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.
Smets
L A
LA
Department of Experimental Therapy, The Netherlands Cancer Institute, (Antoni van Leeuwenhoek Huis), Amsterdam.
Loesberg
C
C
Janssen
M
M
Van Rooij
H
H
D019797:3-Iodobenzylguanidine; D036002:ADP Ribose Transferases; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002474:Cell-Free System / Q000378:metabolism; D002772:Cholera Toxin / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000378:metabolism; D007462:Iodobenzenes / Q000494:pharmacology*; D007939:Leukemia L1210 / Q000201:enzymology; D009536:Niacinamide / Q000494:pharmacology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D014407:Tumor Cells, Cultured
D001549:Benzamides; D007462:Iodobenzenes; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D009536:Niacinamide; D019797:3-Iodobenzylguanidine; D003907:Dexamethasone; C025160:3-aminobenzamide; D002772:Cholera Toxin; D036002:ADP Ribose Transferases