The Involvement of Poly(ADP‐ribose) Polymerase in the Degradation of NAD Caused by γ‐Radiation and N‐Methyl‐N‐Nitrosourea

Abstract

Both N-methyl-N-nitrosourea and y-radiation lower cellular NAD in mouse leukaemia cells (L1210) in a dose-dependent way. The minimum NAD level is reached 2 h after a brief exposure to N-methyl-N-nitrosourea, but within 15 min of y-irradiation. The cells remain metabolically active ; they are able to recover their control NAD levels and are impermeable to trypan blue.Several inhibitors of poly(ADP-ribose) polymerase inhibit the drop in cellular NAD caused by these two agents: 2 mM 5-methylnicotinamide, 1 mM theophylline or 1 mM theobromine inhibit the effect of N-methyl-N-nitrosourea on cellular NAD level; 200 pM thymidine, 500 pM 5-methylnicotinamide, 500 pM theophylline and 500 pM theobromine prevent the lowering of cellular NAD by y-irradiation. The extent to which the drop in cellular NAD is inhibited is dependent on both the concentration of cytotoxic agent and of polymerase inhibitor. Caffeine will inhibit the drop in NAD but only at 10 mM, while nicotonic acid is ineffective even at this dose.The activity of poly(ADP-ribose) polymerase in permeabilized cells immediately after y-radiation increases with dose up to 12 krad, giving a maximal 3.4-fold stimulation of the enzyme activity, whereas the degradation of NAD under conditions optimal for NAD glycohydrolase does not change. The activity of the polymerase shows a close temporal correlation with the NAD drop following both y-radiation and N-methyl-N-nitrosourea. The enzyme activity is maximal when the NAD content is decreasing at the highest rate and has returned to normal levels when it ceases falling.In permeabilized cells we can distinguish poly(ADP-ribose) polymerase and NAD glycohydrolase activity by their differential response to inhibitors. The polymerase is sensitive to 5-methylnicotinamide, theophylline, theobromine and thymidine ; the NAD glycohydrolase is sensitive to 5-methylnicotinamide and theophylline, but not to theobromine and thymidine.We propose that the decrease in cellular NAD level produced by y-radiation and by N-methyl-Nnitrosourea is caused by an increased flux through poly(ADP-ribose) mediated by an increased activity of poly(ADP-ribose) polymerase. This consequently lowers the cellular NAD level. This hypothesis implies an involvement of (ADP-ribose), in the cellular response to cytotoxic drugs.In 1956, Roitt was the first to show that cytotoxic agents inhibit glycolysis due to a lowering of the cellular NAD level [l]. He used the ethyleneimines, a group of alkylating agents, but other workers have observed a marked decrease in cellular NAD following ionizing radiation [2 -41.Abbreviations. ADP-ribose, adenosine(S')diphospho(S)-P-D-ribose; (ADP-ribose),, mixture of ADP-ribose, oligo(ADP-ribose) and poly(ADP-ribose); EGTA, ethylenebis(oxoethylenenitri1o)tetraacetic acid ; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid; Mes, 4-morpholineethanesulphonic acid ; S.D., standard deviation of the mean.Enzymes. NAD glycohydrolase (EC 3.2.2.6); poly(ADP-ribose) polymerase (EC 2.7.7.-).Streptozotocin, the methylnitrosourea deri...

The differential cytotoxicity of RSU 1069: Cell survival studies indicating interaction with DNA as a possible mode of action

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Nicotinamide, a missing link in the early stress response in eukaryotic cells: A hypothesis with special reference to oxidative stress in plants

“…oxidative stress [7] and exposure to various mutagens [8] or to UV light [6]. Activation of PADPRP does not occur at the gene level, but through interaction between PADPRP and damaged DNA [9].…”

Inhibitors of nuclear ADP‐ribosyl transferase retard DNA repair after N‐methyl‐N‐nitroso‐urea

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