Pyridine nucleotide analog interference with metabolic processes in mitogen-stimulated human T lymphocytes.

The differential metabolic effects of three nicotinamide analogs, 6-aminonicotinamide, 3-aminobenzamide, and 5-methylnicotinamide, were analyzed in mitogen-stimulated preparations of human T lymphocytes. Mitogen stimulation with the phorbol ester TPA and a monoclonal antibody to the T3 cell surface antigen caused an increase in cellular NAD and ATP levels and a marked increase in glucose metabolism as demonstrated by an increase in cellular levels of glucose 6-phosphate and a sevenfold increase in radioactive CO2 formation from [l-14C]glucose. 6-Aminonicotinamide had drastic inhibitory effects on the mitogen-stimulated increases in NAD and ATP levels as well as on the metabolism of glucose. Treatment of the mitogen-stimulated cells with 6-aminonicotinamide also caused a marked increase in cellular levels of 6-phosphogluconate, suggesting inhibition of the hexose monophosphate shunt at 6-phosphogluconate dehydrogenase. Radioactive CO2 formation from [6-14C]glucose showed that metabolism through the tricarboxylic acid cycle was not used to compensate for the inhibition of the hexose monophosphate shunt pathway. Treatment of cells with 3-aminobenzamide had the opposite effect of 6-aminonicotinamide in that cellular NAD levels increased, presumable due to inhibition of poly(ADP-ribose) polymerase. 3-Aminobenzamide did not interfere with ATP or glucose 6-phosphate levels and did not cause significant elevations of 6-phosphogluconate. Thus, 6-aminonicotinamide appears to have direct inhibitory effects on the synthesis of both pyridine nucleotides and poly(ADP-ribose), whereas 3-aminobenzamide has its major inhibitory effect on poly(ADP-ribose) synthesis. 5-Methylnicotinamide also interferes with the mitogen-stimulated increase in NAD levels but not as effectively as 6-aminonicotinamide. The alterations in pyridine nucleotide metabolism resulting from treatment with these nicotinamide analogs can produce drastic and diverse alterations in pathways of glucose utilization and energy generation.

Role of nicotinamide adenine dinucleotide and adenosine triphosphate in glucocorticoid-induced cytotoxicity in susceptible lymphoid cells.

The possibility that corticosteroid cytotoxicity could be mediated by activation of poly(ADP-ribose) polymerase and consequent depletion of NAD and ATP was evaluated in steroid-sensitive S49.1 and steroid-resistant S49.143R mouse lymphoma cells and in lymphocytes from a patient with chronic lymphocytic leukemia. All cell types were shown to have the enzyme poly(ADP-ribose) polymerase and to increase activity in response to DNA strand breaks. Incubation of susceptible cells with 1 microM dexamethasone resulted in DNA strand breaks. Susceptible cells also showed a dose-dependent decrease in NAD and ATP that preceded loss of cell viability. These studies suggest that steroid-induced cytotoxicity in susceptible lymphocytes is due to the presence of DNA strand breaks that activate poly(ADP-ribose) polymerase to a sufficient degree to consume cellular pools of NAD with a consequent depletion of ATP and loss of cell viability.

Induction of the pyridine nucleotide synthesis pathway in mitogen-stimulated human T-lymphocytes.

Mitogen stimulation of purified human T-lymphocytes with the phorbol ester 12-O-tetradecanoyl, phorbol-13-acetate (TPA) and a monoclonal antibody to the T3 cell surface antigen caused a 6-11-fold increase in cellular levels of poly(ADP-ribose) polymerase, a 6-20-fold amplification of cellular NAD+ levels and a 3-21-fold increase in NADP+ levels. Treatment of the cells with a combination of the two mitogenic signals also caused a 5-20-fold increase in NMN pyrophosphorylase activity, a 3-14-fold increase in ATP-NMN adenylyl transferase activity, and a 5-13-fold increase in NAD kinase activity. This is the first report showing induction of these three enzymes as part of the mitogenic response in purified human T-lymphocytes. Maximum increases in activity of each of these three enzymes required the combined presence of TPA and monoclonal antibody to human T-cell T3 antigen anti-T3. Analysis of the relative enzyme levels indicates that NMN pyrophosphorylase is the rate-limiting enzyme for NAD synthesis and NAD kinase is the rate-limiting enzyme for NADP synthesis.

Metabolic consequences of DNA damage: DNA damage induces alterations in glucose metabolism by activation of poly (ADP-ribose) polymerase.

In this communication we show that activation of poly(ADP-ribose) polymerase by DNA damage can produce drastic alterations in carbohydrate metabolism. We examined alterations in NAD+, NADP+, ATP and glucose-6-phosphate in L1210 murine leukemia cells, following exposure to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. Treatment of cells with 20 micrograms/ml MNNG produced rapid depletion of NAD+ and ATP. The G-6-P pool showed a biphasic change: first the pool size decreased, then increased to a level greater than that present in control cells. Nicotinamide treatment prevented the total depletion of NAD+ and this in turn helped preserve the ATP pools and prevented the biphasic alteration in G-6-P pool sizes.