Cornelia Loesberg

The effect of calcium on the secretion of factor VIII-related antigen by cultured human endothelial cells.

Cultured human endothelial cells derived from the umbilical cord vein are able to release factor VIII-related antigen into the culture medium. The experiments described in this paper show the presence of two pathways for the secretion of factor VIII-related antigen from endothelial cells. There is a basal release of this antigen, independent of the presence of extracellular calcium ions. This release can be inhibited by cycloheximide and is therefore directly related to de novo protein synthesis. Besides this basal release, there is an extra release of factor VIII-related antigen that can be stimulated by thrombin, the Ca2+-ionophore A23187 or phorbol myristate acetate. As demonstrated by immunofluorescence, the stimulus-inducible release originates from storage granules in the cells. This stimulus-inducible release is dependent on extracellular Ca2+ but independent of intracellular cAMP.

Cell cycle-dependent inhibition of human vascular smooth muscle cell proliferation by prostaglandin E1

We examined the influence of prostaglandins on the initiation of proliferation of growth-arrested human adult aortic and fetal smooth muscle cells. Prostaglandins of the E series (25 nM) exerted a significant (p less than or equal to 0.05) inhibitory effect on DNA synthesis. Inhibition was observed when PGE1 was added in the G1 phase of the cell cycle. PGE1 had no effect when added once DNA synthesis had started. Thus prostaglandins of the E series may inhibit the responsiveness of smooth muscle cells to the mitogenic action of critical growth factors, such as PGDF. This inhibitory response is cell-cycle dependent. Once smooth muscle cells have entered S phase, PGE1 is no longer effective. Our data also suggest that cAMP is involved in the PGE1-induced growth inhibition, since concomitant with PGE1 addition, cAMP levels rose rapidly; addition of the cAMP analogue db-cAMP resulted in a cell-cycle-dependent inhibition pattern comparable to that observed with PGE1.

Isolation of a storage and secretory organelle containing Von Willebrand protein from cultured human endothelial cells

Von Willebrand protein was synthesized and secreted by human endothelial cells in culture. Ca2+ ionophore A23187 and phorbol myristate acetate stimulated the release of Von Willebrand protein from the cultured cells. Stimulated release was accompanied by the disappearance of rod-like structures from the cultured endothelial cells immunostained for Von Willebrand protein, suggesting the existence of a storage organelle for Von Willebrand protein in these cells (Loesberg, C., Gonsalves, M.D., Zandbergen, J., Willems, C., Van Aken, W.G., Stel, H.V., Van Mourik, J.A. and De Groot, P.G. (1983) Biochim. Biophys. Acta 763, 160-168). Cultured human endothelial cells were fractionated on a density gradient of colloidal silica. Von Willebrand protein was found in two organelle populations: a buoyant one sedimenting with a variety of cell organelle marker enzymes, including those of the Golgi apparatus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum and plasma membrane fragments (peak density of this fraction: 1.08 g X ml-1), and a dense one with a peak density of 1.12 g X ml-1. The dense organelles containing Von Willebrand protein were apparently free of other organelles. Stimulating Von Willebrand protein release with phorbol myristate acetate or Ca2+ ionophore A23187 resulted in a decrease or even complete disappearance of Von Willebrand protein from the high-density organelle fraction, implying a role of this organelle in the stimulus-induced release of Von Willebrand protein. The Von Willebrand protein content of the buoyant fraction was lowered to some extent or did not change upon incubation of the cells with ionophore A23187 and phorbol myristate acetate. Restoration of Von Willebrand protein content of the dense organelle fraction after stimulation occurred within 2 days; this was accompanied by recurrence of immunostaining of rod-shaped structures in cells and an increase in cellular Von Willebrand protein. The excretion of restored Von Willebrand protein could be stimulated again.

Intracellular inhibition of mono(ADP-ribosylation) by meta-iodobenzylguanidine: specificity, intracellular concentration and effects on glucocorticoid-mediated cell lysis

meta-Iodobenzylguanidine (MIBG) is a high-affinity substrate for mono(ADP-ribosyl)transferase of cholera toxin and turkey erythrocyte membranes (Loesberg, C., Van Rooij, H. and Smets, L.A.(1990) Biochim. Biophys. Acta 1037, 92-99). In the present study the drug was investigated as a potential inhibitor of intracellular ribosyltransferases by competition with endogenous acceptors. To this end, MIBG was compared with the conventional ADP-ribosylation inhibitors nicotinamide and 3-aminobenzamide in cell-free ribosylation systems and in intact L1210 leukemia cells. Poly(ADP-ribose)polymerase (poly-ADPRP) was assayed by the DNAse-I-induced incorporation of [14C]NAD in nuclei of permeabilized L1210 cells. Mono(ADP-ribosyl)transferase (mono-ADPRT) was assayed as NAD linkage to [125I]iodoguanyltyramine catalysed by turkey erythrocyte membranes or activated cholera toxin. Poly-ADPRP was inhibited by nicotinamide (IC50 = 0.03 mM) and by 3-aminobenzamide (IC50 less than or equal to 0.03 mM) but was insensitive to MIBG. Conversely, mono-ADPRT was inhibited by MIBG (IC50 = approx. 0.1 mM) but not by 3-aminobenzamide and only weakly so by nicotinamide in high concentration (10 mM). In L1210 cells, intracellular levels of nicotinamide equilibrated at 60-70% of the extracellular drug concentrations assayed at 1 and 10 mM. In contrast, MIBG was concentrated 15-fold by nonspecific uptake. The preferential interference of the drugs with endogenous mono- or poly-ADP ribosylations, predicted from inhibitory capacity in vitro and intracellular concentrations, was confirmed by their effect on dexamethasone-induced lysis of L1210 cell lines. Inhibition of endogenous mono-ADPRT with 0.03 mM MIBG or 10 mM nicotinamide induced sensitivity to glucocorticoids in refractory L1210-wt cells. In contrast, inhibition of poly-ADPRP by 3-aminobenzamide or nicotinamide (1 mM each) did not confer susceptibility to refractory cells but enhanced the lytic process in the sensitive subline L1210-H7 or in L1210-wt cells sensitized by MIBG. These results indicate that MIBG is the first substrate for guanidino-specific mono-ADPRT which accumulates in intact mammalian cells and effectively competes with intracellular acceptors for endogenous enzymes.

Meta-iodobenzylguanidine (MIBG), a novel high-affinity substrate for cholera toxin that interferes with cellular mono(ADP-ribosylation)

Meta-iodobenzylguanidine (MIBG) is a guanidine analogue of the neurotransmitter norepinephrine. Radioiodinated [131I]MIBG is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. Moreover, non-radiolabelled MIBG exerts several cell-biological effects, tentatively ascribed to interference with cellular mono(ADP-ribosyl) transferases (Smets, L.A., Bout, B. and Wisse, J. (1988) Cancer Chemother. Pharmacol. 21, 9-13; Smets, L.A., Metwally, E.A.G., Knol, E. and Martens, M. (1988) Leukemia Res. 12, 737-743). In the present study it was investigated whether MIBG could serve as an acceptor for the ribosyl transferase activity of cholera toxin and of erythrocyte membranes. MIBG appeared a substrate for the cholera toxin-catalyzed transfer of the ADP-ribose moiety of NAD to arginine-like residues with the highest affinity for this enzyme reported as yet (Km = 6.5 microM). MIBG was also ADP-ribosylated by the mono(ADP-ribosyl)transferase(s) of turkey erythrocyte membranes. Moreover, the drug appeared a potent affector of the ADP-ribose linkage to membrane proteins by these enzymes. Interference by MIBG was stronger than by related guanyltyramine, the monoamine precursors of MIBG, meta-iodobenzylamine had no effect at all. In contrast, the drug failed to affect endogenous, O-linked poly(ADP-ribose) polymerase, induced in nuclei of S49-leukemia cells by deoxyribonuclease. Since MIBG is the first described drug that specifically interferes with the cellular N-linked mono(ADP-ribosyl) transferase reactions, it may be an important tool to elucidate the physiological role of this posttranscriptional protein modification.

Mitochondrial effects of the guanidino group-containing cytostatic drugs, m-iodobenzylguanidine and methylglyoxal bis (guanylhydrazone)

The involvement of mitochondrial damage in the antiproliferative effects of m-iodobenzylguanidine [MIBG] and methylglyoxal bis (guanylhydrazone) [methylGAG] was studied in human neuroblastoma SK-N-SH, mouse neuroblastoma N1E115 and mouse lymphosarcoma S49 cells. Proliferation of SK-N-SH cells was insensitive to MIBG (100 microM gave 15% inhibition), but sensitive to methylGAG (IC50 = 50 microM). MIBG and methylGAG were approximately equitoxic to N1E115 cells (IC50 of 92 and 87 microM, respectively). S49 cells were most sensitive to both MIBG (IC50 = 11 microM) and methylGAG (IC50 = 5 microM). In isolated sonicated mitochondria, MIBG inhibited respiration a complex I of the respiratory chain (EC50 = 0.5 mM), whereas methylGAG was much less effective (EC50 greater than 15 mM). In intact cells, MIBG at 31 microM impaired mitochondrial respiration and stimulated the glycolytic flux. In contrast, equimolar concentrations of methylGAG had no effect on oxygen consumption, ATP content, glucose consumption and lactate production. MethylGAG significantly increased putrescine levels in N1E115 and S49 cells within 12 hr via inhibition of S-adenosylmethionine decarboxylase. No such effects were seen in SK-N-SH cells for up to 48 hr. Equimolar concentrations of MIBG had no effect on the putrescine levels in the various cell lines, suggesting that MIBG did not inhibit S-adenosylmethionine decarboxylase. It is concluded that the antiproliferative mechanisms of the guanidino compounds are essentially different. MIBG inhibited mitochondrial respiration at complex I with concomitant stimulation of the glycolytic flux but was essentially without effect on polyamine levels. On the other hand, cytotoxicity of methylGAG was not associated with mitochondrial dysfunction.

The Norepinephrine Analog Meta-Iodo-Benzylguanidine (MIBG) as a Substrate for Mono(ADP-Ribosylation)

Human tumors of neuro-ectodermal origin such as pheochromocytoma, neuroblastoma and medullary thyroidoma, have often preserved the capacity of neuronal cells to recapture extracellular norepinephrine. Meta-iodo-benzylguanidine (MIBG; Wieland et al., 1980), is a functional analog of norepinephrine, derived from the neuron-blocking agents bretyllium and guanethidine which competes with biogenic amines for uptake and storage into chromaffin tissues. Radioiodinated [131I]- MIBG has been successfully applied for the scintigraphic visualization of adrenergic tissues (Wieland et al., 1981) and neuroendocrine tumors such as pheochromocytoma (Sisson et al., 1981; Shapiro et al., 1985), neuroblastoma (Treuner et al., 1984; Hoefnagel et al., 1985) and many other tumors of the APUD series (Von Moll et al., 1987). During the clinical application of [131I]-MIBG with radiotherapeutic intent, patients receive milligram amounts of a potentially active amine. We therefore investigated pharmacological properties of this novel radiopharmaceutical.