Tom B. Sculley

mSin1 Is Necessary for Akt/PKB Phosphorylation, and Its Isoforms Define Three Distinct mTORC2s

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that participates in at least two distinct multiprotein complexes, mTORC1 and mTORC2 . These complexes play important roles in the regulation of cell growth, proliferation, survival, and metabolism. mTORC2 is a hydrophobic motif kinase for the cell-survival protein Akt/PKB and, here, we identify mSin1 as a component of mTORC2 but not mTORC1. mSin1 is necessary for the assembly of mTORC2 and for its capacity to phosphorylate Akt/PKB. Alternative splicing generates at least five isoforms of the mSin1 protein , three of which assemble into mTORC2 to generate three distinct mTORC2s. Even though all mTORC2s can phosphorylate Akt/PKB in vitro, insulin regulates the activity of only two of them. Thus, we propose that cells contain several mTORC2 flavors that may phosphorylate Akt/PKB in response to different signals.

The EBNA- 3 gene family proteins disrupt the G2/M checkpoint

The Epstein–Barr nuclear antigens (EBNA), EBNA-3, -4 and -6, have previously been shown to act as transcriptional regulators, however, this study identifies another function for these proteins, disruption of the G2/M checkpoint. Lymphoblastoid cell lines (LCLs) treated with a G2/M initiating drug azelaic bishydroxamine (ABHA) did not show a G2/M checkpoint response, but rather they display an increase in cell death, a characteristic of sensitivity to the cytotoxic effects of the drug. Cell cycle analysis demonstrated that the individual expression of EBNA-3, -4 or -6 are capable of disrupting the G2/M checkpoint response induced by ABHA resulting in increased toxicity, whereas EBNA-2, and -5 were not. EBNA-3 gene family protein expression also disrupted the G2/M checkpoint initiated in response to the genotoxin etoposide and the S phase inhibitor hydroxyurea. The G2 arrest in response to these drugs were sensitive to caffeine, suggesting that ATM/ATR signalling in these checkpoint responses may be blocked by the EBNA-3 family proteins. The function of EBNA-3, -4 and -6 proteins appears to be more complex than anticipated and these data suggest a role for these proteins in disrupting the host cell cycle machinery.

Expression of Epstein-Barr virus nuclear antigens 3, 4, and 6 are altered in cell lines containing B-type virus

A high proportion of HIV-positive sera were found to react with 130- and 180-kDa antigens which were present in the Jijoye cell line. The majority of the HIV-positive sera which detected these antigens also contained antibodies to Epstein-Barr virus (EBV) nuclear antigen 2B (EBNA2B) suggesting a relationship between B-type EBV strains and the expression of the 130K/180K antigens. Cell lines were established by infection of B lymphocytes with different A- and B-type strains of EBV. Incubation of these lines with sera from individuals infected with either A-type or B-type EBV strains demonstrated that the 130K and 180K antigens were only expressed by cell lines containing B-type virus. Sera from individuals infected with A-type EBV did not react with the 180K antigen in any cell lines but could detect EBNAs 3, 4, and 6 antigens in the A-type cell lines. Restriction enzyme analysis of the BamHI E region of the EBV genome revealed marked differences between the A and B types of the virus. These results demonstrate that expression of antigens encoded from the BamHI E region of EBV (EBNAs 3, 4, and 6) are altered in cell lines transformed by B-type strains of EBV.

A-type and B-type Epstein-Barr virus differ in their ability to spontaneously enter the lytic cycle

In this study replication of A-type and B-type Epstein-Barr virus (EBV) strains has been assessed. A-type and B-type type lymphoblastoid cell lines (LCLs) were established by infecting B lymphocytes, isolated from five EBV-seropositive donors, with different A-type and B-type virus isolates. The presence of viral capsid antigens (VCA) in these LCLs was determined by immunofluoresence assay and by immunoblotting. All of the B-type EBV strains were capable of spontaneously generating virus regardless of the origin of the donor cells. In contrast the A-type strains, other than strain IARC-BL36, did not readily produce VCA in any of the different donor lymphocytes used. This study demonstrates another biological difference between the two virus types: their ability to spontaneously enter the lytic cycle.

The Role of Epstein-Barr Virus Subtypes in Human Immunodeficiency Virus-Associated Lymphoma

High grade B cell non-Hodgkins lymphoma (NHL) is an increasingly important problem in individuals infected with the human immunodeficiency virus (HIV). Fifty percent of these tumours harbour the Epstein-Barr virus (EBV) and there is an equal frequency of EBV A and B-subtypes in the tumours. This contrasts with the recent report that only A-type EBV is associated with Hodgkins disease. Such studies are challenging the traditional models of EBV-associated lymphomagenesis and showing the way for further studies in this field. This article reviews the studies of EBV subtypes in HIV-associated NHL and uses this new knowledge to discuss the role of EBV in lymphomagenesis.

A histone deacetylase inhibitor, azelaic bishydroxamic acid, shows cytotoxicity on Epstein-Barr virus-transformed B-cell lines - A potential therapy for posttransplant lymphoproliferative disease

BACKGROUND Posttransplant lymphoproliferative disease (PTLD), driven by the presence of Epstein-Barr virus (EBV), is becoming an increasingly important clinical problem after solid organ transplantation. The use of immunosuppressive therapy leads to the inhibition of the cytotoxic T cells that normally control the EBV latently infected B cells. The prognosis for many patients with PTLD is poor, and the optimal treatment strategy is not well defined. METHOD This study investigates the use of a histone deacetylase inhibitor, azelaic bishydroxamic acid (ABHA), for its ability to effectively kill EBV-transformed lymphoblastoid cell lines. RESULTS In vitro treatment of lymphoblastoid cell lines with ABHA showed that they were effectively killed by low doses of the drug (ID50 2-5 microg/ml) within 48 hr. As well as being effective against polyclonal B-cell lines, ABHA was also shown to be toxic to seven of eight clonal Burkitts lymphoma cell lines, indicating that the drug may also be useful in the treatment of late-occurring clonal PTLD. In addition, ABHA treatment did not induce EBV replication or affect EBV latent gene expression. CONCLUSION These studies suggest that ABHA effectively kills both polyclonal and clonal B-cell lines and has potential in the treatment of PTLD.

Both A type and B type Epstein-Barr virus nuclear antigen 6 interact with RBP-2N.

Using the yeast two-hybrid system, Epstein-Barr virus nuclear antigen 6A (EBNA6A) was found to interact with the RBP-2N isoform of RBP-J kappa. The interaction of EBNA6A and EBNA6B with RBP-2N was compared and the results indicated that EBNA6B was less efficient at interacting with RBP-2N than was EBNA6A. Deletion mutation analysis of EBNA6A identified a region involved in the interaction with RBP-2N, while analysis of RBP-2N identified a domain which interacts with EBNA6A. The region of RBP-2N to which EBNA6A binds has previously been shown to interact with EBNA2.

Prediction of BRCA1 and BRCA2 mutation status using post-irradiation assays of lymphoblastoid cell lines is compromised by inter-cell-line phenotypic variability

Background and purposeAssays to determine the pathogenicity of unclassified sequence variants in disease-associated genes include the analysis of lymphoblastoid cell lines (LCLs). We assessed the ability of several assays of LCLs to distinguish carriers of germline BRCA1 and BRCA2 gene mutations from mutation-negative controls to determine their utility for use in a diagnostic setting.Materials and methodsPost-ionising radiation cell viability and micronucleus formation, and telomere length were assayed in LCLs carrying BRCA1 or BRCA2 mutations, and in unaffected mutation-negative controls.ResultsPost-irradiation cell viability and micronucleus induction assays of LCLs from individuals carrying pathogenic BRCA1 mutations, unclassified BRCA1 sequence variants or wildtype BRCA1 sequence showed significant phenotypic heterogeneity within each group. Responses were not consistent with predicted functional consequences of known pathogenic or normal sequences. Telomere length was also highly heterogeneous within groups of LCLs carrying pathogenic BRCA1 or BRCA2 mutations, and normal BRCA1 sequences, and was not predictive of mutation status.ConclusionGiven the significant degree of phenotypic heterogeneity of LCLs after γ-irradiation, and the lack of association with BRCA1 or BRCA2 mutation status, we conclude that the assays evaluated in this study should not be used as a means of differentiating pathogenic and non-pathogenic sequence variants for clinical application. We suggest that a range of normal controls must be included in any functional assays of LCLs to ensure that any observed differences between samples reflect the genotype under investigation rather than generic inter-individual variation.

Epstein-Barr virus-specific cytotoxic T cell response in cardiac transplant recipients.

PBLs from 10 normal seropositive donors, 15 precardiac transplant patients, and 17 postcardiac transplant patients have been assayed for their ability to mount a cytotoxic T cell response to both A- and B-type EBV. Compared with the results obtained with healthy seropositive donors, pre- and posttransplant patients had significantly weaker T cell responses against both A-type and B-type EBV. Analysis of individual patients showed a preferential T cell response to B-type EBV in 4/15 pre- and 6/17 posttransplant patients and a preferential T cell response to A-type EBV in 1/15 pretransplant and 2/17 posttransplant recipients. PBMCs were obtained from patients and analyzed for the presence of A- and B-type EBV using polymerase chain reaction. EBV types detected in the PBMCs of each individual were correlated with their EBV-specific CTL response. The results obtained indicated that the EBV-specific cytotoxic T cell response of these patients matched the EBV types with which they were infected.

Epstein-Barr virus and HIV play no direct role in persistent generalized lymphadenopathy syndrome.

Persistent generalized lymphadenopathy (PGL) and polyclonal B cell activation are features of infection with HIV. Epstein‐Barr virus (EBV) and HIV are known to activate B cells in vitro, but whether they are important B cell activators in patients infected with HIV is less clear. In this study, lymph node tissue was obtained from 10 patients with PGL and assessed for evidence of EBV and HIV gene sequences. DNA was extracted and specific viral gene sequences identified using the polymerase chain reaction (PCR). EBV sequences were difficult to detect in the PGL tissue, with a signal intensity similar to that of other benign and malignant lymphoid conditions not associated with EBV. HIV sequences were also rare in the PGL tissue, consistent with HIV infection of the small number of peripheral blood cells and nodal T cells likely to be present in such a sample. These findings suggest that the polyclonal B cell activation typical of HIV is not driven by direct EBV or HIV infection of B cells.