Activation of p53 by MDM2 antagonists has differential apoptotic effects on Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt's lymphoma cells.

p53 inactivation is often observed in Burkitt's lymphoma (BL) cells, because of either mutations in p53 gene or an overexpression of the p53-negative regulator MDM2. Epstein-Barr virus (EBV) is present in virtually 100% of BL cases occurring in endemic areas, but in only 10-20% of sporadic cases. In EBV(-) BL cells, reactivation of p53, induced by reducing MDM2 protein level, led to apoptosis. We show here that nutlin-3, a potent antagonist of MDM2, activates the p53 pathway in all BL cell lines harboring wild-type p53, regardless of EBV status. However, nutlin-3 strongly induced apoptosis in EBV(-) or latency I EBV(+) cells, whereas latency III EBV(+) cells were much more resistant. Prior treatment with sublethal doses of nutlin-3 sensitizes EBV(-) or latency I EBV(+) cells to apoptosis induced by etoposide or melphalan, but protects latency III EBV(+) cells. p21(WAF1) which is overexpressed in the latter, is involved in this protective effect, as siRNA-mediated inhibition of p21(WAF1) restores sensitivity to etoposide. Nutlin-3 protects latency III BL cells by inducing a p21(WAF1)-mediated G1 arrest. Most BL patients with wild-type p53 tumors could therefore benefit from treatment with nutlin-3, after a careful determination of the latency pattern of EBV in infected patients.

Apoptosis of tumoral and nontumoral lymphoid cells is induced by both mdm2 and p53 antisense oligodeoxynucleotides.

Following stress signals, the p53 tumor suppressor protein plays a critical role in regulation of cell proliferation, mainly through induction of growth arrest or apoptosis. Therefore, this protein needs to be strictly regulated and numerous studies have shown that the MDM2 protein is an essential element for p53 regulation in normal cells and, most importantly, that overexpression of MDM2 is responsible for p53 inactivation in various types of tumors. A previous study showed that this is the case in some Burkitt lymphoma (BL) cell lines, where enhanced translation of mdm2 messenger RNA results in overexpression of the protein that complexes and inactivates wild-type p53. To further investigate the role of the p53/MDM2 complex in these BL cells, as well as in other lymphoid cells that do not overexpress MDM2, this study used antisense oligodeoxynucleotides directed either against mdm2 or against p53. Results show that the mdm2 antisense oligodeoxynucleotide induces apoptosis of cells that express a high or low level of MDM2 protein, only if they contain wild-type p53. Moreover, apoptosis is independent of the accumulation of p53 following mdm2 antisense treatment. Finally, the p53 antisense oligodeoxynucleotide, which inhibits the expression of wild-type p53, also induces a decrease of the MDM2 level in cells, whether or not they overexpress this protein, and causes apoptosis of these cells. These results indicate that decreasing the MDM2 protein level by directly or indirectly targeting its biosynthesis is a potent tool for the induction of apoptosis.

Intracellular signaling events in CD77-mediated apoptosis of Burkitt's lymphoma cells.

In the hematopoietic system CD77, a glycolipid surface antigen, is restricted to group I Burkitt's lymphoma (BL) cell lines and a subset of germinal center B lymphocytes. Recently, we have reported that recombinant B subunits of Verotoxin, which specifically binds to CD77, induce programmed cell death of CD77+ BL cells. Here, we show that an anti-CD77 monoclonal antibody (38.13) immobilized on tissue culture dishes also induces apoptosis, and we have explored the signal transducing events leading to this cell death. We show that ligation of CD77 antigen causes an increase of the intracellular Ca2+ concentration owing to an influx of extracellular Ca2+ through calcium channels. Chelation of extracellular Ca2+ with EGTA partially prevents anti-CD77-induced apoptosis, indicating that this process is probably Ca2+ dependent. We show that the cross-linking of CD77 provokes an increase of intracellular cAMP levels followed by cAMP-dependent protein kinase activation. We report that BL cells produce ceramide when they are exposed to 38.13 but, unexpectedly, without a concomitant decrease in sphingomyelin or CD77 content. Finally, we provide evidence that C2-ceramide, calcium ionophore, and forskolin (which increases intracellular levels of cAMP) independently induce apoptosis of CD77+ BL cells and, moreover, that C2-ceramide and forskolin strongly synergize to cause cell death. The possible role of CD77-mediated apoptosis in the B cell selection that occurs in germinal centers is discussed.

High concentration of the EBV latent membrane protein 1 in glycosphingolipid-rich complexes from both epithelial and lymphoid cells.

Latent Membrane Protein 1 (LMP1) is an EBV-transforming protein which is detected both in lymphoblastoid cell lines-resulting from EBV-immortalization in vitro- and in undifferentiated nasopharyngeal carcinoma (NPC), an EBV-associated malignancy of epithelial origin. To better define LMP1 subcellular targets, LMP1 distribution was analyzed in cellular glycosphingolipid-rich complexes (GSL-complexes) derived from epithelial and lymphoid cells. These complexes are obtained by extraction of glycosphingolipid-rich membrane domains (GSL-domains), which are clustering sites for heterotrimeric G-proteins and G-protein-associated receptors. LMP1 concentration was enriched 50-fold in GSL-complexes extracted from a NPC tumor line, C15. High concentrations of LMP1 were also observed in GSL-complexes derived from cultured lymphoid and epithelial cells. These data suggest that association with GSL-domains is an important step in LMP1 trafficking and is probably required for some aspects of its biological activity.

Histo-blood group p: biosynthesis of globoseries glycolipids in EBV-transformed B cell lines.

The genetic and biosynthetic basis of the histo-blood group P-system is not fully understood. Individuals with the rare p phenotype do not express the three glycolipid antigens (Pk, P and P1) of this system, probably because of deficiencies in glycosyltransferases involved in their biosynthesis. Iiuka et al. [Iiuka S, Chen SH, Yoshida A (1986) Biochem Biophys Res Commun 137: 1187-95], however, previously reported that detergent extracts from an EBV-transformed B cell line derived from a p individual did express the glycosyltransferase activity (Pk transferase) assumed to be missing in this blood group status. Here, we have reinvestigated the antigen expression and glycosyltransferase activities in two p individuals by analysing EBV-transformed cell lines as well as erythrocytes to confirm the blood group P status. The thin layer chromatography glycolipid profile of extracts from erythrocytes and EBV-transformed B cell lines showed characteristic accumulation of lactosylceramide and absence of Pk and P antigens. Glycosyltransferase activities of the B cell lines were analysed using glycolipid substrates and both extracts were found to contain lactosylceramide synthetase and P transferase activities but to be completely devoid of Pk transferase activity. The presented data indicate that p individuals, in contrast to previous reports, do not express a functional Pk glycosyltransferase.

Sequential changes in glycolipid expression during human B cell differentiation: enzymatic bases.

We have previously reported that human B cell differentiation is accompanied by sequential changes in glycosphingolipid expression. Pre-B cells contain lacto-series type II chain-based glycolipids and GM3 ganglioside; mature/activated B cells do not synthesize lacto-series compounds but express GM3 and globo-series glycolipids (Gb3 and Gb4); terminally differentiated B cells, in addition to these compounds, also contain GM2 ganglioside. At the cell surface, Gb3, Gb4 and GM2 constitute stage-specific antigens. To elucidate the biosynthetic mechanism leading to these modifications we have compared activities of the glycosyltransferases involved in the core structure assembly and the first elongation steps of neo-lacto, ganglio- and globo-series glycolipids. These glycosyltransferase activities have been measured in B cell lines and normal B lymphocytes at various stages of differentiation. We first determined the optimal requirements of the four glycosyltransferases which synthesize Lc3, GM3, Gb4 and GM2 glycolipids in B lymphocytes and then tested these enzymes and the Gb3 synthetase in the selected B cells. The following results were obtained: beta 1-->3 N-Acetylglucosaminyltransferase (Lc3 synthetase) has a high activity in pro- and pre-B cells whereas it is undetectable in more differentiated cells; alpha 2-->3 sialytransferase (GM3 synthetase) is activated from the pre-B cell stage to the terminally differentiated myeloma cells; alpha 1-->4 galactosyltransferase (Gb3 synthetase) is only detected in cells representing the late stages of B cell differentiation; beta 1-->3 N-Acetylgalactosaminyltransferase (Gb4 synthetase) is only found in some lymphoblastoid cell lines, representative of activated B cells whereas the beta 1-->4 N-Acetylgalactosaminyltransferase (GM2 synthetase) has a high activity in these lymphoblastoid cell lines and in terminally differentiated myeloma cells. These results suggest that the sequential shifts in the three major glycosphingolipid series observed during B cell differentiation are mostly due to sequential activations of the corresponding glycosyltransferases.

Sequential shifts in the three major glycosphingolipid series are associated with B cell differentiation.

Cell surface glycolipid expression as well as total glycolipid content of various B cell lines, representative of different B cell stages, and normal B lymphocytes were examined. Glycolipids, made up of a carbohydrate chain attached to a lipid called ceramide, are classified in four main 'series'. These series are defined according to the identity and chemical bonding of the sugars closest to the ceramide moiety. The pre-B cell lines contained lacto-series type II chain-based glycolipids and II3-alpha-N-acetyl-neuraminosyllactosylceramide (GM3) ganglioside. Upon differentiation, the lacto-series synthesis was shut down whereas compounds of the globo-series appeared: resting lymphocytes and lymphoblastoid cell lines (LCL) expressed GM3, globotriaosylceramide (Gb3), and globoside (Gb4). At a later stage of B cell differentiation, biosynthesis of the ganglio-series was extended and myeloma cells expressed II3-alpha-N-acetyl-neuraminosylgangliotriosylceramide (GM2). At the cell surface, in addition to Gb3, that we previously described as specifically expressed on Burkitt's lymphoma cells and on a subset of germinal centre tonsillar B cells, two glycolipids seemed specific of certain B cell lines: Gb4 was strongly positive on six out of eight LCLs and on the low buoyant density fraction of tonsillar B lymphocytes, whereas GM2 ganglioside was only detected on the two myeloma cell lines. These results, demonstrating the stage-dependent expression of certain glycolipids, suggest that these carbohydrate molecules could play functional roles during B cell differentiation.

Analysis of phenotypic and functional changes during ganglioside-induced inhibition of human T cell proliferation.

Glycosphingolipids added to the cell culture medium can be incorporated into the plasma membrane and interfere with the growth of certain cell types. In the past years, previous reports have shown that gangliosides, a class of glycosphingolipids bearing sialic acid can inhibit antigen or mitogen induced T cell proliferative responses in vitro. We report here that the inhibition of PHA induced proliferation by the trisialoganglioside GT1b was not reversed by addition of exogenous IL-1, IL-2, TPA and calcium ionophore. Furthermore, GT1b did not affect IL-2 production by activated T cells. In addition, GT1b ganglioside could also decrease strongly the expression of the T cell antigens CD3, CD2, CD4, CD8 and the alpha/beta T cell receptor antigenic complex whereas it did not affect HLA-class I antigens. By contrast, GT1b modulated only partially membrane expression of activation antigens such as CD25 (Tac) and transferrin receptor and increased the expression of HLA-class II antigens. Moreover CD25 messenger RNA induction was not affected by GT1b treatment of PHA-stimulated T cells. Our results demonstrate that gangliosides, in spite of their anti-proliferative capacity and their modulation effect on T cell antigen membrane expression, do not prevent the progression of T cells into early stages of the activation process.

Identification of a subset of normal B cells with a Burkitt's lymphoma (BL)-like phenotype.

Fresh biopsy cells from cases of Burkitt's lymphoma (BL) display a homogeneous cell surface phenotype. The cells were found to be reactive with the pan B cell marker B1, and consistently co-expressed the BL-associated glycolipid antigen, BLA, and the common acute lymphoblastic leukemia antigen, CALLA, but lacked the B cell "activation" antigens characteristically expressed on EB virus-transformed normal B cells. Microscopic and cell sorter analysis of cells isolated from a series of fresh normal tonsils have identified a subpopulation of normal B cells carrying the same cell surface markers. That BLA and CALLA could be co-expressed on individual B cells was demonstrated by two-color immunofluorescence (IF) of tonsils in suspension, and immunoperoxidase (IP) staining of serial tonsil sections. These BLA+, CALLA+, "activation" antigen- cells were further characterized as B1+, sIgM+, sIgD-, C3d/EB virus receptor+ and were susceptible to virus-induced transformation in vitro. IF studies on Percoll-fractionated tonsillar cell populations and direct examination of IP-stained tonsil semi-thin sections indicated that the BLA+, CALLA+ cells were localized in germinal centers. Their morphological characteristics matched those of BL cells, and their location within germinal centers was consistent both with the known phenotype of germinal center tonsillar B cells and with the description of BL as a proliferation of centroblasts. We suggest that this population of tonsillar germinal center B cells provides the normal counterpart of BL tumor cells.

Induction of cell differentiation in Burkitt lymphoma lines. BLA: a glycolipid marker of B-cell differentiation.

We have previously described an MAb referred to as 38.13 which reacts with a glycolipid membrane antigen (named BLA) on Burkitt lymphoma (BL) lines. The BLA antigen and other B-cell differentiation markers have been studied on BL lines treated with sodium butyrate, agents from the phorbol-diester series (with or without differentiating properties) and teleocidin. With all differentiation inducers tested, expression of BLA as well as of surface IgM decreased on the induced cells whereas that of BI increased and that of HLA DR remained stable. No BL cells studied reacted with anti-IgD, with B2 or with LBI MAbs, either before or after induction. An EBV-negative line (BJAB) was compared to its Epstein-Barr virus (EBV)-converted subline (BJAB/B95). Both EBV-positive and EBV-negative lines gave comparable results. These data demonstrate that BL cells could be moved along their differentiation pathway by chemical inducers and suggest that BLA represents a new glycolipid marker of early B-cell differentiation.

A monoclonal antibody with anti-Burkitt lymphoma specificity. I. Analysis of human haematopoietic and lymphoid cell lines.

38-13 is a hybridoma-produced monoclonal rat IgM which appears to define a Burkitt's lymphoma-associated antigen (BLA). In this paper, we described the reactivity of 38-13 with a panel of human haematopoietic and lymphoid cell lines. In indirect immunofluorescence (IF) assays, 15 of 26 Burkitt's lymphoma (BL) lines studied were clearly stained with 38-13 (from 13 to 100% positive cells) by microscope, with varying numbers of heavily labelled cells. In these positive cell lines, fluorescence-activated cell-sorter (FACS) analysis demonstrated that BLA was actually present on all the cells. Positive BL included Epstein-Barr virus (EBV) genome-carrying lines and EBV-negative ones; thus, BLA is not related to the presence of EBV. Most of the 15 BL cells that reacted with 38-13 contained a typical t(8;14) translocation, but had variant translocations such as t(2;8) and t(8;22). The cells were derived from BL patients of different geographical origins and clinical features. Four BL lines were poorly stained and seven were negative with 38-13 in IF assays. The 32 EBV-positive lymphoblastoid cell-lines (LCL) studied were negative. In three line pairs, consisting of a tumor line and an LCL from the same patient, only the BL line was demonstrated to react with 38-13. A series of non-BL cells, including haematopoietic, lymphoid and solid tumor lines, all failed to react with 38-13. Various attempts to modulate the expression of BLA on BL cells were unsuccessful. However, it cannot be ruled out that BLA is actually a transient B-cell differentiation marker.

Natural killer cell activity in human bone marrow recipients: early reappearance of peripheral natural killer activity in graft-versus-host disease.

Natural killer (NK) cell activity toward K562 target cells and antibody-dependent cell-mediated cytotoxicity (ADCC) toward L1210 cell sensitized with anti-L1210 antisera were sequentially tested in peripheral blood lymphocytes (PBLs) from 24 human bone marrow (BM) recipients. Although consistently decreased before the transplant, NK cell activity was restored in all of the patients tested that argues for a bone marrow origin of NK progenitors in humans. In patients without graft-versus-host disease (GVHD), peripheral NK cell activity remained low during the 1st month after the transplant, then rapidly increased and reached normal values usually between days 30 and 50. By contrast, peripheral ADCC appeared earlier restored (since day 13), suggesting that NK and ADCC are two distinct effector mechanisms. When restored, peripheral NK cell activity remained within normal range, except in seven cases with a drastic fall in NK cell values contemporary with a severe viral infection, mainly with cytomegalovirus (CMV). NK cells are thus suggested to play an important role in the control of viral infections in these deeply immunodepressed patients. In patients with acute GVHD, strikingly high NK values were observed early after the transplant, and during the 1st month a strong correlation did exist between high NK values and acute GVHD occurrence. These results suggest that cells involved in GVHD mechanism are able to exert NK cell activity at some stages of their maturation. The assessment of NK cell activity could be an attractive routine procedure for monitoring the prophylaxis of GVHD in human BM recipients.