Biomedical subjects

J Wiels

Publications and source records attributed to J Wiels.

At least 19 recordsLinked to original sources

PubMed2001

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.

Apoptosis↗

PubMed2000

Molecular basis for the p phenotype. Identification of distinct and multiple mutations in the alpha 1,4-galactosyltransferase gene in Swedish and Japanese individuals.

p phenotype individuals lack both P(k) (Gb3) and P (Gb4) glycolipid antigens of the P blood group system. To explore the molecular basis for this phenotype, DNA sequences of Gb3 synthase (alpha1, 4-galactosyltransferase; alpha1,4Gal-T) in six p phenotype individuals from Japan and Sweden were analyzed. A missense mutation P251L and a nonsense mutation W261stop in three and one Japanese indivuiduals, respectively, and missense mutations M183K and G187D in one each of two Swedish p individuals were found, indicating that p individuals from Japan and Sweden have distinct and multiple homozygous point mutations in the coding region. In the function analysis of the mutated alpha1,4Gal-Ts by the transfection of the expression vectors, P251L and M183K mutations showed complete loss of enzyme function, and W261stop and G187D mutations resulted in the marginal activity. BLAST analysis of homologous sequences of alpha1, 4Gal-T revealed that three residues, Met(183), Gly(187), and Pro(251), at which missense mutations were found, were highly conserved among all species examined, suggesting their importance for the function of alpha1,4Gal-T.

Amino Acid Sequence↗

PubMed2000

Cloning and expression of the histo-blood group Pk UDP-galactose: Ga1beta-4G1cbeta1-cer alpha1, 4-galactosyltransferase. Molecular genetic basis of the p phenotype.

The molecular genetic basis of the P histo-blood group system has eluded characterization despite extensive studies of the biosynthesis of the P(1), P, and P(k) glycolipids. The main controversy has been whether a single or two distinct UDP-Gal:Galbeta1-R 4-alpha-galactosyltransferases catalyze the syntheses of the structurally related P(1) and P(k) antigens. The P(1) polymorphism is linked to 22q11.3-ter. Data base searches with the coding region of an alpha4GlcNAc-transferase identified a novel homologous gene at 22q13.2 designated alpha4Gal-T1. Expression of full coding constructs of alpha4Gal-T1 in insect cells revealed it encoded P(k) but not P(1) synthase activity. Northern analysis showed expression of the transcript correlating with P(k) synthase activity and antigen expression in human B cell lines. Transfection of P(k)-negative Namalwa cells with alpha4Gal-T1 resulted in strong P(k) expression. A single homozygous missense mutation, M183K, was found in six Swedish individuals of the rare p phenotype, confirming that alpha4Gal-T1 represented the P(k) gene. Sequence analysis of the coding region of alpha4Gal-T1 in P(1)+/- individuals did not reveal polymorphisms correlating with P(1)P(2) typing.

Amino Acid Sequence↗

PubMed2000

Molecular cloning of globotriaosylceramide/CD77 synthase, a glycosyltransferase that initiates the synthesis of globo series glycosphingolipids.

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (alpha1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed alpha1, 4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.

Amino Acid Sequence↗

PubMed1999

Evidence of LMP1-TRAF3 interactions in glycosphingolipid-rich complexes of lymphoblastoid and nasopharyngeal carcinoma cells.

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV) protein expressed in EBV-transformed B lymphocytes and in approximately 50% of nasopharyngeal carcinomas (NPCs). LMP1 signaling involves several cellular signaling intermediates, especially TNF receptor-associated factors (TRAFs). We have shown previously that LMP1 is highly concentrated in a cell fraction called glycosphingolipid-rich membrane complexes (GSL complexes). We report here that parallel accumulation of LMP1 and TRAF3, but not TRAF1 or TRADD, was observed in GSL complexes from lymphoblastoid and LMP1-positive NPC cells. In contrast, TRAF3 was not concentrated in GSL complexes from LMP1-negative cells. Binding of LMP1 and TRAF3 in GSL complexes was demonstrated in lymphoblastoid and NPC cells, by co-immunoprecipitation with both anti-LMP1 and anti-TRAF3 antibodies.

Animals↗

PubMed1999

High expression of MDM2 protein and low rate of p21(WAF1/CIP1) expression in SCID mice Epstein Barr virus-induced lymphoproliferation.

To study the prevalence of p53 inactivation and MDM2/p21(WAFI/CIP1) expression in severe combined immunodeficient (SCID) mice Epstein-Barr virus (EBV)-induced lymphoproliferation, 19 samples obtained after ip injection of peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors or lymphoblastoid cell lines (LCL) were analyzed. In all samples tested, overexpression of Ki-67 antigen was shown by immunohistochemistry, indicating a high proliferative index of SCID mice EBV-induced lymphoproliferation. P53 mutations were screened by functional assay in yeast in 14 samples. With this test, a p53-inactivating mutation was found in only one case; the remaining cases exhibited a wild-type p53 pattern. However, an accumulation of p53 protein was detected by immunohistochemistry in six of 19 samples. P21 expression was found in seven of 19 samples but was not correlated with the rate of p53 protein in tumors. In contrast, high levels of nuclear accumulation of MDM2 were found in all samples by immunohistochemistry. These results suggest that a high Ki-67 proliferative index in SCID mice EBV-induced lymphoproliferation is not due to the inactivation of p53 by mutation, but could be associated with an overexpression of MDM2, which would act by a p53-independent mechanism.(J Histochem Cytochem 47:1315-1321, 1999)

Animals↗

PubMed1998

The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein.

Previously, we showed that the addition of human erythrocyte glycosphingolipids (GSLs) to nonhuman CD4(+) or GSL-depleted human CD4(+) cells rendered those cells susceptible to HIV-1 envelope glycoprotein-mediated cell fusion. Individual components in the GSL mixture were isolated by fractionation on a silica-gel column and incorporated into the membranes of CD4(+) cells. GSL-supplemented target cells were then examined for their ability to fuse with TF228 cells expressing HIV-1LAI envelope glycoprotein. We found that one GSL fraction, fraction 3, exhibited the highest recovery of fusion after incorporation into CD4(+) nonhuman and GSL-depleted HeLa-CD4 cells and that fraction 3 contained a single GSL fraction. Fraction 3 was characterized by MS, NMR spectroscopy, enzymatic analysis, and immunostaining with an antiglobotriaosylceramide (Gb3) antibody and was found to be Gal(alpha1-->4)Gal(beta1-->4)Glc-Cer (Gb3). The addition of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells recovered fusion, but the addition of galactosylceramide, glucosylceramide, the monosialoganglioside, GM3, lactosylceramide, globoside, the disialoganglioside, GD3, or alpha-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4/CXCR4-dependent HIV-1 fusion.

Acetylation↗

PubMed1998

Overexpression of MDM2, due to enhanced translation, results in inactivation of wild-type p53 in Burkitt's lymphoma cells.

Numerous studies have indicated that inactivation of p53 is one of the essential requirements for the unrestrained growth of tumoral cells. When the status of the p53 gene was examined in various types of lymphoid malignancies, mutations in p53 have been predominantly detected in Burkitt's lymphoma (BL) cells, therefore suggesting that alteration of p53 could specifically contribute to the malignant phenotype of these tumoral cells. In addition to mutations, functional inactivation of p53 can also occur through interaction of the wild-type gene product with various viral or cellular proteins. The cellular MDM2 protein, for example, is able to inhibit p53 tumor suppressor function by concealing its transactivation domain. Mdm2 gene amplification has been described in several types of sarcomas, resulting in overexpression of the MDM2 protein. In this study, we have examined the status of MDM2 and p53 in 20 BL cell lines. Four were found to contain wild-type p53 and to overexpress MDM2 protein. Within these BL cells, both molecules are physically associated since they can be co-precipitated and p53 is inactivated as cells neither arrest in G1 nor enter apoptosis following gamma-radiation. We also report that the high level of the MDM2 protein in BL cells is neither associated with an amplification of the mdm2 gene nor with an elevated level of RNA or an increased protein stability, but is rather due to an enhanced translation ability of the mdm2 RNA. These results indicate that in certain BL cells, overexpression of MDM2 protein regulated at the posttranscriptional level, induces an escape from p53-controlled cell growth.

Burkitt Lymphoma↗

PubMed1997

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.

Apoptosis↗

PubMed1997

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.

Animals↗

PubMed1996

Multiple pathways of Fas-induced apoptosis in primary culture of hepatocytes.

Fas (Apo1/CD95) is a member of the tumour necrosis factor/nerve growth factor receptor superfamily and mediates apoptosis in various cell types (for review sec [1]). Although this apoptotic activity has been clearly related to homeostasis in the immune system and pathological situations in non-lymphoid organs, the Fas signaling pathway remains mostly elusive. We and others previously showed that Fas-induced apoptosis of primary culture hepatocytes requires either an inhibitor of translation or a protein kinase inhibitor, suggesting that two distinct pathways of Fas signaling exist in hepatocytes. We report here that activation of ICE-like and CPP32-like cysteine proteases are required for Fas-mediated apoptosis, but that these pathways involve different subclasses of serine proteases and are selectively modulated by inhibitors of protein tyrosine kinases. These results confirm that distinct pathways can lead to Fas-induced apoptosis in hepatocytes. Further understanding of these pathways could facilitate the rational design of anti-apoptotic drugs in liver diseases associated with massive Fas-mediated hepatocyte apoptosis, including fulminant hepatitis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

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.

Antigens, Surface↗

PubMed1995

Differential regulation of glycosphingolipid biosynthesis in phenotypically distinct Burkitt's lymphoma cell lines.

Earlier studies have shown that Burkitt's lymphoma (BL) cell lines can be divided into 2 major groups: group I, which retain the original BL biopsy phenotype with expression of CD10 and CD77 antigens and lack of B-cell activation markers, and group III, which, after several in vitro passages, progress toward an "LCL-Like" phenotype with loss of CD10 and C77 expression and up-regulation of B-cell activation antigens. In previous studies we have shown that several glycolipid molecules constitute stage-specific antigens for B cells and that sequential shifts in the 3 major glycolipid series are observed during B-cell differentiation, these changes being mostly due to sequential activations of the corresponding glycosyltransferases. In the present work, 10 BL cell lines with group I or group III phenotype have been examined for cell surface expression of 5 glycolipid antigens (LacCer, GM3, Gb3/CD77, Gb4 and GM2), total glycolipid content and enzymatic activities of 4 glycosyltransferases (GM3, Gb3, Gb4 and GM2 synthetases). We now report that group I and group III BL cells differ in their glycolipid metabolism and express either mostly globoseries or ganglioseries compounds. Indeed, Gb3 is the major glycolipid of group I cells, whereas GM3 and GM2 are the 2 major components of group III cells, and these phenotypic differences are mainly due to differential activities of the corresponding glycosyltransferases: group I cells have high Gb3 synthetase activities and low or no GM3 and GM2 synthetase activities, whereas group III cells have high GM3 and GM2 synthetase activities and low Gb3 synthetase activities. Finally, we also show that, unlike LCL, group III BL cells do not synthesize Gb4.

Burkitt Lymphoma↗

PubMed1995

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.

B-Lymphocytes↗

PubMed1995

Expression of the glycolipid globotriaosylceramide (Gb3) in testicular carcinoma in situ.

Changes in the cell membrane glycolipid composition and metabolism are frequently associated with carcinogenesis. The accumulation of globo-series glycolipids is the most notable change of the germ cell glycolipid composition observed in testicular tumours. In this study, the expression of the globo-series core-structure, globotriaosylceramide (Gb3) was investigated in the preinvasive stage of testicular germ cell tumours, carcinoma in situ (CIS). Seventeen tissue specimens with CIS and 12 samples of overt testicular tumours were immunostained with anti-Gb3 monoclonal antibody 38-13. The accumulation of Gb3 was detected in 12 CIS samples (70.6%) and in 8 invasive tumour samples (66.7%), including seminoma, non-seminoma and a combined germ cell tumour. Our findings indicate that the composition of glycolipids shift at the common preinvasive stage of testicular germ cell tumours and confirm that Gb3 is a tumour-associated antigen of testicular germ cell neoplasia.

Carcinoma in Situ↗

PubMed1995

The fate of human CD77+ germinal center B lymphocytes after rescue from apoptosis.

Germinal center (GC) B lymphocytes, defined by various criteria, have been shown to spontaneously undergo apoptosis in vitro unless they receive a positive signal. This rescue signal seems to be a multi-component process which involves not only the B cell receptor but also other cell surface receptors such as the CD40 antigen. In previous studies, we have shown that expression of the CD77 antigen is restricted to GC B lymphocytes and that CD77+ cells readily enter programmed cell death when cultured in vitro. In order to better characterize the CD77+ B lymphocytes, we have investigated the fate of these cells after rescue from apoptosis. Survival of CD77+ cells was achieved either with a combination of anti-CD40 mAb and IL4 (the CD40 system developed by Banchereau et al., (1991) Science 251, 70-72) or EBV infection. After 4 days of culture, similar phenotypic and functional changes of the CD77+ lymphocytes were observed in both systems: CD77 antigen was down-regulated, CD23 antigen which was originally negative became strongly expressed and the expression of CD38 and CD20 remained constant. Furthermore, large quantities of soluble CD23 were produced by the surviving cells. These results indicate that CD77 antigen is expressed by GC B cells which are highly susceptible to enter apoptosis but which are not doomed to die.

Antibodies, Monoclonal↗

PubMed1993

Apoptosis induced in Burkitt's lymphoma cells via Gb3/CD77, a glycolipid antigen.

Gb3/CD77 is a glycolipid antigen, specifically expressed on two different B-cell populations, Burkitt's lymphoma and a subset of tonsillar B-lymphocytes located in germinal centers, which could be the normal counterpart of Burkitt cells. Both Gb3/CD77(+) populations have recently been shown to enter programmed cell death (apoptosis) readily. Here we show that verotoxin, also called Shiga-like toxin, which is known to bind to the carbohydrate moiety of Gb3/CD77, induces cell death in Gb3/CD77(+) Burkitt's lymphoma cells, not only by inhibiting protein synthesis as classically described but also through an additional mechanism, namely apoptosis. Furthermore a recombinant B-subunit of verotoxin, which carries only the binding property of the holotoxin, also induces apoptosis in Gb3/CD77(+) cells. Gb3/CD77 could thus represent the first example of a glycolipid antigen able to transduce a signal leading to apoptosis.

Antigens, CD↗

PubMed1991

CD77: an antigen of germinal center B cells entering apoptosis.

We have previously reported that a neutral glycolipid (globotriosylceramide; Gb3) was specifically expressed on Burkitt's lymphoma cells and on a subset of germinal center tonsillar B lymphocytes. Recently the Gb3 molecule was recognized as a new B cell differentiation antigen and now defines the CD77 cluster. Here we report an extensive phenotypic and functional characterization of the tonsillar CD77+ B lymphocytes. These cells have a low buoyant density and are thus purified using a Percoll gradient. They express various B cell antigens such as CD19, CD20, CD21, CD22 and CD40, as well as the adhesion molecules LFA-1, LFA-3 and CD44. They are positive for surface IgM and negative for surface IgD. Although these results suggest a phenotype of activated B cells, the CD77+ cells are negative for the classical activation antigens: CD23 (the low-affinity Fc receptor for IgE), CD25 [the interleukin (IL) 2 receptor alpha chain] and CD71 (the transferrin receptor). Proliferation and protein synthesis of CD77+ cells was measured after stimulation with a range of mitogens and IL. None of the agents tested are able to induce proliferation and protein synthesis with the exception of a combination of recombinant IL 4 plus anti-CD40 antibody. When examined by electron microscopy, CD77+ B lymphocytes present a morphology similar to that of cells undergoing programmed cell death, also called apoptosis (i.e. chromatin condensation, nuclear fragmentation, membrane blebbing). As shown by direct examination of DNA, these CD77+ cells are indeed in the process of apoptosis. Treatment of the CD77+ cells by recombinant IL 4 and anti-CD40 antibody prevents apoptosis. All these results suggest that the CD77 molecule defines a B lymphocyte maturation pathway, specific for germinal center, where the cells undergo programmed cell death.

Antibodies, Monoclonal↗