A new development of triterpene acid-containing extracts from Viscum album L. displays synergistic induction of apoptosis in acute lymphoblastic leukaemia

Objectives: Aqueous Viscum album L. extracts are widely used for anti‐cancer therapies. Due to their low solubility, triterpenes (which are known to act on cancers), do not occur in aqueous extracts in significant amounts. Using cyclodextrins, we have found it possible to solubilize mistletoe triterpene acids and to determine their effects on acute lymphoblastic leukaemia (ALL) in vitro and in vivo. Materials and methods: A C.B‐17/SCID model of pre‐B ALL (NALM‐6) was used to test efficacy and mechanisms of treatment with lectin‐ and triterpene acid containing preparations in vivo. Cytotoxicity of increasing concentrations of V. album L. preparations was assessed in vitro. Apoptosis was determined using mitochondrial membrane potential measurements, annexin V/PI, western blot analyses and caspase inhibitor assays. Results: Solubilized triterpene acid‐ or lectin‐containing V. album L. extracts inhibited cell proliferation and demonstrated cytotoxic properties in vitro. Annexin V/PI and mitochondrial membrane potential assays indicated that dose‐dependent induction of apoptosis was the main mechanism. Combination (viscumTT) of lectin‐ (viscum) and triterpene‐containing (TT) extracts resulted in greatest induction of apoptosis. Furthermore, caspase activity demonstrated that these extracts were able to induce apoptosis through both caspase‐8 and ‐9 dependent pathways. In vivo experimentation showed that treatment of mice with viscumTT combination prolonged mean survival to 50.5 days compared to 39.3 days in the phosphate‐buffered saline group. Conclusion: Here for the first time, we have demonstrated that either solubilized triterpene acids or lectins and combinations thereof, induce dose‐dependent apoptosis in the ALL cell line NALM‐6 via caspase‐8 and ‐9 dependent pathways.     

Trans fatty acids induce apoptosis in human endothelial cells.

The present study was designed to investigate the hypothesis that trans fatty acids can induce apoptosis of human umbilical vein endothelial cells (HUVEC). To test this hypothesis apoptosis was measured in HUVEC treated with 0.1, 1.0 or 5.0 mM trans elaidic acid (t-18:1) or linoelaidic acid (t,t-18:2) for 24 hours. For the detection of apoptosis, TdT-mediated dUTP nick end labelling assay (TUNEL), cell binding of annexin V and propidium iodide uptake were measured. Active Caspase-3 and cleaved PARP (poly-ADP-ribose polymerase) were also measured in the cell lysate. Moreover, cellular ability to produce ROS (reactive oxygen species) was measured by DCF fluorescence Both acids studied induce both early (annexin-positive cells) and late stages of apoptosis (cells stained by propidium iodide) in a dose-dependent manner. Also the appearance of TUNEL-positive cells was induced by both trans fatty acids tested, in a dose dependent manner. Both trans acids induce apoptosis through their effect on Caspase-3 activity and on intracellular ROS production. It is worth emphasising that linoelaidic acid proved to be a more potent inducer of apoptosis and ROS production in endothelial cells than elaidic acid. The present studies suggest that trans fatty acids may play a role in damaging and death of vascular endothelial cells in atherosclerosis.     

Biochemical, histochemical and cell biological investigations on the actions of mistletoe lectins I, II and III with human breast cancer cell lines

Cytotoxicity of the mistletoe lectins I, II and III towards six human breast cancer cell lines was assessed using the Mossman assay. In addition, binding of the three mistletoe lectins to the separated membrane glycoproteins of these cell lines, the binding and uptake of these lectins into the cells in tissue culture and the binding of the lectins to histological preparations of these cell lines were analysed. The results indicate that there are quantitative differences concerning the toxicity of these three lectins towards the different cell lines. Furthermore, the lectin binding pattern in the cell lines differed. In Western blots, several membrane glycoproteins were labelled by the lectins. Our results indicate subtle differences between the three lectins with regard to the parameters mentioned above; however, the toxicity of all three lectins from mistletoe is so similar that they all seem suitable for the construction of immunotoxins.Dedication: This work is dedicated to one of the discoverers (amongst many other important contributions) ofHelix pomatia agglutinin, which plays an important role in metastasis research, Professor Dr G. Uhlenbruck on the occasion of his 65th birthday.     

Immortalized human endothelial cells have a decreased response to IL-1 secondary to an IL-1 receptor defective expression restored by corticosteroids

A human endothelial cell line is a convenient tool for exploring cell physiology and testing drugs and toxics. Several attempts have been made using SV40 to immortalize endothelial cells. We used human umbilical vein endothelial cells (HUVEC) transformed with a construct made of promoter of the vimentin gene and SV40 Tag. The proliferation of immortalized vascular endothelial cells (IVEC), as measured by [methyl-³H]thymidine incorporation, was compared to that of HUVEC in the presence of endothelial cell growth factor and cytokines: tumor necrosis factor- (TNF-), interleukin-1 (IL-1) and interferon- (IFN-). Inhibition of [methyl-³H]thymidine incorporation by IL-1 was lower than that observed with HUVEC, while TNF- reduced the proliferation of IVEC and HUVEC to similar extents. Induction of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin by TNF-, measured by a radiometric technique, was similar in IVEC and HUVEC, while the induction of E-selectin by IL-1 on IVEC was limited and significantly different from that observed on HUVEC (p<0.001). The number of ¹²⁵I-IL-1 binding sites on IVEC is 3-fold less than on HUVEC and the IL-1 receptor number was reduced. Dexamethasone treatment of IVEC restored their reactivity to IL-1 and corrected the IL-1 binding and the receptor number. These results showed that the introduction of SV40 gene not only immortalized the cell but also altered IL-1 receptor expression. This alteration may be improved by addition of corticosteroids to the cell culture, which extends the possibility of using IVEC as a model of endothelial cells.     

Cell adhesion markers are expressed by a stable human endothelial cell line transformed by the SV40 large T antigen under vimentin promoter control

Markers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII-related antigen. Treatment of IVEC cells with Interleukin-1β (IL-1 β) at 10 U.ml^?1 activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 μM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT-PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium. © 1993 Wiley-Liss, Inc.     

N-methyl-N'-nitro-N-nitrosoguanidine activates multiple cell death mechanisms in human fibroblasts

Response to genotoxic stress may trigger the activation of distinct mechanisms that serve to promote cell death, including apoptosis and necrosis. In this study we examined the response of human fibroblasts, either proficient or deficient for the damage-activated protein kinase ataxia telangiectasia-mutated (ATM), to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Analysis of both long- and short-term viability shows that both ATM-proficient YZ-5 and ATM-deficient EBS-7 fibroblasts display a cytotoxic response to MNNG. Consistent with activation of apoptosis in response to MNNG, we observed increased caspase-3 cleavage and activity, appearance of fragmented nuclei, and increased staining with annexin V in both ATM-proficient and -deficient fibroblasts. Flow cytometry demonstrated that these cell lines also display a nonapoptotic cell death in response to MNNG. This form of cell death is associated with activation of poly-ADP ribose polymerase (PARP), and analysis of PARP activity indicated increased protein poly(ADP-ribosylation) in YZ-5 when compared to EBS-7. This PARP activity was accompanied by apoptosis-inducing factor release and translocation from the mitochondria to the nucleus. Finally, the PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) or the caspase-3 inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone dramatically diminished the cytotoxic response to MNNG, reinforcing the roles for apoptotic and nonapoptotic cell death in human fibroblasts treated with MNNG. From these findings, we conclude that MNNG induces a heterogeneous death response in human fibroblasts.     

A phosphoramidon-sensitive metalloprotease induces apoptosis of human endothelial cells by Group B Streptococcus

We explored Group B Streptococcus (GBS)-induced apoptosis in human umbilical vein endothelial cells (HUVEC) and the role of phosphoramidon, a zinc metalloprotease inhibitor, in this process. GBS 90186 strain (serotype V, a blood isolate) and concentrated supernatant (CS) were used to investigate the viability and morphological alterations in HUVEC by Trypan blue uptake, electrophoresis in 2 % agarose gel and scanning electron microscopy assays. Apoptosis before and after phosphoramidon-treatment were verified by flow cytometry using annexin V-FITC labeling. Differences were considered significant when P < 0.05 using unpaired Student’s t test. GBS and CS induced HUVEC death by apoptosis (76.5 and 32 %, respectively) with an increasing pro-apoptotic Bax expression and decreasing anti-apoptotic Bcl-2 expression. Caspase-3 was activated during GBS-induced endothelial apoptosis. Phosphoramidon reduced 89.3 and 100 % of GBS and CS cell death by apoptosis, respectively. Some GBS strains may induce cell death by apoptosis with involvement of metalloproteases and signaling through the intrinsic pathway of apoptosis, which may contribute to GBS survival during sepsis of adults and neonates.     

Cleavage of Poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis

Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a target of caspases during apoptosis: its cleavage onto 89- and 24-kDa fragments is considered to be a hallmark of the apoptotic mode of cell death. Another hallmark is the activation of endonuclease which targets internucleosomal DNA. The aim of the present study was to reveal cell cycle phase specificity as well as the temporal and sequence relationships of PARP cleavage vis-à-vis DNA fragmentation in two model systems of apoptosis, one induced by DNA damage via cell treatment with camptothecin (CPT) (mitochondria-induced pathway) and another by the cytotoxic ligand tumor necrosis factor alpha (TNF-alpha) (cell surface death receptor pathway). PARP cleavage was detected immunocytochemically using antibody which recognizes its 89-kDa fragment (PARP p89) while DNA fragmentation was assayed by in situ labeling of DNA strand breaks. The frequency and extent of PARP cleavage as well as DNA fragmentation were measured by mutiparameter flow and laser scanning cytometry. PARP cleavage, selective to S phase cells, was detected 90 min after administration of CPT. PARP cleavage in the cells treated with TNF-alpha was not selective to any cell cycle phase and was seen already after 30 min. DNA fragmentation trailed PARP cleavage by about 30 min and showed a similar pattern of cell cycle specificity. PARP p89 was present in nuclear chromatin but at least in the early phase of apoptosis it did not colocalize with DNA strand breaks. The rate of cleavage of PARP molecules in individual cells whether induced by CPT or TNF-alpha was rapid as reflected by the paucity of cells with a mixture of cleaved and noncleaved PARP molecules. In contrast, DNA fragmentation proceeded stepwise before reaching the maximal number of DNA strand breaks. Although time windows for PARP cleavage vs DNA fragmentation were different at early stages of apoptosis, a good overall correlation between the cytometric assays of apoptotic cells identification based on these events was observed in both CPT- and TNF-alpha-treated cultures.     

Proteomes of umbilical vein and microvascular endothelial cells reflect distinct biological properties and influence immune recognition

Human umbilical vein endothelial cells (HUVEC) are widely used as a source of endothelial cells (EC). However, HUVEC characteristics cannot be extrapolated to other types of EC, particularly microvascular ECs. Our objective was to compare the proteomes of microvascular ECs and HUVEC. Proteomes of HUVEC and human microvascular pulmonary EC (HMVEC-P) and dermal EC (HMVEC-D) from healthy Caucasian donors were compared by 2D DIGE and MS. Fatty acid binding proteins 4 and 5 were among the 159 and 30 proteins spots found to have at least twofold change in expression between HUVEC and HMVEC-D and between HUVEC and HMVEC-P samples, respectively. Eight protein spots showed twofold changed expression between HMVEC-D and HMVEC-P samples. Ingenuity? analysis revealed that proteins differentially expressed between HUVEC and HMVEC-D samples interact with retinoic acid. In vitro tubulogenesis assays showed a differential effect of retinoic acid between HUVEC and HMVEC. Moreover, serum IgG from patients with a rare vascular disease, systemic sclerosis, showed distinct reactivity profiles in HUVEC and HMVEC-D protein extracts. The proteome profiles of HUVEC and microvascular EC differ noticeably, which reflects distinct biological properties and influence immune recognition.     

Molecular characterization of the recombinant A-chain of a type II ribosome-inactivating protein (RIP) from Viscum album coloratum and structural basis on its ribosome-inactivating activity and the sugar-binding properties of the B-chain

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the Achain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.     

Hyperglycemia alters PI3k and Akt signaling and leads to endothelial cell proliferative dysfunction

Diabetes mellitus is a major risk factor for the development of vascular complications. We hypothesized that hyperglycemia decreases endothelial cell (EC) proliferation and survival via phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathways. We cultured human umbilical vein ECs (HUVEC) in 5, 20, or 40 mM d-glucose. Cells grown in 5, 20, and 40 mM mannitol served as a control for osmotic effects. We measured EC proliferation for up to 15 days. We assessed apoptosis by annexin V and propidium iodide staining and flow cytometry, analyzed cell lysates obtained on culture day 8 for total and phosphorylated PI3k and Akt by Western blot analysis, and measured Akt kinase activity using a GSK fusion protein. HUVEC proliferation was also tested in the presence of pharmacological inhibitors of PI3k-Akt (wortmannin and LY294002) and after transfection with a constitutively active Akt mutant. ECs in media containing 5 mM d-glucose (control) exhibited log-phase growth on days 7-10. d-Glucose at 20 and 40 mM significantly decreased proliferation versus control (P < 0.05 for both), whereas mannitol did not impair EC proliferation. Apoptosis increased significantly in HUVEC exposed to 40 mM d-glucose. d-Glucose at 40 mM significantly decreased tyrosine-phosphorylated PI3k, threonine 308-phosphorylated-Akt, and Akt activity relative to control 5 mM d-glucose. Pharmacological inhibition of PI3k-Akt resulted in a dose-dependent decrease in EC proliferation. Transfection with a constitutively active Akt mutant protected ECs by enhancing proliferation when grown in 20 and 40 mM d-glucose. We conclude that d-glucose regulates Akt signaling through threonine phosphorylation of Akt and that hyperglycemia-impaired PI3k-Akt signaling may promote EC proliferative dysfunction in diabetes.     

Effect of stent coating alone on in vitro vascular smooth muscle cell proliferation and apoptosis

Synthetic polymers, like methacrylate (MA) compounds, have been clinically introduced as inert coatings to locally deliver drugs that inhibit restenosis after stent. The aim of the present study was to evaluate the effects of MA coating alone on vascular smooth muscle cell (VSMC) growth in vitro. Stainless steel stents were coated with MA at the following doses: 0.3, 1.5, and 3 ml. Uncoated/bare metal stents were used as controls. VSMCs were cultured in dishes, and a MA-coated stent or an uncoated bare metal stent was gently added to each well. VSMC proliferation was assessed by bromodeoxyuridine (BrdU) incorporation. Apoptosis was analyzed by three distinct approaches: 1) annexin V/propidium iodide fluorescence detection; 2) DNA laddering; and 3) caspase-3 activation and PARP cleavage. MA-coated stents induced a significant decrease of BrdU incorporation compared with uncoated stents at both the low and high concentrations. In VSMCs incubated with MA-coated stents, annexin V/propidium iodide fluorescence detection showed a significant increase in apoptotic cells, which was corroborated by the typical DNA laddering. Apoptosis of VSMCs after incubation with MA-coated stents was characterized by caspase-3 activation and PARP cleavage. The MA-coated stent induced VSMC growth arrest by inducing apoptosis in a dose-dependent manner. Thus MA is not an inert platform for eluting drugs because it is biologically active per se. This effect should be taken in account when evaluating an association of this coating with antiproliferative agents for in-stent restenosis prevention.     

Exogenous superoxide mediates pro-oxidative, proinflammatory, and procoagulatory changes in primary endothelial cell cultures

Endothelial dysfunction/activation underlies the development of long-term cardiovascular complications and atherosclerosis. The aim of this study was to examine a direct role for exogenous sublethal flux of superoxide on endothelial cell dysfunction. Human umbilical vein endothelial cells (HUVEC) were exposed to superoxide generated by 0.1 mM xanthine and 4 mU/ml xanthine oxidase for 15 min and essential endothelial functions were examined. Superoxide dismutase and/or catalase was used as scavenger for O(2)(-)/H(2)O(2) to determine the key culprit. HUVEC detachment was determined by neutral red uptake and apoptosis by annexin V binding. Inflammation was estimated by IL-8 mRNA expression and cellular adhesion molecules (CAM). eNOS and iNOS message and eNOS protein served as an indirect measure for NO. Procoagulable state was evaluated by estimating the intracellular tissue factor. Activation of endothelial NADPH oxidase was determined by lucigenin chemiluminescence. Sublethal superoxide dose evoked: (1) proinflammatory state manifested by increased IL-8 mRNA expression and CAM on the endothelial surface, (2) HUVEC apoptosis and activated endothelial NADPH oxidase, (3) increase in intracellular tissue factor, and (4) decrease in eNOS mRNA and protein and up-regulation of iNOS mRNA. We conclude that extracellular low flux of superoxide exhibits pleiotropic characteristics, triggering activation/dysfunction of endothelial cells.     

Development of annexin V mutants suitable for labeling with Tc(i)-carbonyl complex

(99m)Tc-annexin V can be used to image organs undergoing cell death during cancer chemotherapy and organ transplant rejection. We investigated whether the novel Tc-carbonyl labeling method would be suitable for annexin V. Two mutant molecules of annexin V, called annexin V-122 and annexin V-123, were constructed with N-terminal extensions containing either three or six histidine residues. These molecules were expressed cytoplasmically in E. coli and purified with a final yield of 33 mg of protein/L of culture. Analysis by SDS-PAGE, isoelectric focusing, gel filtration chromatography, and mass spectrometry confirmed the purity and homogeneity of the protein preparations. Both mutant proteins retained full binding affinity for cell membranes with exposed phosphatidylserine. Using the Tc-carbonyl reagent, both proteins could be labeled with (99m)Tc to specific activities of at least 10-20 microCi/microg with full retention of bioactivity. The radiolabeled proteins were stable when incubated with phosphate-buffered saline or serum in vitro, and there was no transchelation of label to serum proteins during in vitro incubation. In conclusion, annexin V can be modified near its N-terminus to incorporate sequences that form specific chelation sites for (99m)Tc-carbonyl without altering its high affinity for cell membranes.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Oligoclonal in vitro response of CD4 T cells to vesicles of mistletoe extracts in mistletoe-treated cancer patients

 Mistletoe (Viscum album) extracts are widely used in adjuvant cancer therapy. We have investigated the in vitro responsiveness of T cells from mistletoe-treated cancer patients and untreated healthy donors to various preparations of mistletoe extracts. Proliferation of peripheral blood mononuclear cells from treated but not from untreated patients was observed in response to therapeutically used mistletoe extracts prepared from apple (mali) or pine (pini) host trees. The strongest proliferation was induced by a vesicle preparation of mali extract. Activation was strongly inhibited by interleukin-10. Using a newly developed flow-cytometry assay, we determined that cell growth was restricted to CD4 T cells. Analysis with a panel of monoclonal antibodies against the variable region of the T cell receptor β chain (Vβ) revealed an oligoclonal pattern of CD4 T cell activation. These results indicate that therapeutic administration of mistletoe extracts sensitizes a restricted set of CD4 T lymphocytes in mistletoe-treated patients. Received: 25 May 1996 / Accepted: 9 January 1997     

Optimization of use of UEA-1 magnetic beads for endothelial cell isolation

Most endothelial cells (EC) in the body belong to the microvasculature. Isolation and subsequent culture of these microvessel EC contributes greatly to our understanding of the heterogeneity and vascular specificity that exist between one organ site and another. However, a major obstacle is the overgrowth of contaminating cells (fibroblasts, pericytes, smooth-muscle cells) in cultures. Since 1990 the use of magnetic beads in combination with either a lectin, Ulex europaeus agglutinin-1 (UEA-1), or a monoclonal antibody has represented a powerful tool for the isolation/purification of microvessel EC. In the former case, operative conditions remain to be optimized to obtain pure cultures of EC.We have performed studies to optimize conditions of use for magnetic beads coated with UEA-1. Incubating beads with cells, the influences are studied of time, temperature, cell concentration, and number of beads per target cell for two cell types, human umbilical vein EC (HUVEC) and skin fibroblasts (HSF), either isolated or mixed. The effect of the last parameter was also checked on the behavior of cells undergoing proliferation after isolation. Results, expressed as isolation efficiency (from 40% to 90%) allowed us to select a 15-min incubation time at 4°C with rotary agitation, an optimal concentration of 4 x 10⁵ cells/ml, and an optimal cell:bead ration of 1:3. From a mixed cell population and in these conditions, even very low HUVEC:HSF proportions of 2.5:97.5 allowed us to obtain a pure HUVEC population in subsequent culture.Abbreviations UEA-1 Ulex europaeus agglutinin-1 - EC endothelial cells - HUVEC human umbilical vein endothelial cells - HSF human skin fibroblasts - MPC magnetic particle concentrator - IE isolation efficiency     

Wheat germ agglutinin and other selected lectins increase synthesis of decay-accelerating factor in human endothelial cells.

Decay accelerating factor (DAF) is a cell-surface phosphatidylinositol-anchored protein that protects the cell from inadvertent complement attack by binding to and inactivating C3 and C5 convertases. We have measured DAF on human umbilical vein endothelial cells (HUVEC) by immunoradiometric assay after its removal by phosphatidylinositol-specific phospholipase C or Nonidet P-40 detergent extraction and have previously demonstrated that DAF synthesis can be stimulated by phorbol ester activation of protein kinase C. We now report that although stimulation (4-48 h) of HUVEC with various cytokines, including TNF, IL-1, and IFN-gamma, did not alter DAF levels, wheat germ agglutinin (WGA) (5-50 micrograms/ml), a lectin specific for binding N-acetyl neuraminic acid and N-acetyl glucosamine residues, increased DAF levels fivefold when incubated with HUVEC for 12 to 24 h. The lectins Con A and PHA also stimulated DAF expression twofold, whereas a number of others including Ulex europaeus, Bandeiraea simplicifolia lectin I, and Ricinus communis agglutinin I, which bind to endothelial cells, were inactive. The increase in DAF by WGA was inhibited by N-acetyl glucosamine (10-50 mM) but by neither N-acetyl neuraminic acid nor removal of surface N-acetyl neuraminic acid with neuraminidase. However, succinylated WGA, which has unaltered affinity for N-acetyl glucosamine but not longer binds N-acetyl neuraminic acid, was inactive. These data suggest that the binding of WGA to sugar residues alone is not sufficient to trigger DAF expression and that occupation of additional, specific sites are required. The increase in DAF levels on HUVEC was blocked by inhibitors of RNA and protein synthesis. We conclude that continuous occupation by WGA of specific binding sites on HUVEC triggers events leading to DAF synthesis. This unique, long term stimulation of endothelial cells by lectins may be relevant to cell:cell interactions at the endothelium.