Inhibition of poly(ADP-ribosyl)ation does not prevent lymphocyte entry into the cell cycle.

The enzyme poly(ADP-ribosyl)transferase (ADPRT) becomes activated soon after a mitogenic stimulus is applied to lymphocyte cultures. It has also been reported that ADPRT inhibitors prevent cell proliferation when added to cultures at the same time as the mitogen. While this has been ascribed to the need to seal physiologically present DNA strand breaks before cells enter S phase, the presence of DNA strand breaks in quiescent human lymphocytes has been recently questioned. We demonstrate here that non-toxic concentrations of ADPRT inhibitors do not affect lymphocyte blastization and proliferation, as measured by thymidine incorporation and cytofluorimetry. We therefore suggest that ADPRT activation is required for late functions which are not needed for cell cycle progression.

Influence of benzamide on killing and mutation of density-inhibited V79 cells by MNNG.

Density-inhibited V79 cells when held for 24 h in complete medium after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) show improved survival levels and decreased mutant frequencies at all dose levels, compared to cells not so held. However, when benzamide, an inhibitor of poly(ADP-ribose) synthesis was present during this 24-h holding, the improvement in survival and decrease in mutant frequencies were not observed. Rather, compared to the control, the cells became more sensitive to MNNG and mutant frequency also increased significantly for all doses studied.

Evidence of a regulatory role of the level of poly (ADP-ribose) in chromosomal proteins in metallothionein gene expression by glucocorticoids but not by heavy metals.

The induction capacity of dexamethasone, a synthetic glucocorticoid, for the synthesis of metallothionein was about the same as that of 3-aminobenzamide, which is an inhibitor of ADP-ribosylation of chromosomal proteins, in cultured mouse mammary tumor cells. Both inductions of metallothionein were temporally correlated with a decrease in the amount of endogenous poly (ADP-ribose) on nonhistone high-mobility-group 14 and 17 proteins. In contrast, the extent of cadmium-induced metallothionein synthesis was 2-3-times that of dexamethasone or 3-aminobenzamide. However, cadmium had essentially no effect on de-ADP-ribosylation of these proteins.

Poly-ADP-ribosylated histones: potent DNA suppressors.

When rat liver nuclear chromatin was sonicated in buffer containing 0.35 M (NH4)2SO4 to release the engaged RNA polymerases, a potent inhibitor was also released. This inhibitor elicited dramatic inhibition of RNA synthesis regardless of whether the free or engaged RNA polymerase was used. On further analysis, it became apparent that the site of inhibition was on the DNA template, not on the enzyme. This inhibitor could be extracted into 0.25 N HCl by the standard procedure for the isolation of histones. This acid-soluble inhibitor, showing typical histone band on gel, was RNase A and DNase I resistant, but was sensitive to both pronase and snake venom phosphodiesterase digestion, as well as to 0.1 N KOH hydrolysis. Furthermore, when [14C]adenine labeled poly-ADP-ribosylated histones were digested by snake venom phosphodiesterase, the release of radioactivity was in parallel to the loss of inhibitor activity. We conclude that the inhibitor substances are poly-ADP-ribosylated histones and propose that the poly-ADP-ribosylated histones rather than the histones are the natural suppressors of the gene.

Decrease in ADP-ribosylation of HeLa non-histone proteins from interphase to metaphase.

Variations for non-histones in the ADP-ribosylating activities of interphase and metaphase cells were investigated. 32P-Labeled nicotinamide adenine dinucleotide ([32P]NAD), the specific precursor for the modification, was used to radioactively label proteins. Permeabilized interphase and mitotic cells, as well as isolated nuclei and chromosomes, were incubated with the label. One-dimensional and two-dimensional gels of the proteins of total nuclei and chromatin labeled with [32P]NAD showed more than 100 modified species. Changing the labeling conditions resulted in generally similar patterns of modified proteins, though the overall levels of incorporation and the distributions of label among species were significantly affected. A less complex pattern was found for nuclear scaffolds. The major ADP-ribosylated proteins included the lamins and poly(ADP-ribose) polymerase. Inhibitors of ADP-ribosylation were effective in preventing the incorporation of label by most non-histones. Snake venom phosphodiesterase readily removed protein-bound 32P radioactivity. A fundamentally different distribution of label from that of interphase nuclei and chromatin was found for metaphase chromosome non-histones. Instead of 100 or more species, the only major acceptor of label was poly(ADP-ribose) polymerase. This profound change during mitosis may indicate a structural role for ADP-ribosylation of non-histone proteins.

A comparison of the toxic and SCE-inducing effects of inhibitors of ADP-ribosyl transferase in Chinese hamster ovary cells.

The effects of inhibitors of ADP-ribosyl transferase (ADPRT), an enzyme believed to function in the maintenance of normal cellular functions, on specific genotoxic endpoints were examined in Chinese hamster ovary cells. Two classes of inhibitors, which differed both in their mechanism and efficiency of inhibition, were compared. Each of the inhibitors was found to increase the sister-chromatid exchange (SCE) frequency, reduce cloning efficiency, slow cell growth and potentiate methyl nitrosourea (MNU)-induced toxicity. With nicotinamide and two of its analogs, the relative ability to induce genotoxic endpoints generally followed the reported level of enzymatic inhibition. That is, benzamide was the most effective at inducing SCE and reducing cell survival and growth measurements, followed by nicotinamide and 1-methyl nicotinamide. However, no single quantitative relationship was found between the SCE and reduced cell survival caused by these agents. Theophylline, a representative methylxanthine, was the most toxic on the basis of concentration, but less effective than benzamide at inducing SCE. A strong association was found between the ability of the inhibitors to reduce cell survival, affect cell growth and potentiate the toxic effects of MNU. Therefore, although no consistent quantitative relationship could be demonstrated between SCE induction and the other endpoints for all ADPRT inhibitors, a general relationship was found between the toxic properties of nicotinamide and its analogs and their ability to induce SCE.

[Poly (ADP-ribose), ADP-ribosylation of proteins and regulation of cell activity]. / Poli(ADF-riboza), ADF-ribozilirovanie belkov i reguliatsiia kletochnoi aktivnosti.

The nature of a before unknown biological activity of NAD as a substrate in protein modification reaction is considered. Upon enzymatic digestion of NAD its adenosinediphosphate ribose (ADPR) part is transferred to acceptor proteins. ADPR in its mono- or polymeric form is covalently linked to proteins at the expense of NAD's high energy bound. Negatively charged ADPR, in association with a protein, is able to alter the charge, conformation and biological activity of the latter. The reaction is important in structural rearrangements of chromatin, in the synthesis and repair of DNA, in cell growth and differentiation and in the mechanisms of actions of actions of bacterial toxins.