Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase.

Deoxyadenosine has been implicated as the toxic metabolite causing profound lymphopenia in immunodeficient children with a genetic deficiency of adenosine deaminase (ADA), and in adults treated with the potent ADA inhibitor deoxycoformycin. However, the biochemical basis for deoxyadenosine toxicity toward lymphocytes remains controversial. The present experiments have examined in detail the sequential metabolic changes induced in nondividing human peripheral blood lymphocytes by incubation with deoxyadenosine plus deoxycoformycin, or with 2-chlorodeoxyadenosine (CdA), an ADA resistant deoxyadenosine congener with anti-leukemic and immunosuppressive properties. The lymphotoxic effect of deoxyadenosine and CdA required their phosphorylation, and was inhibited by deoxycytidine. As early as 4 h after exposure to the deoxynucleosides, strand breaks in lymphocyte DNA began to accumulate, and RNA synthesis decreased. These changes were followed by a significant fall in intracellular NAD levels at 8 h, a drop in ATP pools at 24 h, and cell death by 48 h. Incubation of the lymphocytes with 5 mM nicotinamide, a NAD precursor and an inhibitor of poly(ADP-ribose) synthetase, prevented NAD depletion. The nicotinamide treatment also rendered the lymphocytes highly resistant to deoxyadenosine and CdA toxicity, without altering dATP formation or the accumulation of DNA strand breaks. The poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide exerted a similar although less potent effect. These results suggest that NAD depletion, probably triggered by poly(ADP-ribose) formation, is the principle cause of death in normal resting human lymphocytes exposed to deoxyadenosine plus deoxycoformycin, or to CdA.

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Mechanism of deoxyadenosine and 2-chlorodeoxyadenosine toxicity to nondividing human lymphocytes.

DNA strand breaks, NAD metabolism, and programmed cell death

In the past five years, poly(ADP-ribosyl)ation has developed greatly with the help of molecular biology and the improvement of biochemical techniques. In this article, we describe the physico-chemical properties of the enzymes responsible for the synthesis and degradation of poly(ADP-ribose), respectively poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase. We then discuss the possible roles of this polymer in DNA repair and replication as well as in cellular differentiation and transformation. Finally, we put forward various hypotheses in order to better define the function of this polymer found only in eucaryotes. (Mol Cell Biochem122: 171–193, 1993)

Molecular and biochemical features of poly (ADP-ribose) metabolism

Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo.

Poly(adenosinediphosphoribose) polymerase inhibitors stimulate unscheduled deoxyribonucleic acid synthesis in normal human lymphocytes.

Amplification of pyridine nucleotide pools in mitogen-stimulated human lymphocytes

A series of nicotinamide analogs were evaluated for their ability to inhibit L1210 cell poly(adenosine diphosphoribose) polymerase, and also for their ability to potentiate the cytocidal effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), nitrogen mustard, and γ-irradiation in L1210 cells growing in culture and in vivo. In vitro, nicotinamide, 5-methylnicotinamide, 6-aminonicotinamide, and benzamide effectively inhibited L1210 cell poly(adenosine diphosphoribose) polymerase; 1-methylnicotinamide, nicotinic acid, and benzoic acid did not. In culture, 6-aminonicotinamide potentiated the cytocidal effect of BCNU; however, it did not significantly potentiate the effects of nitrogen mustard or γ-irradiation. In vivo, both 6-aminonicotinamide and nicotinamide potentiated the cytocidal effect of BCNU; however, the concentrations of nicotinamide required for this effect were 10- to 20-fold higher than those of 6-aminonicotinamide. None of the analogs significantly potentiated the in vivo effect of nitrogen mustard or γ-irradiation. Treatment of L1210-bearing mice with varying combinations of BCNU and 6-aminonicotinamide produced a synergistic increase in life span; in some cases, the combination led to the production of long-term disease-free survivors.

https://cancerres.aacrjournals.org/content/canres/42/11/4382.full.pdf

Synergistic antileukemic effect of 6-aminonicotinamide and 1,3-bis(2-chloroethyl)-1-nitrosourea on l1210 cells in vitro and in vivo.

Poly(ADP-ribose) is synthesized from NAD+ in the nuclei of eukaryotic cells and the activity of the chromatin-bound enzyme, poly(ADP-ribose) polymerase, is markedly stimulated by DNA strand breaks1–7. Recent studies suggest that poly(ADP-ribose) is associated with the repair of DNA damage8–10 and the process of cellular differentiation11,12. Hence, abnormalities in the metabolism of poly(ADP-ribose) could be aetiologically related to some of the human genetic disorders of DNA repair, especially those that are characterized by congenital and developmental anomalies. Defects in poly(ADP-ribose) metabolism could arise from abnormalities in the enzyme poly(ADP-ribose) polymerase, abnormalities in levels of its substrate NAD+, the proteins that act as acceptors for poly(ADP)-ribosylation or polymer stability. We have examined cells from patients with genetic disorders of DNA repair to determine whether they contain the enzyme poly(ADP-ribose) polymerase, whether the enzyme increases its activity in response to DNA damage and whether the cells maintain normal levels of NAD+. We demonstrate here that cells from patients having Fanconi's anaemia have lower NAD+ levels than cells from normal donors. This abnormality may contribute to the disorders of DNA repair and congenital anomalies that characterize this disease13,14.

Abnormal NAD+ levels in cells from patients with Fanconi's anaemia.

Poly(ADP-ribose) polymerase is a chromosomal enzyme that is completely dependent on added DNA for activity. The ability of DNA molecules to activate the polymerase appears to be enhanced by the presence of DNA damage. In the present study, we used SV 40 DNA and SV 40 minichromosomes to determine whether different types of DNA damage and different chromosomal components affect stimulation of polymerase activity. Treatment of SV 40 minichromosomes with agents or conditions that induced single-strand breaks increased their ability to stimulate poly(ADP-ribose) synthesis. This stimulation was enhanced by addition of histone H1 at a ratio of 1 microgram of histone H1 to 1 microgram of DNA. Higher ratios of histone H1 to DNA suppressed the ability of SV 40 minichromosomes containing single-strand breaks to stimulate enzyme activity. Treatment of SV 40 minichromosomes or SV 40 DNA with HaeIII restriction endonuclease to produce double-strand breaks markedly stimulated poly(ADP-ribose) polymerase activity. The stimulation of poly(ADP-ribose) polymerase by double-strand breaks occurred in the absence of histone H1 and was further enhanced by adding histone H1 up to ratios of 2 to 1 relative to DNA. At higher ratios of histone H1 to DNA, the presence of the histone continued to enhance the poly(ADP-ribose) synthesis stimulated by double-strand breaks.

Donna M. Catino

Papers

Poly(adenosinediphosphoribose) polymerase inhibitors stimulate unscheduled deoxyribonucleic acid synthesis in normal human lymphocytes.

Amplification of pyridine nucleotide pools in mitogen-stimulated human lymphocytes

Synergistic antileukemic effect of 6-aminonicotinamide and 1,3-bis(2-chloroethyl)-1-nitrosourea on l1210 cells in vitro and in vivo.

Abnormal NAD+ levels in cells from patients with Fanconi's anaemia.

Activation of poly(adenosine diphosphate ribose) polymerase by SV40 minichromosomes: effects of DNA damage and histone H1