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

The ADP-ribosyl moiety of NAD+ is consumed in reactions catalyzed by three classes of enzymes: poly(ADP-ribose) polymerase, protein mono(ADP-ribosyl)transferases, and NAD+ glycohydrolases. In this study, we have evaluated the selectivity of compounds originally identified as inhibitors of poly(ADP-ribose) polymerase on members of the three classes of enzymes. The 50% inhibitory concentration (IC50) of more than 20 compounds was determined in vitro for both poly(ADP-ribose) polymerase and mono(ADP-ribosyl)transferase A in an assay containing 300 microM NAD+. Of the compounds tested, benzamide was the most potent inhibitor of poly(ADP-ribose) polymerase with an IC50 of 3.3 microM. The IC50 for benzamide for mono(ADP-ribosyl)transferase A was 4.1 mM, and similar values were observed for four additional cellular mono(ADP-ribosyl)transferases. The IC50 for NAD+ glycohydrolase for benzamide was approximately 40 mM. For seven of the best inhibitors, inhibition of poly(ADP-ribose) polymerase in intact C3H1OT1/2 cells was studied as a function of the inhibitor concentration of the culture medium, and the concentration for 50% inhibition (culture medium IC50) was determined. Culture medium IC50 values for benzamide and its derivatives were very similar to in vitro IC50 values. For other inhibitors, such as nicotinamide, 5-methyl-nicotinamide, and 5-bromodeoxyuridine, culture medium IC50 values were 3-5-fold higher than in vitro IC50 values. These results suggest that micromolar levels of the benzamides in the culture medium should allow selective inhibition of poly(ADP-ribose) metabolism in intact cells. Furthermore, comparative quantitative inhibition studies should prove useful for assigning the biological effects of these inhibitors as an effect on either poly(ADP-ribose) or mono(ADP-ribose) metabolism.     

Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture.

Primary cultures of embryonic chick skeletal myogenic cells were used as an experimental model to examine the possible role of mono(ADP-ribosyl)ation reactions in myogenic differentiation. Initial studies demonstrated arginine-specific mono(ADP-ribosyl)transferase activity in the myogenic cell cultures. We then examined the effect of a novel inhibitor of cellular arginine-specific mono(ADP-ribosyl)transferases, meta-iodobenzylguanidine (MIBG), on differentiation of cultured embryonic chick skeletal myoblasts. MIBG reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (15-60 microM) concentrations of MIBG blocked myoblast fusion, the differentiation-specific increase in creatine phosphokinase activity, and both DNA and protein accumulation in myogenic cell cultures. Meta-iodobenzylamine, an analog of MIBG missing the guanidine group, had no effect. Low concentrations of methylglyoxal bis-guanylhydrazone, a substrate for cholera toxin with a higher Km than MIBG, also had no effect, but higher concentrations reversibly inhibited fusion. These findings suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis. In addition, the total arginine-specific mono(ADP-ribosyl)transferase activity increased with differentiation in the myogenic cell cultures, and this increase was also blocked by MIBG treatment. Because high levels of activity were found in the membrane fraction derived from later, myotube cultures, the membrane fraction from 96-h cultures was incubated with [32P]NAD+ and subjected to electrophoresis and autoradiography. Three proteins, migrating at 21, 20, and 17 kDa, that were ADP-ribosylated in the absence, but not the presence, of MIBG were identified. These proteins may be endogenous substrates for this enzyme.     

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

Two classes of enzymes, poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferases, catalyze covalent attachment of multiple or single residues, respectively, of the ADP-ribose moiety of NAD+ to various proteins. In order to find good inhibitors of poly(ADP-ribose) synthetase free of side actions and applicable to in vivo studies, we made a large scale survey using an in vitro assay system, and found many potent inhibitors. The four strongest were 4-amino-1,8-naphthalimide, 6(5H)- and 2-nitro-6(5H)-phenanthridinones, and 1,5-dihydroxyisoquinoline. Their 50% inhibitory concentrations, 0.18-0.39 microM, were about two orders of magnitude lower than that of 3-aminobenzamide that is currently most popularly used. A common structural feature among all potent inhibitors, including 1-hydroxyisoquinoline, chlorthenoxazin, 3-hydroxybenzamide, and 4-hydroxyquinazoline, in addition to the four mentioned above, was the presence of a carbonyl group built in a polyaromatic heterocyclic skeleton or a carbamoyl group attached to an aromatic ring. Most of the inhibitors exhibited mixed-type inhibition with respect to NAD+. Comparative studies of the effects on poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase from hen heterophils revealed high specificity of most of the potent inhibitors for poly(ADP-ribose) synthetase. On the other hand, unsaturated long-chain fatty acids inhibited both enzymes, and saturated long-chain fatty acids and vitamin K1 acted selectively on mono(ADP-ribosyl)transferase. The finding of many inhibitors of ADP-ribosyltransferases, especially poly(ADP-ribose) synthetase, supports the view that ADP-ribosylation of proteins may be regulated by a variety of metabolites or structural constituents in the cell.     

Poly(ADP-ribose) metabolism in X-irradiated Chinese hamster cells: its relation to repair of potentially lethal damage

Nicotinamide-adenine dinucleotide (NAD+) is the substrate used by cells in poly(ADP-ribose) synthesis. X-irradiation of log-phase Chinese hamster cells caused a rapid decrease in NAD+ levels which was linearly dependent on radiation dose. The activity of ADP-ribosyl transferase ( ADPRT ) also increased linearly with radiation dose. The decrease of NAD+ was slower, and the increase in ADPRT activity was less pronounced, in a radiation sensitive line, V79- AL162 /S-10. An inhibitor of ADPRT , m-aminobenzamide, largely prevented the depletion of cellular NAD+ and reduced the rate at which ADPRT activity disappeared during post-irradiation incubation. Post-irradiation treatment with hypertonic buffer or with medium containing D2O--which inhibit repair of radiation-induced potentially lethal damage--enhanced the depletion of NAD+ and prevented the reduction in ADPRT activity following irradiation. The characteristics of the effects of treatment with hypertonic buffer on NAD+ metabolism were qualitatively similar to the effects that such treatment has on radiation-induced cell killing. These results suggest that poly(ADP-ribose) synthesis after irradiation plays a role in the repair of potentially lethal damage.     

Failure of Poly(ADP-Ribose) Polymerase Cleavage by Caspases Leads to Induction of Necrosis and Enhanced Apoptosis

Activation of poly(ADP-ribose) polymerase (PARP) by DNA breaks catalyzes poly(ADP-ribosyl)ation and results in depletion of NAD+ and ATP, which is thought to induce necrosis. Proteolytic cleavage of PARP by caspases is a hallmark of apoptosis. To investigate whether PARP cleavage plays a role in apoptosis and in the decision of cells to undergo apoptosis or necrosis, we introduced a point mutation into the cleavage site (DEVD) of PARP that renders the protein resistant to caspase cleavage in vitro and in vivo. Here, we show that after treatment with tumor necrosis factor alpha, fibroblasts expressing this caspase-resistant PARP exhibited an accelerated cell death. This enhanced cell death is attributable to the induction of necrosis and an increased apoptosis and was coupled with depletion of NAD+ and ATP that occurred only in cells expressing caspase-resistant PARP. The PARP inhibitor 3-aminobenzamide prevented the NAD+ drop and concomitantly inhibited necrosis and the elevated apoptosis. These data indicate that this accelerated cell death is due to NAD+ depletion, a mechanism known to kill various cell types, caused by activation of uncleaved PARP after DNA fragmentation. The present study demonstrates that PARP cleavage prevents induction of necrosis during apoptosis and ensures appropriate execution of caspase-mediated programmed cell death.     

ADP-Ribosylation of Highly Purified Rat Brain Mitochondria

Highly purified synaptic and nonsynaptic mitochondria were prepared from rat brain, and their ADP-ribosyl transferase and NAD glycohydrolase activities were investigated. Data show that there is no significant difference in ADP-ribosyl transferase activity between these two types of subcellular preparations. However, NAD glycohydrolase activity appeared to be much higher in nonsynaptic mitochondria. The specific activity of both enzymes was investigated in the presence of the inhibitor nicotinamide or its analogue 3-aminobenzamide or other adenine nucleotides, such as ATP or ADP-ribose. The inhibitory effect of nicotinamide or 3-aminobenzamide on ADP-ribosyl transferase appears rather weak compared with their effect on NAD glycohydrolase activity. However, ADP-ribose and ATP appeared more effective in inhibiting ADP-ribosyl transferase. Our results provide evidence for the existence of ADP-ribosyl transferase activity in rat brain mitochondria. When NAD glycohydrolase was inhibited totally by nicotinamide, the transfer of ADP-ribose from NAD to mitochondrial proteins still occurred. The chain length determinations show that the linkage of ADP-ribose to mitochondrial proteins is oligomeric.     

Cellular NAD+ and ATP levels in alkylation-induced cytotoxicity enhanced by an inhibitor of poly(ADP-ribose) synthesis

Alkylating agents cause a marked depletion of cellular NAD+ levels by activating nuclear ADP-ribosyl transferase (ADPRT), which utilizes NAD+ as a substrate in the synthesis of poly(ADP-ribose). As a consequence of NAD+ depletion, it is possible that cellular ATP pools could be depleted. Because of this, exogenously supplied NAD+ had been proposed as a way to counteract some of the effects of an alkylator. We found that exogenously supplied NAD+ significantly increased intracellular levels of NAD+ in MMS- and MNNG-treated V79 Chinese hamster cells. Cytotoxicity was not changed by the exogenously supplied NAD+, however. 3-Aminobenzamide (3-ABA), an ADPRT inhibitor, prevented the depletion of intracellular NAD+ by MMS or MNNG treatment and potentiated cytotoxicity. As was the case without 3-ABA, exogenously supplied NAD+ plus 3-ABA did not change the cytotoxicity, even though NAD+ levels were increased. Intracellular ATP levels were also measured and were found to be unaffected following MMS treatment, and only slightly depleted following MNNG treatment. Exogenously supplied NAD+ raised these levels above those for their respective controls. Because survival was unaffected by elevated levels of NAD+ and ATP, our results suggest that depletion of cellular NAD+ pools following MMS and MNNG treatment is not a critical factor in determining cytotoxicity for these V79 cells. The energy reserves of V79 cells, at doses of MMS or MNNG which kill 99% of the cells, are apparently adequate to maintain normal levels of ATP.     

N-acetyl-p-benzoquinone imine induces Ca2+ release from mitochondria by stimulating pyridine nucleotide hydrolysis.

The mechanism of N-acetyl-p-benzoquinone imine (NAPQI)-induced release of Ca2+ from rat liver mitochondria was investigated. The addition of NAPQI or 3,5-Me2-NAPQI (a dimethylated analogue of NAPQI with only oxidizing properties) to mitochondria resulted in the rapid and extensive oxidation of NADH and NADPH. High-performance liquid chromatographic analysis of mitochondrial pyridine nucleotides revealed that the formation of NAD+ and NADP+ was followed by a time-dependent net loss of total pyridine nucleotides as a result of their hydrolysis, with the formation of nicotinamide. Preincubation of the mitochondria with cyclosporin A completely prevented the quinone imine-stimulated release of sequestered Ca2+ from mitochondria. Cyclosporin A did not affect the ability of NAPQI or 3,5-Me2-NAPQI to oxidize NAD(P)H but prevented the quinone imine-induced hydrolysis of the pyridine nucleotides. Although there was no detectable change in total protein-bound ADP-ribose content during quinone imine-induced Ca2+ release from mitochondria, meta-iodobenzylguanidine, a competitive inhibitor of protein mono(ADP-ribosylation), prevented Ca2+ release by NAPQI and 3,5-Me2-NAPQI; meta-iodobenzylguanidine did not inhibit the quinone imine-induced NAD(P)H oxidation and only partially blocked hydrolysis of the oxidized pyridine nucleotides. It is concluded that NAPQI causes the oxidation of mitochondrial NADH and NADPH, and stimulates Ca2+ release as a result of the further hydrolysis of the oxidized pyridine nucleotides and protein mono(ADP-ribosylation).     

Stimulation of poly(ADP-ribose) synthesis by free radicals in C3H10T1/2 cells: relationship with NAD metabolism and DNA breakage

We have studied the effect of H2O2 and O2- produced by xanthine and xanthine oxidase on NAD catabolism, poly(ADP-ribose) synthesis, and production of DNA single-strand breaks in C3H10T1/2 cells. The results show a correlation between the induction of DNA single-strand breaks, the decrease of NAD pool, and the accumulation of polymer. New techniques, based on affinity chromatography and reversed-phase high pressure liquid chromatography, have allowed an accurate determination of polymer contents and showed a 20-fold stimulation of polymer biosynthesis induced by active oxygen species. Inhibition experiments performed with 3-aminobenzamide have shown that the decrease in NAD levels after exposure of cells to active oxygen species was caused by stimulation of poly(ADP-ribosyl)ation and of another cellular process.     

Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes

Micromolar concentrations of extracellular beta-NAD+ (NAD(e)+) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar NAD(e)+ generates both inositol 1,4,5-trisphosphate (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NAD(e)+-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y(11) receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y(11)-transfected 1321N1 astrocytoma cells: micromolar NAD(e)+ promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y(11) but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y(11), and the down-regulation of P2Y(11) expression by short interference RNA prevented NAD(e)+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that beta-NAD(e)+ is an agonist of the P2Y(11) purinoceptor and that P2Y(11) is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.     

Membrane-bound form of ADP-ribosyl cyclase in rat cortical astrocytes in culture

ADP-ribosyl cyclase activities in cultured rat astrocytes were examined by using TLC for separation of enzymatic products. A relatively high rate of [3H]cyclic ADP-ribose production converted from [3H]NAD+ by ADP-ribosyl cyclase (2.015+/-0.554 nmol/min/mg of protein) was detected in the crude membrane fraction of astrocytes, which contained approximately 50% of the total cyclase activity in astrocytes. The formation rate of [3H]ADP-ribose from cyclic ADP-ribose by cyclic ADP-ribose hydrolase and/or from NAD+ by NAD glycohydrolase was low and enriched in the cytosolic fraction. Although NAD+ in the extracellular medium was metabolized to cyclic ADP-ribose by incubating cultures of intact astrocytes, the presence of Triton X-100 in the medium for permeabilizing cells increased cyclic ADP-ribose production three times as much. Isoproterenol and GTP increased [3H]cyclic ADP-ribose formation in crude membrane-associated cyclase activity. This isoproterenol-induced stimulation of membrane-associated ADP-ribosyl cyclase activity was confirmed by cyclic GDP-ribose formation fluorometrically. This stimulatory action was blocked by prior treatment of cells with cholera toxin but not with pertussis toxin. These results suggest that ADP-ribosyl cyclase in astrocytes has both extracellular and intracellular actions and that signals of beta-adrenergic stimulation are transduced to membrane-bound ADP-ribosyl cyclase via G proteins within cell surface membranes of astrocytes.     

Inhibition of translational initiation in eukaryotic cells by calcium ionophore

Ca2+ has been recently reported to be required for high rates of translational initiation in GH3 pituitary cells (Chin, K.-V., Cade, C., Brostrom, C.O., Galuska, E.M., and Brostrom, M.A. (1987) J. Biol. Chem. 262, 16509-16514). In the present investigation low concentrations of the Ca2+ ionophores, A23187 and ionomycin, were found to rapidly suppress the Ca2+-dependent component of protein synthesis in GH3 cells. More ionophore was required to inhibit amino acid incorporation into protein as extracellular Ca2+ was increased. Pre-existing inhibitions of protein synthesis produced by low concentrations of ionophore at low extracellular Ca2+ concentrations were reversed by adjustment to high extracellular Ca2+. Treatment with ionophore reduced the cellular contents of polysomes and 43 S preinitiation complex to values equivalent to those found for Ca2+-depleted cells. Average ribosomal transit times were unaffected by ionophore, and treated cells retained the ability to accumulate polysomes when incubated with cycloheximide. Cell types, such as HeLa and Chinese hamster ovary, that normally display only a modest Ca2+-dependent component of protein synthesis, manifested a strong underlying Ca2+ dependence in amino acid incorporation and polysome formation following treatment with low concentrations of ionophore. Protein synthesis in GH3 or HeLa cells during recovery from heat shock and arsenite treatment was not affected by cellular Ca2+ depletion or ionophore treatment. On the basis of these results, Ca2+ ionophore is proposed to inhibit Ca2+-dependent translational initiation through facilitating the mobilization of sequestered intracellular Ca2+.     

Effect of 3-aminobenzamide on the process of ultraviolet-induced DNA excision repair

The effect of 3-aminobenzamide, a potent inhibitor of poly(ADP-ribosyl)ation, on UV-induced DNA excision repair was investigated. HeLa cells were treated with DNA replication inhibitors, hydroxyurea (HU) and 1-beta-D-arabinofuranosyl cytosine (araCyt), before and after ultraviolet light (UV) irradiation, to accumulate DNA single-strand breaks. The activity of poly(ADP-ribosyl)ation measured in the permeable cell system of HeLa cells was enhanced in a UV dose-dependent manner after the combined treatment with HU and araCyt in vivo. However, DNA repair synthesis in vitro was not affected by addition of 1 mM 3-aminobenzamide or nicotinamide, while incorporation of [3H]NAD in the same system was completely inhibited. Furthermore, neither the magnitude of UV-induced DNA single-strand breaks accumulated by the combined treatment of HU and araCyt nor the rate of their rejoining after release from the HU and araCyt block were influenced even in the presence of 10 mM 3-aminobenzamide. As the cytotoxicity of UV irradiation was significantly potentiated by 5 mM 3-aminobenzamide, these results suggest that poly(ADP-ribosyl)ation is involved in a process other than DNA excision repair induced by UV irradiation.     

Reduction of mono(ADP-ribosyl)ation of 20 kDa protein with maturation in rat testis: involvement of guanine nucleotides

When the homogenate prepared from immature rat testes was incubated with [32P]NAD, several proteins (90, 39 and 20 kDa) were ADP-ribosylated in the absence of bacterial toxins. This observation suggested the existence of an endogenous ADP-ribosyltransferase and substrates. The data that the digested product by phosphodiesterase of ADP-ribosylated 20 kDa protein was 5'-AMP suggested that 20 kDa protein was mono(ADP-ribosyl)ated. In addition, the mono(ADP-ribosyl)ation of 20 kDa protein was enhanced by guanine nucleotides such as GTP, GDP and GTP[gamma S], and decreased by the concentrations of 10 mM Mg2+. In contrast, the incorporation of ADP-ribose moiety from NAD to both 90 and 39 kDa proteins was not changed by guanine nucleotides. On the other hand, mono(ADP-ribosyl)ation of 20 kDa protein was not observed in the homogenate prepared from other tissues of the same rats. Furthermore, we found that mono(ADP-ribosyl)ation of 20 kDa protein was decreased with the maturation of the rats and that an endogenous mono(ADP-ribosyl)transferase and 20 kDa protein were located in the nuclei.     

Paracrine roles of NAD+ and cyclic ADP-ribose in increasing intracellular calcium and enhancing cell proliferation of 3T3 fibroblasts

CD38 is a bifunctional ectoenzyme synthesizing from NAD(+) (ADP-ribosyl cyclase) and degrading (hydrolase) cyclic ADP-ribose (cADPR), a powerful universal calcium mobilizer from intracellular stores. Recently, hexameric connexin 43 (Cx43) hemichannels have been shown to release cytosolic NAD(+) from isolated murine fibroblasts (Bruzzone, S., Guida, L., Zocchi, E., Franco, L. and De Flora, A. (2001) FASEB J. 15, 10-12), making this dinucleotide available to the ectocellular active site of CD38. Here we investigated transwell co-cultures of CD38(+) (transfected) and CD38(-) 3T3 cells in order to establish the role of extracellular NAD(+) and cADPR on [Ca(2+)](i) levels and on proliferation of the CD38(-) target cells. CD38(+), but not CD38(-), feeder cells induced a [Ca(2+)](i) increase in the CD38(-) target cells which was comparable to that observed with extracellular cADPR alone and inhibitable by NAD(+)-glycohydrolase or by the cADPR antagonist 8-NH(2)-cADPR. Addition of recombinant ADP-ribosyl cyclase to the medium of CD38(-) feeders induced sustained [Ca(2+)](i) increases in CD38(-) target cells. Co-culture on CD38(+) feeders enhanced the proliferation of CD38(-) target cells over control values and significantly shortened the S phase of cell cycle. These results demonstrate a paracrine process based on Cx43-mediated release of NAD(+), its CD38-catalyzed conversion to extracellular cADPR, and influx of this nucleotide into responsive cells to increase [Ca(2+)](i) and stimulate cell proliferation.     

Selective probing of ADP-ribosylation reactions with oxidized 2'-deoxy-nicotinamide adenine dinucleotide

A homogeneous preparation of an arginine-specific mono(ADP-ribosyl)transferase from turkey erythrocytes effectively utilized 2'-deoxy-NAD+ for the 2'-deoxy(ADP-ribose) modification of arginine methyl ester with an apparent Km of 27.2 microM and a Vmax of 36.4 mumol min-1 (mg of protein)-1. The adduct formed was also used as a substrate by an avian erythrocyte arginine(ADP-ribose)-specific hydrolase that generated free 2'-deoxy(ADP-ribose). In contrast, 2'-deoxy-NAD+ was not a substrate in the initiation or elongation reaction catalyzed by highly purified poly(ADP-ribose) polymerase from calf thymus. However, 2'-deoxy-NAD+ was a potent noncompetitive inhibitor of NAD+ in the elongation reaction catalyzed by the polymerase, with an apparent Ki of 32 microM. These results indicate that 2'-deoxy-NAD+ may be utilized to specifically identify protein acceptors for endogenous mono(ADP-ribosyl)transferases in complex biological systems that may contain a high activity of poly(ADP-ribose) polymerase, i.e., cell nuclei preparations.     

Extracellular NAD(+) induces calcium signaling and apoptosis in human osteoblastic cells

ADP-ribosyl cyclase/CD38 is a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD(+) (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing second messenger cyclic ADP ribose (cADPR). Furthermore, CD38 mediates cADPR influx inside a number of cells, thereby inducing Ca(2+) mobilization. Intracellularly, cADPR releases Ca(2+) from ryanodine-sensitive pools, thus activating several Ca(2+)-dependent functions. Among these, the inhibition of osteoclastic-mediated bone resorption has been demonstrated. We found that HOBIT human osteoblastic cells display ADP-ribosyl cyclase activity and we examined the effects of CD38 stimulation on osteoblasts function. Extracellular NAD(+) induced elevation of cytosolic calcium due to both Ca(2+) influx from the extracellular medium and Ca(2+) release from ryanodine-sensitive intracellular stores. Culturing these cells in the presence of NAD(+) caused a complete growth arrest with a time-dependent decrease of cell number and the appearance of apoptotic nuclei. The first changes could be observed after 24 h of treatment and became fully evident after 72-96 h. We propose a role of extracellular NAD(+) in bone homeostatic control.     

细胞周期及DNA损伤剂引起的细胞NAD含量变化及其意义

本文观察了FL细胞中ADP-核糖基转移酶(ADPRT)底物NAD含量的细胞周期性变化及其与DNA复制之间的关系。FL细胞NAD含最在G_1期最高,而在S期DNA合成高峰后0—3小时(S/G_2期)达到最低点。ADPRT抑制剂3 AB能够抑制NAD含量的细胞周期性变化,而且S期DNA合成亦受到抑制,并呈现S期延长,提示ADP-核糖基化作用可能参与DNA复制过程。本文还观察了三种DNA损伤剂MNNG、MMS及4NQO对处于细胞周期不同时相的FL细胞NAD含量的影响,以及ADPRT抑制剂3 AB及尼克酰胺对此影响的作用。证明ADPRT抑制剂可以特异地抑制DNA损伤性NAD含量下降而对正常FL细胞NAD含量及代谢抑制剂2,4-DNP所致的NAD含量下降没有影响。从而有可能建立一个以测量细胞内NAD含量为指标的简便、快速、特异的检测DNA损伤因子的方法。     

Poly(ADP-ribose) and the recovery from damage in Chinese hamster cells due to 5-bromodeoxyuridine photolysis

Exposure of Chinese hamster cells to near-u.v. light, following the uniform incorporation of 5-bromodeoxyuridine (BrdUrd) into their DNA, resulted in cell killing that was close to exponential. An inhibitor of poly(ADP-ribose) synthesis, 3-aminobenzamide (3-ABA), enhanced the cytotoxic effect of this treatment when present for 2 h at 20 mM after light exposure. The dose modifying factor was 1.4. Under conditions that resulted in a sigmoidal survival curve (a 30 min BrdUrd pulse in S phase, followed 90 min later by light exposure) the effect of 3-ABA was to remove the shoulder of the survival curve with very little change in its final slope. Using various inhibitors of ADP-ribosyl transferase (ADPRT) the enhanced cell killing was found to correlate with the inhibitors' relative potency. Cellular NAD+, the substrate for poly(ADP-ribose) synthesis, was rapidly depleted after exposure. This depletion was largely prevented by 3-ABA; the activity of ADPRT increased with the fluence of near-u.v. light; and the concentration of cellular NAD+ decreased with exposure. ADPRT activity was maximal immediately after exposure to near u.v. light and then decayed to pre-exposure levels within 30 min (37 degrees C). The enhanced cytotoxicity of BrdUrd + near-u.v. light, when followed by 3-ABA treatment, disappeared at a rate similar to that of the decay in ADPRT activity. We conclude from these results that poly(ADP-ribose) synthesis is important for the recovery from BrdUrd photolysis damage in DNA. Because this damage and its repair are relatively specific (e.g. compared to ionizing radiation) and relatively easy to manipulate, it could serve as a model system for the study of the role of poly(ADP-ribose) in the repair of DNA damage.