The transcription factor Egr1 is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin.

Recent studies are reviewed indicating that the transcription factor early growth response-1 (Egr1) is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin. The downstream pathways of these factors display multiple nodes of interaction with each other, suggesting the existence of a functional network of suppressor factors that serve to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr1 is oncogenic in prostate cancer. In the majority of these cancers, PTEN or p53 is inactive. It is suggested that these defects in the suppressor network allow for the unopposed induction of TGFbeta1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, and explain the role of Egr1 in prostate cancer. Egr1 is a novel and logical target for intervention by gene therapy methods, and targeting methods are discussed.

The early growth response gene EGR-1 behaves as a suppressor gene that is down-regulated independent of ARF/Mdm2 but not p53 alterations in fresh human gliomas.

PURPOSE: EGR-1 is an immediate early gene with diverse functions that include the suppression of growth. EGR-1 is down-regulated many cancer cell types, suggesting a tumor suppressor role, and may critically involve the p53 pathway. The aim of this work was to measure the expression of EGR-1 and the p16/INK4a/ARF-Mdm2-p53 pathway status in fresh human gliomas. EXPERIMENTAL DESIGN: Thirty-one human gliomas with different grades of malignancy were investigated for Egr-1 mRNA and the protein expression, frequency, and spectrum of p53 gene mutations, mdm2 gene amplification, and p16/INK4a/ARF allele loss. RESULTS: The amplification of Mdm2 and the deletion of the p16/INK4a gene was found in 3 and 5 cases, respectively, whereas mutations of p53, including two novel mutations, were observed in 10 other cases. The three types of changes occurred strictly mutually exclusively, emphasizing that these genes operate in a common pathway critical to glioma progression. EGR-1 mRNA was significantly down-regulated in astrocytomas (14.7 +/- 5.1%) and in glioblastomas (33.6 +/- 10.0%) versus normal brain. Overall, EGR-1 mRNA was strongly suppressed (average, 15.2 +/- 13.9%) in 27 of 31 cases (87%), independent of changes in p16/INK4a/ARF and Mdm2; whereas 4 of 31 cases with residual EGR-1 expression as well as the highest EGR-1 variance segregated with p53 mutations. Immunohistochemical analyses confirmed the suppression of EGR-1 protein. CONCLUSIONS: These results indicate that EGR-1 is commonly suppressed in gliomas independent of p16/INK4a/ARF and Mdm2 and that suppression is less crucial in tumors bearing p53 mutations, and these results implicate an EGR-1 growth regulatory mechanism as a target of inactivation during tumor progression.

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.

Cavernous angiomas of the nervous system in Italy: clinical and genetic study.

We performed a clinical and genetic study of patients affected by cavernous angiomas (CA) of the nervous system. We examined initial signs and symptoms in sporadic and familial cases. We obtained clinical, neuroimaging and genetic data on 15 Italian patients with CA of the nervous system with positive, doubtful or apparently negative family history. Genetic markers surrounding three different gene regions (7q, 3q and 7p) were analysed. In one small family, genetic linkage was consistent with all chromosome loci. In another family with the unusual association of cerebral and spinal CA, linkage with chromosome 7q and, likely, 7p was excluded, while linkage with locus 3q was possible. Our results indicate that Italian families with CA may show genetic heterogeneity. Non-specific and subtle onset symptoms hide the presence of CA within families. Patients with multiple CA may have silent cerebral lesions confirming the low penetrance of clinical signs in spite of radiological ones.

Expression of metabotropic glutamate receptors in murine thymocytes and thymic stromal cells.

RT-PCR combined with immunoblotting showed the expression of group-I (mGlu1 and 5) and group-II (mGlu2 and 3) metabotropic glutamate receptors in whole mouse thymus, isolated thymocytes and TC-1S thymic stromal cell line. Cytofluorimetric analysis showed that mGlu-5 receptors were absent in CD4(-)/CD8(-) but present in more mature CD4(+) CD8(+) and CD4(+)CD8(-) thymocytes. mGlu-1a receptors showed an opposite pattern of expression with respect to mGlu5, whereas mGlu2/3 receptor expression did not differ between double negative and double positive cells. mGlu receptors expressed in both thymic cell components were functional, as indicated by measurements of polyphosphoinositide hydrolysis or cAMP formation. These data suggest a possible role for mGlu receptor signalling in the thymus.

Wilms' tumor gene expression by normal and malignant human B lymphocytes.

Very little is known about Wilms' tumor gene (WT1) expression in B cells and its importance for growth regulation and differentiation. We have investigated WT1 expression in fresh B lymphocytes and in a panel of B-cell lines of normal and malignant origin, including both Epstein-Barr virus (EBV) genome negative and EBV carrying cell lines. WT1 is constitutively activated in all lymphoblastoid cell lines (LCL) derived from EBV immortalization of lymphocytes from normal donors in vitro. These cell lines are distinguished for the presence of activated B-cell markers and an unrestricted expression of viral latent genes. In contrast, WT1 expression is abrogated in normal B lymphocytes and in all Burkitt tumor derived cell lines, irrespective of the EBV genome carrying status and their phenotype pattern. A single step RT-PCR for simultaneous detection of the four spliced transcript isoforms has been applied to confirm their expression. Analysis of variant relative proportions suggested the maintenance of a balanced expression of the isoforms in LCL, as reported in non tumorous tissues. These data, together with the evidence that the replication in vitro of lymphoblastoid cells is not affected by WT1 activation following viral immortalization, support the hypothesis that gene inactivation, in addition to disrupted alternate splicing, may play a role in growth control derangements.

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).

The BFRF1 gene of Epstein-Barr virus encodes a novel protein.

Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are approximately 100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in the BamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription of BFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.

Human herpes virus-6 seroprevalence and leukaemias: a case-control study. GIMEMA (Gruppo Italiano Malattie Ematologiche dell' Adulto).

The relationships between acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL), chronic myeloid leukaemia (CML) and refractory anaemia with excess of blasts (RAEB) and human herpes virus (HHV)-6 antibody level were investigated in a multicentre case-control study. An association between increased HHV-6 seropositivity and geometric mean titre ratio with AML was shown: P for trend = 0.022, adjusted odds ratio 1.20, 95% confidence interval 1.07-1.33 respectively. No association was found between HHV-6 and ALL, CML or RAEB.

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.

Transcription of latent and replicative Epstein-Barr-virus genes in bone-marrow and peripheral-blood mononuclear cells of healthy donors.

Reverse-transcriptase polymerase chain reaction has been used to analyze the expression of 2 latent genes (EBNA-1 and LMP-1) and one replicative gene (BZLF-1) of Epstein-Barr virus in mononuclear cells from bone marrow and peripheral blood of healthy donors. EBV-gene transcription was detected in 8 out of 15 bone-marrow samples. Among these, 5 allowed the detection of latency-associated transcripts in the absence of BZLF-1 expression. Only one sample showed positivity for expression of both latent and lytic genes. In 2 cases, BZLF-1 was the only transcript detected. In peripheral blood, 4 out of 7 samples showed evidence of EBNA-1 transcription; LMP-1 was expressed in 5 samples, and in 2 cases concomitant expression of EBNA-1 and BZLF-1 was detected. These results provide a direct demonstration by RT-PCR of EBV-gene transcription in bone-marrow-resident viral infected cells and suggest, in contrast to previous studies on peripheral blood, that LMP-1 and BZLF-1 are frequently transcribed also in absence of EBV-related disease. The heterogeneous viral gene expression found makes it difficult to define a pattern of viral latency in vivo which coincides with that described for lymphoblastoid or Burkitt's-lymphoma cell lines at different stages of differentiation.

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.

EGR-1, the reluctant suppression factor: EGR-1 is known to function in the regulation of growth, differentiation, and also has significant tumor suppressor activity and a mechanism involving the induction of TGF-beta1 is postulated to account for this suppressor activity.

The transcription factor EGR-1 is a potential regulator of over 30 genes and plays a role in growth, development, and differentiation and, in addition, has significant transformation suppression activity. The regulatory properties are reviewed and a hypothesis for the transformation suppression activity is proposed. EGR-1 contains three "zinc-finger" motifs in the C-terminal portion of the molecule that constitute the DNA-binding domain and interact with the promoters by virtue of two classes of GC-rich elements: single GC-elements (GCEs) with the consensus 5'-T-G-C-G-T/g-G/A-G-G-C/a/t-G-G/T-3' and overlapping sites consisting of an Sp-1 binding site and the GCE consensus or close homolog of these sequences. The Wilm's tumor suppressor gene product WT1 interacts with the same GCE and, owing in part to four alternate splice products, interacts with a broader range of GC-rich elements with the consensus 5'-GNGNGGGNG-3' and 5'-TCCTCCTCCTCCTC-3'. WT1 commonly but not invariably acts as repressor of transcription, whereas EGR-1, in the absence of overlapping Sp-1 binding sequences, is often an activator. The well-known rapid response of the EGR-1 gene following mitogenic stimulation together with the occurrence of GCEs in the promoters of many growth factors and protooncogenes suggests a role of EGR-1 in growth. Moreover, EGR-1 is constitutively expressed in several viral-transformed systems. On the other hand, studies of model and human tumor lines reveal that EGR-1 has significant growth and transformation suppression roles. Recent studies show that this effect can be accounted for by the ability of EGR-1 to induce the expression and secretion of TGF-beta1, a potent growth suppressor of many cell types, by binding to a single GCE of the TGF-beta1 promoter. Although the effects of EGR- at overlapping Sp1/EGR-1 DNA binding sites are not predictable, known cases fall into two loose groups. Sp1 is usually activating and increasing concentrations of EGR-1 lead to displacement that results in either inhibition of transactivation or EGR-1-dependent transactivation. Moreover, recent studies suggest that displaced Sp1 binds to and activates the endogenous Egr-1 gene, thereby leading to "facilitated inhibition" of Sp1 function by the resulting increased EGR-1. This effect may augment the growth suppressive function of EGR-1 based on induction of TGF-beta1.

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.

Epstein-Barr virus DNA is present both in CD10/CD77 positive and negative subsets of human tonsillar lymphocytes.

We report the fractionation of freshly isolated subsets of tonsillar lymphocytes according to cell density and double sorting for the differential expression of CD10 and CD77, and their analysis for the presence of Epstein Barr virus genome by nested PCR. All the subsets of tonsillar lymphocytes gave unequivocal evidence for the presence of EBV DNA, when obtained from EBV seropositive individuals. Confirmation of all cases examined demonstrates that B lymphocytes from the germinal centers of tonsils are also involved in carrying the EBV infection in vivo.

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.

The transcriptional factor Egr-1 is synthesized by baculovirus-infected insect cells in an active, DNA-binding form.

The Egr-1 (zfp-6) gene encodes a zinc-finger-containing nuclear protein that is rapidly and transiently induced in quiescent cells treated with mitogens. We have constructed baculovirus vectors that synthesize mouse Egr-1 protein initiating at two putative ATG start sites. The ATG site producing the larger protein (Mr, 80,000) is similar, if not identical, to Egr-1 synthesized by serum-stimulated quiescent mouse fibroblasts, thus identifying the likely site for translation. The protein synthesized by the insect cells is active as assayed by its ability to bind to a specific DNA sequence that has been identified as an Egr-1 binding site. The insect cell system will allow further studies of the structure and function of the Egr-1 product, a protein that appears to be an important "master switch" for other genes.

Epstein-Barr virus DNA sequences in precursor monocyte-macrophage cell lines established from the bone marrow of children with maturation defects of haematopoiesis.

Epstein-Barr virus (EBV) DNA sequences were detected in four established monoblast or early monocytic cell lines (CM-S, ROV-S, CV-S and AD-S) obtained from bone marrow of children suffering from maturation defects of haematopoiesis. EBV is present in these cells in a latent state. The viral DNA in these cell lines was analysed by Southern blot hybridization, using a set of cloned EBV DNA fragments from the EBV strain B95-8 as probes. A common spectrum of highly related but distinguishable EBV DNA restriction enzyme sequences was found, suggesting some genomic diversity. Propagation of the cells in long-term culture revealed a gradual decrease of EBV copies per cell in all lines with some minor changes in the restriction pattern of the EBV DNA. These findings demonstrate that human precursor monocyte cells may be susceptible to infection by EBV.

Imbalance of the Epstein-Barr virus-host relationship in AIDS-related complex patients.

Patients affected by AIDS related-complex (ARC) showed several immunological abnormalities that could lead to a disregulation of immunosurveillance against viral latent infections. We report Epstein-Barr virus (EBV) reactivation was found in seven of eight ARC patients and in two of seven affected by persistent generalized lymphoadenopathy. These patients showed either elevated levels of circulating EBV-positive transformed cells and/or depressed EBV-specific T cell cytotoxicity as assessed by the regression assay. Natural killer cell activity was found to be decreased and correlated with evidence of circulating EBV-infected cells and with impaired EBV-specific immune control. Our data demonstrate that loss of control of EBV latency in the infected host by specific immune mechanisms increases the risk for EBV reactivation and emergence of clones with unlimited growth potential. A role of EBV as a cofactor in the development of ARC is suggested.