Christopher D. Gregory

Induction of bcl-2 expression by epstein-barr virus latent membrane protein 1 protects infected B cells from programmed cell death

Epstein-Barr virus (EBV) not only induces growth transformation in human B lymphocytes, but has more recently been shown to enhance B cell survival under suboptimal conditions where growth is inhibited; both effects are mediated through the coordinate action of eight virus-coded latent proteins. The effect upon cell survival is best recognized in EBV-positive Burkitts lymphoma cell lines where activation of full virus latent gene expression protects the cells from programmed cell death (apoptosis). Here we show by DNA transfection into human B cells that protection from apoptosis is conferred through expression of a single EBV latent protein, the latent membrane protein LMP 1. Furthermore, we demonstrate that LMP 1 mediates this effect by up-regulating expression of the cellular oncogene bcl-2. The interplay between EBV infection and expression of this cellular oncogene has important implications for virus persistence and for the pathogenesis of virus-associated malignant disease.

Human CD14 mediates recognition and phagocytosis of apoptotic cells

Cells undergoing programmed cell death (apoptosis) are cleared rapidly in vivo by phagocytes without inducing inflammation. Here we show that the glycosylphosphatidylinositol-linked plasma-membrane glycoprotein CD14 (refs 2, 3) on the surface of human macrophages is important for the recognition and clearance of apoptotic cells. CD14 can also act as a receptor that binds bacterial lipopolysaccharide (LPS), triggering inflammatory responses. Overstimulation of CD14 by LPS can cause the often fatal toxic-shock syndrome,. Here we show that apoptotic cells interact with CD14, triggering phagocytosis of the apoptotic cells. This interaction depends on a region of CD14 that is identical to, or at least closely associated with, a region known to bind LPS. However, apoptotic cells, unlike LPS, do not provoke the release of pro-inflammatory cytokines from macrophages. These results indicate that clearance of apoptotic cells is mediated by a receptor whose interactions with ‘non-self’ components (LPS) and ‘self’ components (apoptotic cells) produce distinct macrophage responses.

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.

Homology between a human apoptosis specific protein and the product of APG5, a gene involved in autophagy in yeast

Apoptosis specific proteins (ASP) are expressed in the cytoplasm of cultured mammalian cells of various lineages following induction of apoptosis. The cDNA encoding ASP has been cloned from a human expression library and has significant homology to the Saccharomyces cerevisiae APG5 gene which is essential for yeast autophagy. The ASP gene, known as hAPG5, can be transcribed to give mRNAs of 3.3 kbp, 2.5 kbp and 1.8 kbp which are present at comparable levels in viable and apoptotic cells, demonstrating that protein expression must be regulated at the translational level. These data indicate a possible relationship between apoptosis and autophagy and suggest evolutionary conservation in mammalian apoptosis of a degradative process present in yeast.

Viruses and cancer risks: Outgrowth of Epstein-Barr virus-positive Burkitt's lymphoma in the immune host

This work examines ways in which Epstein-Barr (EB) virus-positive Burkitts lymphoma (BL) cells achieve outgrowthin vivo in the face of prevailing EB virus-specific cytotoxic T-cell surveillance. Earlier work has shown that some, but not all, BL cell linesin vitro are insensitive to virus-specific T-cell cytolysis and the present study identifies two mechanisms whereby the tumour cells might evade detection. First, BL-cell lines which stably retain the original tumour cell phenotype on serial passagein vitro show very low expression of two cell adhesion-related molecules, LFA-1 and ICAM 1, and are negative for a third such molecule, LFA-3; these molecules are thought to play a crucial role in the non antigen-dependent phase of effector: target cell conjugation which precedes antigen-specific recognition and target cell lysis. Secondly, those same BL cell lines display an unusually restricted pattern of EB virus latent gene expression with at least two potentially important target proteins for the T-cell response, namely EBNA 2 and LMP, not detectably expressed.

Apoptosis in Hematopoiesis and Leukemogenesis

The number of cells in any tissue is dependent on the balance between cell gain by proliferation and cell loss by differentiation and/or cell death (Figure 1). The importance of this balancing act is demonstrated particularly well within the hematopoietic system, where it is thought that in the order of billions of cells are produced every day to replace cell loss. Thus, it is reasonable to suggest that defects in this cell-gain—cell-loss balance are likely to contribute to a number of pathologies of the blood. Enhanced survival, restricted differentiation, and uncontrolled proliferation together conspire to promote the development of lymphomas and leukemias.

Altered Growth Phenotype of a Burkitt’s Lymphoma Line Following the Introduction and Stable Expression of the EBNA 2A Gene

Using recombinant retrovirus-mediated transfer, the coding region for the Epstein-Barr virus-encoded EBNA 2A gene was selectively introduced and stably expressed in the Louckes Burkitt lymphoma cell line. Transfected cells displayed the following altered growth characteristics when compared with the parental line: (i) maintenance of proliferation at reduced serum concentrations, (ii) improved seeding efficiency, (iii) production of a unique growth-enhancing factor, (iv) increased sensitivity to the unique factor. These growth-related changes were accompanied by the appearance of large amounts of soluble CD23 fragments in medium conditioned by Louckes cells expressing EBNA 2A. Tumor-promoting phorbol esters were found to induce similar phenotypic change in the Louckes parental line as seen on the introduction of the EBNA 2A gene. Possible mechanisms by which EBNA 2A provides a growth advantage to B lymphoma cells are discussed.

Isolation of a Normal B Cell Subset with a Burkitt-Like Phenotype & Examination of its Interaction with EB Virus

Our understanding of the pathogenesis of Burkitt’s Lymphoma (BL) and, in particular, the role played by Epstein-Barr (EB) virus has been hampered by the hitherto unsuccessful attempts to identify the normal B cell subpopulation from which this tumour arises. Previous studies relying upon long established tumour-derived cell lines have reported marked heterogeneity in surface phenotype between individual tumours (1–3), leading to the suggestion that BL might be derived from a relatively broad “window” of target cells on the B cell differentiation pathway. Our recent work has shown that this is not the case (4). Analysis of fresh biopsy cells from EB-virus genome-positive cases of BL has shown that the tumour cells display a consistent and homogeneous surface phenotype with co-expression of the pan B cell marker B1 (CD20), the common acute lymphoblastic leukaemia antigen, CALLA (CD10) and the glycolipid BL-associated antigen, BLA.