Fifty-five consecutive cases of Hodgkin’s lymphoma (HL), collected between 1996 and 1998 from Cairo, Egypt, were histologically subtyped, phenotyped, and then studied for the presence of Epstein–Barr virus (EBV). We used immunohistochemical stains for EBV latent membrane protein 1 (LMP-1) and in situ hybridization stains for EBV-encoded small RNA (EBER-1) transcripts. Forty-five cases (82%) had classic HL (cHL), and ten cases (18%) had nodular lymphocyte predominant HL (NLPHL), with each group expressing its typical phenotype. LMP-1 stains were positive in 63% and 0% of cHL and NLPHL cases, respectively. EBER-positive Reed–Sternberg cells and variants were also present in 62% and 0% of each group, respectively. The cHL cases showed variable EBER positivity: nodular sclerosis, 58%; mixed cellularity, 100%; lymphocyte depletion, 100%; and unclassifiable, 67%. Our findings are similar to those from other developing countries and point towards a pathogenic role of EBV in cHL.
Classic Hodgkin’s lymphoma; Nodular lymphocyte predominant Hodgkin’s lymphoma; Epstein–Barr virus; EBER-1; LMP-1
Aims—To analyse the latent membrane protein-1 (LMP-1) gene in a series of patients with Epstein-Barr virus (EBV) positive LMP expressing ordinary and HIV associated Hodgkin's disease to detect possible genetic alterations and particularly the existence of deletions near the 3′ end of the gene.
Methods—Expression of the EBV LMP-1 was assessed using immunohistochemistry in 186 cases of Hodgkin's disease and 31 cases of HIV associated Hodgkin's disease. Genomic DNA was extracted from frozen lymph node biopsy specimens from 25 cases of Hodgkin's disease and 11 of HIV associated Hodgkin's disease, all of whom expressed the LMP-1 protein within diagnostic Hodgkin and Reed-Sternberg (HRS) cells, and amplified by polymerase chain reaction (PCR) using primers specific for the different LMP-1 regions.
Results—LMP-1 expression was observed in 106 of 186 Hodgkin's disease cases and in all 31 HIV associated Hodgkin's disease cases. Molecular analysis of the LMP-1 gene showed a high degree of genetic heterogeneity in the carboxy-terminal domain compared with the prototype B95-8 EBV strain, specially in the patients with HIV associated Hodgkin's disease. Variation in the size of the repeated region was found in 17 of 25 Hodgkin's disease and nine of 11 HIV associated Hodgkin's disease cases. Deletions of 30 base pairs near the 3′ end of the gene were detected in all cases of HIV associated Hodgkin's disease and in six Hodgkin's disease. In one case of Hodgkin's disease a larger deletion was observed. In all patients with LMP-1 deletion mutants, 50-90% of the diagnostic HRS cells expressed the LMP-1 protein.
Conclusions—The presence of the 30 base pair deletion in all cases of HIV associated Hodgkin's disease supports previous studies that reported aggressive histological and clinical behaviour in tumours harbouring this deletion. This deletion may prolong the half-life of the protein which would explain the high levels of LMP-1 expressing HRS cells in those cases carrying LMP-1 deletions. That the 30 base pair deletion was present in all of the HIV associated Hodgkin's disease specimens suggests that impairment of immune function is a stringent requirement for the expansion of malignant cells infected by EBV strains containing the deleted LMP-1 gene.
Epstein-Barr virus; latent membrane protein-1; Hodgkin's disease; HIV
Epstein-Barr virus (EBV) infection leads to Hodgkin’s disease (HD) in some immunocompetent hosts. The malignant Reed-Sternberg cells of HD only express a limited array of subdominant EBV antigens to evade preexisting immune responses to EBV. The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed by HD and various EBV-associated malignancies, have been proposed as a potential target for CTL-based therapy. However, the precursor frequency for LMP-specific CTL is generally low in healthy EBV-infected hosts, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and the oncogenic potential. In the present study, we report that transitively expressing an inhibitor of A20, a key negative regulator of inflammatory signaling pathways, together with the LMP antigens (truncated LMP1 and full-length LMP2) greatly enhances maturation and cytokine production of human (h) monocyte-derived dendritic cells (DCs). As a consequence, LMP1/2-expressed, A20-silenced hDCs have an enhanced potency to prime LMP-specific T cell response. When the in vitro primed T cells are adoptively transferred into tumor-xenografted, severe combined immunodeficient (SCID) mice, some of the xenografted tumors approach complete regression. Thus, the study may provide an available resource of LMP-specific T cells for T cell immunotherapy.
A20; dendritic cells; cytotoxic T lymphocyte; Epstein-Barr virus; latent membrane proteins
AIMS: To evaluate the expression of c-myc and bcl-2 oncogene products in Reed-Sternberg cells in Hodgkin's disease, especially in relation to Epstein-Barr virus infection and expression of EBV encoded latent membrane protein (LMP). METHODS: Tissues from 33 cases of Hodgkin's disease were studied for the presence of EBV DNA by polymerase chain reaction (PCR) and DNA in situ hybridisation (DISH), for the presence of EBER-1 and EBER-2 EBV RNA by RNA in situ hybridisation (RISH); and for the presence of LMP, bcl-2, and c-myc proteins by immunohistochemical staining. RESULTS: A substantial number of Reed-Sternberg cells expressed bcl-2 in 20 of 29 (69%) and c-myc in 30 of 32 (94%) Hodgkin's disease samples. In 18 of the 25 (72%) cases Reed-Sternberg cells expressed both oncogene products. Of these 18 cases, 10 (56%) were EBV-PCR positive; eight (44%) were EBV-PCR negative. CONCLUSIONS: Reed-Sternberg cells in Hodgkin's disease frequently express both bcl-2 and c-myc oncogene products, suggesting that these oncogenes may act in concert in the pathogenesis of the disease. Moreover, the expression of c-myc and bcl-2 proteins in Reed-Sternberg cells is independent of EBV and LMP status.
AIMS--To demonstrate Epstein-Barr virus (EBV) encoded nuclear antigen 1 (EBNA-1) gene expression in EBV associated disorders using a new monoclonal antibody (1H4-1) on routinely processed tissues. METHODS--The pressure cooker antigen retrieval method was used for the immunohistochemical demonstration of EBNA-1 gene expression in formalin fixed, EBV positive tissues from Hodgkin's disease, infectious mononucleosis, HIV associated non-Hodgkin's lymphomas, post-transplant lymphomas, and undifferentiated nasopharyngeal carcinoma (NPC). EBV encoded EBNA-2, latent membrane protein 1 (LMP-1) and BZLF-1 gene expression was also examined using commercially available monoclonal antibodies. RESULTS--Of the 34 EBER in situ hybridisation positive cases of Hodgkin's disease examined, none expressed EBNA-1 in the Reed-Sternberg cells. These cells were nevertheless strongly LMP-1 positive in all cases. Strong EBNA-1 staining was seen in all cases of EBER positive HIV associated non-Hodgkin's lymphoma (five of five), nasopharyngeal carcinoma (five of five), infectious mononucleosis (three of three), and post-transplant lymphoma (one of one). These cases also expressed LMP-1, EBNA-2 and BZLF-1, but at differing levels. CONCLUSION--The pressure cooker antigen retrieval procedure is a sensitive and reliable adjunct to immunohistochemistry, especially with antibodies which are otherwise ineffective on routinely processed tissues. The EBNA-1 gene is not expressed at detectable levels in the malignant cells of Hodgkin's disease, but is consistently expressed in other EBV associated disorders. This finding has important implications for the role of EBNA-1 in the biology of EBV.
Up to 40% of Hodgkin lymphoma (HL) cases are associated with the Epstein-Barr virus (EBV). Clonal viral genomes can be found in the HL tumor cells, the Hodgkin Reed-Sternberg cells (HRS). The latent infection results in expression of the viral oncogenes LMP1 and LMP2A which contribute to generate the particular phenotype of the HRS cells. EBV does not only undergo epigenetic changes of its genome during latency, but also induces epigenetic changes in the host genome. The presence of EBV may alter the composition and activity of the immune cells surrounding the HRS cells. EBV favours a Th1 reaction, but this attempt at a cell mediated immune response appears to be ineffective. The presence of EBV in HL is associated with several clinicopathological characteristics: It is more frequent in cases with mixed cellular histology, in males, in children and older adults, and in developing countries, while the young-adult onset HL of nodular sclerosis type in industrialized countries is typically EBV-negative. Countries in the Mediterranean area often show an intermediate epidemiological pattern. Recent studies suggest a genetic predisposition to develop EBV-associated HL. Circulating EBV-DNA may serve as a biomarker to monitor response to therapy, and eventually, EBV will become a target for therapeutic intervention also in HL.
The B-lymphotropic Epstein-Barr virus (EBV) encodes two isoforms of latent membrane protein 2 (LMP2), LMP2A and LMP2B, which are expressed during latency in B cells. The function of LMP2B is largely unknown, whereas LMP2A blocks B-cell receptor (BCR) signaling transduction and induction of lytic EBV infection, thereby promoting B-cell survival. Transfection experiments on LMP2B in EBV-negative B cells and the silencing of LMP2B in EBV-harboring Burkitt's lymphoma-derived Akata cells suggest that LMP2B interferes with the function of LMP2A, but the role of LMP2B in the presence of functional EBV has not been established. Here, LMP2B, LMP2A, or both were overexpressed in EBV-harboring Akata cells to study the function of LMP2B. The overexpression of LMP2B increased the magnitude of EBV switching from its latent to its lytic form upon BCR cross-linking, as indicated by a more-enhanced upregulation and expression of EBV lytic genes and significantly increased production of transforming EBV compared to Akata vector control cells or LMP2A-overexpressing cells. Moreover, LMP2B lowered the degree of BCR cross-linking required to induce lytic EBV infection. Finally, LMP2B colocalized with LMP2A as demonstrated by immunoprecipitation and immunofluorescence and restored calcium mobilization upon BCR cross-linking, a signaling process inhibited by LMP2A. Thus, our findings suggest that LMP2B negatively regulates the function of LMP2A in preventing the switch from latent to lytic EBV replication.
Recent studies have suggested that Epstein-Barr virus (EBV) may play a role in the aetiology of Hodgkin's disease. To determine the role of EBV in childhood Hodgkin's disease in different geographical areas, immunohistochemical staining and in situ hybridisation were used to analyse latent membrane protein 1 (LMP 1) and small nuclear non-transcribed RNAs (EBER-1) respectively. Testing for EBV within the Reed-Sternberg and Hodgkin's cells was carried out in childhood Hodgkin's disease from 10 different countries. The proportion of LMP 1 positive cases varied significantly, being 50% of cases from the United Kingdom (38/75), South Africa (9/18), Egypt (7/14), and Jordan (8/16), 60% from the United Arab Emirates (6/10), 70% from Australia (11/16), 81% from Costa Rica (34/42), 88% from Iran (7/8), 90% from Greece (20/22), and 100% of the 56 cases from Kenya. A sensitive polymerase chain reaction based EBV strain typing technique was established using archival tissues. EBV strain type 1 was shown to be predominant in childhood Hodgkin's disease from the United Kingdom, South Africa, Australia, and Greece. Type 2 was predominant in Egypt. EBV strain types 1 and 2 were both detected in some cases of childhood Hodgkin's disease in the United Kingdom, Costa Rica, and Kenya. The high incidence of EBV and the presence especially in developing countries of dual infection with both strain types 1 and 2 may reflect socioeconomic conditions leading to malnutrition induced immunological impairment. The possibility of HIV infection also needs to be explored.
AIMS--To evaluate whether there is any correlation between the expression of Epstein-Barr virus (EBV) latent membrane protein (LMP) and oncoprotein bcl-2 in the lymph node biopsy specimens of a Chinese patient with EBV-related reactive lymphoproliferation who later developed T cell lymphoma after a short period of time. METHODS--Immunohistochemistry, with a standard alkaline phosphatase antialkaline phosphatase (APAAP) method and New Fuchsin as a chromogen, was used for single staining of bcl-2 or LMP. Double immunostaining combining APAAP and indirect immunofluorescence was performed for dual labelling of LMP and bcl-2. RESULTS--bcl-2 was expressed in 10-30% of cells in the first lymph node biopsy specimen (EBV-associated lymphoproliferative disorder) and 30-50% of cells in the second lymph node biopsy specimen (T cell lymphoma). LMP was expressed in the first biopsy specimen but not in the second. Double immunostaining results showed that around 78% of LMP positive cells were bcl-2 negative and 94% bcl-2 positive cells were LMP negative. Among the very small fraction of LMP and bcl-2 double positive cells, the intensity of bcl-2 staining was heterogeneous and was not always stronger than that observed in LMP negative bcl-2 positive cells. CONCLUSIONS--The expression of bcl-2 protein is independent of LMP protein status in vivo. Several mechanisms may be involved in EBV associated lymphomagenesis, and bcl-2 induction may occur independently of LMP expression.
The latent membrane protein-1 (LMP1) encoded by Epstein-Barr virus (EBV) is an oncoprotein which acts by constitutive activation of various signalling pathways, including NF-κB. In so doing it leads to deregulated cell growth intrinsic to the cancer cell as well as having extrinsic affects upon the tumour microenvironment. These properties and that it is a foreign antigen, lead to the proposition that LMP1 may be a good therapeutic target in the treatment of EBV associated disease. LMP1 is expressed in several EBV-associated malignancies, notably in Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC). However, the viral protein is only detected in approximately 30%-50% of NPC samples, as such its role in carcinogenesis and tumour maintenance can be questioned and thus its relevance as a therapeutic target.
In order to explore if LMP1 has a continuous function in established tumours, its activity was inhibited through expression of a dominant negative LMP1 mutant in tumour cell lines derived from transgenic mice. LMP1 is the tumour predisposing oncogene in two different series of transgenic mice which separately give rise to either B-cell lymphomas or carcinomas. Inhibition of LMP1 activity in the carcinoma cell lines lead to a reduction in clonagenicity and clone viability in all of the cell lines tested, even those with low or below detection levels of LMP1. Inhibition of LMP1 activity in the transgenic B-cell lines was incompatible with growth and survival of the cells and no clones expressing the dominant negative LMP1 mutant could be established.
LMP1 continues to provide a tumour cell growth function in cell lines established from LMP1 transgenic mouse tumours, of both B-cell and epithelial cell origin. LMP1 can perform this function, even when expressed at such low levels as to be undetectable, whereby evidence of its expression can only be inferred by its inhibition being detrimental to the growth of the cell. This raises the possibility that LMP1 still performs a pro-oncogenic function in the 50% to 70% of NPC tumours wherein LMP1 protein expression cannot be detected. This reinforces the basis for pursuing LMP1 as a therapeutic target in EBV associated LMP1-expressing malignancies.
Almost all researchers agree on the lack of Bob-1 expression in Hodgkin/Reed-Sternberg (H/RS) cells in classic Hodgkin lymphoma (CHL), and utilize this marker as a diagnostic tool in conjunction with other markers to differentiate between lymphocyte predominance Hodgkin lymphoma (LPHL) and CHL.
To study the immunohistochemical (IHC) expression of Bob-1 in Egyptian CHL and to correlate this expression with Epstein-Barr virus (EBV) viral load.
Materials and methods
Paraffin sections of randomly selected 18 CHL cases were included: 2 lymphocyte rich (LR), 4 mixed cellularity (MC), 10 nodular sclerosis (NS) and 2 lymphocyte depletion (LD). All cases were immunostained for Bob-1. EBV was evaluated by EBV early RNA transcripts in situ hybridization (EBER ISH) and immunostaining for EBV latent membrane protein-1 (LMP-1).
Sixty seven percent of cases (12/18) were positive for EBV by ISH and/or immunostaining for LMP-1. Moderate to strong nuclear Bob-1 was observed in 94% of cases. The positivity ranged between 25–100%. Bob-1 immunoreactivity was strongly associated with EBV positivity (p < 0.001).
This study proves nuclear IHC expression of Bob-1 on H/RS in CHL implying the difficulties in applying this marker to differentiate between LPHL and CHL. Does this difference between Western and Egyptian CHL reflect genetic and/or environmental factors, or simply no difference exists as most researchers are concentrated on the Western population and no comparative studies have been done. Studies from other countries might answer this question.
We have examined expression of the Epstein–Barr virus (EBV) latent membrane protein-1 (LMP1) in the malignant Hodgkin and Reed–Sternberg (HRS) cells of Hodgkin's disease (HD) and its impact on response to treatment and on survival. Paraffin tissue from 100 adult immunocompetent patients with HD were analysed using immunohistochemistry to identify LMP1 expression. According to the Rye classification, 8% of patients had lymphocyte predominance (LP) subtype, 48% had nodular sclerosis (NS) disease, 37% were of the mixed cellularity (MC) subtype and 7% were of the lymphocyte depletion (LD) subtype. During the five year follow-up period 27 patients died and 74 patients achieved a complete remission. Patients with LD subtype were older (P = 0.03), less frequently achieved complete remission (P = 0.01), had shorter disease-free survival (P = 0.01) and overall survival (P = 0.002) compared with the other subtypes of HD. LMP1 expression was found in the tumour cells of 26% of cases of HD. LMP1 expression was less common in NS disease than in the other subtypes (P = 0.05), whereas an association between MC subtype and LMP1 expression was not found (P = 0.22). Using the log-rank test there were no differences in overall survival or disease-free survival based on EBV status either when all patients were analysed or when LD and LP subtypes were excluded. However, the presence of EBV was associated with significantly longer disease-free survival in the subgroup of patients ≤ 30 years old (P = 0.02) and in those patients ≤ 34 years old (P = 0.05). In contrast, there was a trend for shorter disease-free survival for EBV-positive patients in the subgroup > 35 years old, but this difference was not statistically significant (P = 0.11). A similar trend was observed in patients > 50 years old. Analysis of the impact of LMP1 expression in patients who had different stage and B symptoms status showed that expression of EBV was associated with longer disease-free survival (P = 0.019) in early stage (1 + 2) patients without B symptoms. Significant differences in the other subgroups based on EBV status was not found. Factors adversely affecting the likelihood to achieve a complete remission were: absence of LMP1 expression (OR 6.4, 95% Cl 1.07–38.5, P = 0.04), age (OR 1.68, 95%Cl 1.15–2.5, P = 0.007) and subtype of HD (OR 3.32, 95%Cl 1.11–9.94, P = 0.03). Age and subtype of HD had an independent impact on overall survival (P = 0.01). We conclude that expression of LMP1 in HRS cells has a favourable impact on prognosis for HD patients, but that this effect may be restricted to young adult patients and those with early stage disease. © 2001 Cancer Research Campaign http://www.bjcancer.com
AIMS--To determine if there is an association between Epstein-Barr virus (EBV) infection and Hodgkin's disease. METHODS--Fifty cases of Hodgkin's disease and 25 reactive lymph nodes were screened for the presence of EBV-RNA (EBER) using in situ hybridisation, and for the expression of EBV encoded latent membrane protein 1 (LMP-1) by immunohistochemistry. RESULTS--In 42% of the cases of Hodgkin's disease, EBER was detected in the nuclei of the malignant cells, and in LMP-1 expression was found 36%. Both EBER and LMP-1 positivity were seen in 34% of the cases. An additional finding was the presence of LMP-1 on follicular dendritic cells in residual germinal centres in two cases of Hodgkin's disease. EBER was not detected in these germinal centres. In reactive lymph nodes only occasional EBER positive, small, lymphoid cells were found, without LMP-1 expression. CONCLUSIONS--These results show a strong correlation between the presence of EBER and the LMP-1 expression in the Reed-Sternberg cells. They corroborate a role for EBV in at least some cases of Hodgkin's disease. LMP-1 is probably presented as an immune complex in the germinal centres, as part of an immune response against EBV.
Aims: Epstein-Barr virus (EBV) immortalises B cells in vitro and is associated with several malignancies. Most phenotypic effects of EBV are mediated by latent membrane protein 1 (LMP1), which interacts with tumour necrosis factor receptor associated factors (TRAFs) to activate NF-κB. This study examines TRAF1 and LMP1 expression in EBV associated lymphoproliferations.
Methods: TRAF1 expression was investigated in 26 Hodgkin lymphomas (HL; 18 EBV+, eight EBV−), seven EBV+ Burkitt lymphomas (BL), two infectious mononucleosis (IM) tonsils, and lymphoreticular tissue from eight chronic virus carriers. Seven anaplastic large cell lymphomas and 10 follicular B cell lymphomas were also studied. Colocalisation of TRAF1 and LMP1 was studied by immunofluorescent double labelling and confocal laser microscopy.
Results: TRAF1 colocalises with LMP1 in EBV infected cells in IM. EBV positive lymphocytes from chronic virus carriers were negative for TRAF1 and LMP1. In HL biopsies, TRAF1 was strongly expressed independently of EBV status, whereas all BL cases were TRAF1−. In EBV+ HL cases, TRAF1 colocalised with LMP1. Eight of 10 follicular lymphomas expressed TRAF1 in centroblast-like cells. Four of seven anaplastic large cell lymphomas weakly expressed TRAF1.
Conclusions: These results suggest that in non-neoplastic lymphocytes, TRAF1 expression is dependent on the presence of LMP1, and that in IM B cells in vivo, LMP1 associated signalling pathways are active. In HL, TRAF1 is expressed independently of EBV status, probably because of constitutive NF-κB activation. The function of TRAF1 in HL remains to be determined.
Epstein-Barr virus; tumour necrosis factor receptor associated factor 1; latent membrane protein 1; infectious mononucleosis; Hodgkin lymphoma; Burkitt lymphoma
Background—In vitro the Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) has been shown to upregulate expression of matrix metalloproteinase 9 (MMP-9), a member of a family of zinc dependent endopeptidases that is believed to facilitate tumour invasion and metastasis by degradation of the extracellular matrix.
Aim—To test whether the expression of MMP-9 in Hodgkin's disease correlates with EBV status and survival and to investigate whether LMP-1 expression affects MMP-9 concentrations in the Hodgkin's disease cell line, L428.
Methods—MMP-9 expression was measured by means of immunohistochemistry in a series of Hodgkin's disease tumours and this expression was correlated with EBV status and survival. The influence of LMP-1 on MMP-9 expression was also investigated in the Hodgkin's disease cell line, L428.
Results—MMP-9 expression was demonstrated in the malignant Hodgkin and Reed-Sternberg cells of all (n = 86) formalin fixed, paraffin wax embedded Hodgkin's disease tumours examined. Although the intensity of MMP-9 immunostaining varied between cases, there was no correlation between MMP-9 expression and EBV status or survival. MMP-9 expression was also detected in a variety of non-malignant cells, including fibroblasts. MMP-9 was detected by zymography in the L428 and KMH2 Hodgkin's disease cell lines, whereas low or undetectable amounts of MMP-9 were found in the L591 Hodgkin's disease cell line. Induction of LMP-1 expression in the Hodgkin's disease cell line L428 did not result in a detectable increase in the values of MMP-9 as measured by zymography.
Conclusions—These results demonstrate that MMP-9 is consistently expressed by the Hodgkin and Reed-Sternberg cells of Hodgkin's disease tumours and by the Hodgkin's disease cell lines, L428 and KMH2. However, this expression does not appear to be related either to LMP-1 values or to survival.
matrix metalloproteinase 9; Hodgkin's disease; Epstein-Barr virus; latent membrane protein 1
Epstein-Barr virus (EBV) infection and latency has been associated with malignant diseases including nasopharyngeal carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and immune deficiency associated lymphoproliferative diseases. EBV-encoded latent membrane protein 2A (LMP2A) recruits Lyn and Syk kinases via its SH2-domain binding motifs, and modifies their signaling pathways. LMP2A transgenic mice develop hyperproliferative bone marrow B cells and immature peripheral B cells through modulation of Lyn kinase signaling. LMP2A/λ-MYC double transgenic mice develop splenomegaly and cervical lymphomas starting at 8 weeks of age. We reasoned that targeting Lyn in LMP2A-expressing B cells with dasatinib would provide a therapeutic option for EBV-associated malignancies. Here, we show that dasatinib inhibits B cell colony formation by LMP2A transgenic bone marrow cells, and reverses splenomegaly and tumor growth in both a pre-tumor and a syngeneic tumor transfer model of EBV-associated Burkitt lymphoma. Our data support the idea that dasatinib may prove to be an effective therapeutic molecule for the treatment of EBV-associated malignancies.
Burkitt lymphoma; dasatinib; Epstein-Barr virus (EBV); latent membrane protein 2A (LMP2A); Lyn; post-transplant lymphoproliferative diseases (PTLD)
Nasopharyngeal carcinoma (NPC) is an epithelial malignancy, which commonly occurs in Southern China, Taiwan, North Africa and Southeast Asia. Nasopharyngeal carcinoma is strongly associated with Epstein-Barr virus infection. The p53 tumour suppressor protein is rarely mutated in NPC suggesting that the inactivation of p53 pathway in NPC could be due to the presence of EBV proteins. The aim of this work was to determine the effects of EBV proteins namely LMP1 and LMP2A on the expression levels of p53 protein.
In this work we found that LMP1, but not LMP2A, decreased p53 protein levels. Overexpression of LMP1 resulted in increased ubiquitination of p53 suggesting that the decreased p53 protein levels by LMP1 was due to increased degradation of the protein. The reduction of p53 protein levels was independent of the PI3K-Akt pathway.
LMP1, but not LMP2A, reduced p53 protein levels through the increase in the polyubiquitination of p53 protein and was independent of the PI3K-Akt pathway.
Nasopharyngeal carcinoma (NPC), Epstein-Barr virus, Latent membrane protein, p53 tumour suppressor gene, ubiquitination
We report the case of an Epstein-Barr virus (EBV)- and human immunodeficiency virus-serum negative patient suffering from repeatedly relapsing classical Hodgkin’s Lymphoma (cHL) associated with a histological picture of plasma cell-hyaline vascular (PC-HV) form of Castleman’s disease (CD). The CD30- and CD15- positive, Reed-Sternberg/Hodgkin cells, only occasionally expressed the CD20 molecule, but not leukocyte common antigen and latent membrane protein-1. Single-strand polymerase chain reaction failed to detect human herpesvirus 8 or EBV in the involved tissues. At the time of second relapse in July 2005, the clinical picture was characterized by a palpable right hypogastric mass, disclosed at physical exam, in the absence of other enlarged peripheral lymph nodes, subjective symptoms or laboratory profile alterations. Combined hybrid-(18)F-fluorodeoxyglucose positron emission-computerized tomography (18F-FDG PET/CT) showed increased radionuclide uptake in multiple external iliac lymph nodes [standardized uptake value (SUV) of 7.4] and non-palpable left supraclavicular lymph nodes (SUV of 5.8). Relapsing cHL in the context of mixed PC-HV CD was documented in two of three surgically excised abdominal lymph nodes never previously enlarged or involved by any lymphoproliferative disease. Because of the limited disease extension and failure to induce continuous remission with previous conventional chemoradiotherapy, the patient was treated with six rituximab injections. This immunotherapy induced significant reduction in size of supraclavicular lymph nodes as evident at ultrasound (US) scan (<1 vs. 2.5 cm, post- vs. pretherapy), which was confirmed by the 18F-FDG PET/CT in October 2005, despite no modification in SUV of 4.2. 18F-FDG PET/CT also disclosed no radionuclide uptake by abdominal lymph nodes. Thus, a second course of four additional rituximab injections was given and subsequent 18F-FDG PET/CT indicated persistent, but reduced incorporation of radionuclide compared to the pretherapy value (SUV of 2.7) in the supraclavicular area and confirmed a normal metabolic activity in the iliac external lymph nodes. Because of uncertain persistent disease in the supraclavicular nodal site, involved-field radiotherapy (RT) was delivered in that area as consolidation treatment. After completion of rituximab and RT for 16 and 14 months respectively, US and 18F-FDG PET/CT exams were indicative of complete remission. This case is in concordance with previously published data suggesting that rituximab immunotherapy might be a valid option in the treatment of CD and also have a role in the management of relapsing cHL.
Castleman’s disease; Hodgkin’s lymphoma; rituximab; human herpesvirus-8 infection; interleukine-6
Latent membrane protein (LMP) 1 and LMP2A encoded by Epstein-Barr virus (EBV) are associated with the development of malignancies, but their expression in extranodal NK/T-cell lymphoma, nasal type (ENKTL) and the relationship with clinical characteristics of this disease remain poorly understood. In the present study, we examined the expression of LMP1 and LMP2A in ENKTL, and investigated the correlations between LMP1 and LMP2A expression with clinicopathological characteristics of ENKTL patients.
Paraffin sections of surgically removed samples from 16 ENKTL patients were analyzed by immunohistochemistry and the related clinicopathological data were collected and analyzed.
Elevated expression (immunohistochemistry score ≥ 4) of LMP1 and LMP2A was detected in the tumor cells of ENKTL. High LMP1 expression was associated with positive B symptoms (p = 0.012), while high LMP2A expression was related to gender (p = 0.029). The expression of both LMP1 and LMP2A showed significant correlations with patients’ overall survival (p = 0.049, p = 0.036).
LMP1 and LMP2A may be prognostic indicators of survival in patients with ENKTL.
LMP1; LMP2A; EBV; ENKTL; Prognosis; Tumor marker
The latent membrane protein (LMP) of Epstein-Barr virus (EBV) forms patches associated with the vimentin intermediate filament system in EBV-transformed lymphoblastoid cell lines, EBV-infected Burkitt's lymphoma cells, and LMP-transfected, EBV-negative Burkitt's lymphoma cells. By gene transfer, LMP induces the expression of vimentin and B-cell activation antigens in EBV-negative Burkitt's lymphoma cells. We have now expressed LMP in an EBV-positive Burkitt's lymphoma cell line, Daudi, which does not express any LMP or vimentin. In these Daudi transfectants, LMP still formed plasma membrane patches in the absence of vimentin. LMP did not resist nonionic detergent extraction in Daudi cells as it does in vimentin-expressing cells. LMP still retained functional activity as judged by induction of B-cell activation antigens. These data indicate that LMP can form plasma membrane patches and induce B-lymphocyte activation independent of vimentin association.
Interferon regulatory factor 7 (IRF7) is one of the transcriptional factors for the activation of type I Interferon (IFN) genes. It is known that IRF7 and the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) are highly expressed in EBV type III latency cells, and LMP1 induces mRNA expression of IRF7. In this study, the expression pattern of endogenous IRF7 was observed in several B cell lines with or without EBV infection by immunofluorescence staining. IRF7 was localized in the cytoplasm of EBV-negative B cells and EBV type I latency B cell lines. However, IRF7 was located both in the cytoplasm and nucleus of EBV type III latency cell lines. In the Jijoye cell (type III latency cell), IRF7 was colocalized with LMP1 in the cytoplasm in a capping configuration, and their interaction was confirmed by co-immunoprecipitation of LMP1 and IRF7. This colocalization was confirmed by co-transfection of IRF7 and LMP1 plasmids in EBV-negative B cells. These results suggest that the IRF7 and LMP1 interact with each other, and this may relate to the mechanism whereby LMP1 exerts functional effects in B-lymphocytes.
Interferon Regulatory Factor-7; Epstein-Barr Virus; Herpesvirus 4, Human; Latent Membrane Protein 1; EBV-associated membrane antigen, Epstein-Barr virus
JunB is a member of the Jun family of proteins that are components of the AP-1 transcription factor complex. AP-1 is involved in cell proliferation and apoptosis. Recent evidence suggests that Hodgkin and Reed– Sternberg cells overexpress JunB and that JunB facilitates constitutive CD30 expression by binding to an AP-1 site in the CD30 promoter. In this study we surveyed JunB expression in a variety of CD30+ lymphoma types including 42 cases of anaplastic large cell lymphoma, 36 classical Hodgkin lymphoma, 15 cutaneous anaplastic large cell lymphoma, and 11 CD30+ diffuse large B-cell lymphoma. In addition, seven cases of nodular lymphocyte-predominant Hodgkin lymphoma and 42 diffuse large B-cell lymphoma, known to be CD30−, were analyzed. JunB expression was assessed using tissue microarrays, immunohistochemistry and a monoclonal antibody specific for JunB. Expression of JunB was observed in 41 of 42 cases of anaplastic large cell lymphoma, including all 21 cases positive for anaplastic lymphoma kinase and 20 of 21 (95%) negative for anaplastic lymphoma kinase. JunB was also expressed in all cases of classical Hodgkin lymphoma, cutaneous anaplastic large cell lymphoma and CD30+ diffuse large B-cell lymphoma, and in lymphomatoid papulosis. By contrast, all nodular lymphocyte-predominant Hodgkin lymphomas and diffuse large B-cell lymphomas that were CD30− were also JunB−. We conclude that JunB is expressed in virtually all CD30+ lymphomas and is a potential target for experimental therapy in patients with these tumors.
JunB; CD30; anaplastic large cell lymphoma; Hodgkin lymphoma; lymphomatoid papulosis
Despite the identification of Epstein-Barr virus (EBV) in tumors of Burkitt’s lymphoma (BL) over 40 years ago, the exact contribution of EBV to BL is undefined. EBV encodes for multiple proteins in latent B cells that affect B cell survival and activation. One such protein, latent membrane protein 2A (LMP2A), protects B cells from numerous pro-apoptotic stimuli. Therefore, we tested if LMP2A protects B cells from apoptosis induced by aberrant c-MYC expression that precedes and dominates BL. We crossed LMP2A-transgenic mice (LMP2A-Tg) in which all B cells express LMP2A to a transgenic mouse that expresses a BL translocation of myc (λ-MYC-Tg mice). LMP2A promotes proliferation and protects B cells from MYC-induced apoptosis in λ-MYC-Tg mice. LMP2A also accelerates the development of lymphoma in LMP2A/λ-MYC-Tg mice. Finally, LMP2A increases the expression of Bcl-XL in both pre-tumor B cells and tumor cells, suggesting a mechanism for LMP2A mediated B cell survival in the presence of MYC. These results support a hypothesis that EBV LMP2A promotes tumor development by protecting pre-tumor B cells that would normally apoptose after the c-myc translocation.
Epstein-Barr virus (EBV) encodes two integral membrane proteins in latently infected growth-transformed cells. One of these, LMP1, can transform rodent fibroblasts and induce markers of B-lymphocyte activation. The second, LMP2, colocalizes with LMP1 in a constitutive patch in the EBV-transformed B-lymphocyte plasma membrane. The experiments reported here demonstrate that LMP2 may biochemically interact with LMP1 and that LMP2 closely associates with and is an important substrate for a B-lymphocyte tyrosine kinase in EBV-transformed B lymphocytes or in B-lymphoma cells in which LMP2 is expressed by gene transfer. LMP2 is also serine and threonine phosphorylated. LMP2 localizes to a peripheral membrane (presumably plasma membrane) patch in transfected B-lymphoma cells and colocalizes with much of the cellular tyrosine-phosphorylated proteins. LMP2 undergoes tyrosine phosphorylation in anti-LMP2 or antiphosphotyrosine immunoprecipitates from transfected B-lymphoma cells or EBV-transformed B lymphocytes. The first 167 of the 497 amino acids of LMP2 retain full ability to associate with and act as a substrate for a tyrosine kinase. A 70-kDa phosphotyrosine cell protein associates with LMP2 in transfected cells or in EBV-transformed B lymphocytes and could be a mediator of the effects of LMP2.
Background and Objectives
Epstein-Barr Virus (EBV) Latent Membrane Protein 1 (LMP1) is linked to a variety of malignancies including Hodgkin's disease, lymphomas, nasopharyngeal and gastric carcinoma. LMP1 exerts its transforming or oncogenic activity mainly through the recruitment of intracellular adapters via LMP1 C-terminal Transformation Effector Sites (TES) 1 and 2. However, LMP1 is also reported to elicit significant cytotoxic effects in some other cell types. This cytotoxic effect is quite intriguing for an oncogenic protein, and it is unclear whether both functional aspects of the protein are related or mutually exclusive.
Methodology and Principal Findings
Using different ectopic expression systems in both Madin-Darby canine kidney (MDCK) epithelial cells and human embryonic kidney HEK-293 cells, we observe that LMP1 ectopic expression massively induces cell death. Furthermore, we show that LMP1-induced cytotoxicity mainly implies LMP1 C-terminal transformation effector sites and TRADD recruitment. However, stable expression of LMP1 in the same cells, is found to be associated with an increase of cell survival and an acquisition of epithelial mesenchymal transition phenotype as evidenced by morphological modifications, increased cell mobility, increased expression of MMP9 and decreased expression of E-cadherin. Our results demonstrate for the first time that the cytotoxic and oncogenic effects of LMP1 are not mutually exclusive but may operate sequentially. We suggest that in a total cell population, cells resistant to LMP1-induced cytotoxicity are those that could take advantage of LMP1 oncogenic activity by integrating LMP1 signaling into the pre-existent signaling network. Our findings thus reconcile the apparent opposite apoptotic and oncogenic effects described for LMP1 and might reflect what actually happens on LMP1-induced cell transformation after EBV infection in patients.