BA92 is indistinguishable from the Z29 isolate, and can be included in the variant B group of HHV-6. A seroepidemiologic analysis of the antibody response to BA92 of normal individuals as well as patients affected by diseases potentially associated to HHV-6 infection has shown an overall seroprevalence of 81%, and that no variations in seroprevalence or in antibody geometric mean titer are observed assaying the sera also against G.S., U1102, or Z29 infected cells, respectively. These findings indicate: (1) HHV-6 infection is widely diffuse in Italy; (2) it is not possible to discriminate between the viral variants by the currently available IF assays, and (3) no conclusions can be drawn on the potential association of HHV-6 with any of the diseases examined.