Synthesis Of Cyclic ADP-ribose Research Articles

Implications of NAD+ boosters in translational medicine.

Nicotinamide adenine dinucleotide (NAD+ ) is an essential metabolite in energy metabolism as well as a co-substrate in biochemical reactions such as protein deacylation, protein ADP-ribosylation and cyclic ADP-ribose synthesis mediated by sirtuins, poly (ADP-ribose) polymerases (PARPs) and CD38. In eukaryotic cells, NAD+ is synthesized through three distinct pathways, which offer different strategies to modulate the bioavailability of NAD+ . The therapeutic potential of dietarily available NAD+ boosters preserving the NAD+ pool has been attracting attention after the discovery of declining NAD+ levels in ageing model organisms as well as in several age-related diseases, including cardiometabolic and neurodegenerative diseases. Here, we review the recent advances in the biology of NAD+ , including the salubrious effects of NAD+ boosters and discuss their future translational strategies.

CD38 enhances the proliferation and inhibits the apoptosis of cervical cancer cells by affecting the mitochondria functions.

Cervical cancer is one of the most common malignant tumors in women all over the world. The exact mechanism of occurrence and development of cervical cancer has not been fully elucidated. CD38 is a type II transmembrane glycoprotein, which was found to mediate diverse activities, including signal transduction, cell adhesion, and cyclic ADP-ribose synthesis. Here, we reported that CD38 promoted cell proliferation and inhibited cell apoptosis in cervical cancer cells by affecting the mitochondria functions. We established stable cervical cancer cell lines with CD38 over-expressed. CCK8 assay and colony formation assay indicated that CD38 promoted cervical cancer cell proliferation. Nude mouse tumorigenicity assay showed that CD38 significantly promotes tumor growth in vivo. CD38 also induced S phase accumulation in cell cycle analysis and suppressed cell apoptosis in cervical cancer cells. Meanwhile, flow cytometry analysis of mitochondria functions suggested that CD38 decreased intracellular Ca2+ levels in cervical cancer cells and CD38 was involved in down-regulation of ROS levels and prevented mitochondrial apoptosis in cervical cancer cells. The percentage of cells with loss of mitochondrial membrane potential (Δψm) in CD38-overexpressed cervical cancer cells was less than control groups. Furthermore, we found an up-regulation of MDM2, cyclinA1, CDK4, cyclinD1, NF-kB P65, c-rel, and a downregulation of P53, P21, and P38 by Western blot analysis. These results indicated that CD38 enhanced the proliferation and inhibited the apoptosis of cervical cancer cells by affecting the mitochondria functions.

CD38 in the nucleus accumbens and oxytocin are related to paternal behavior in mice

BackgroundMammalian sires participate in infant care. We previously demonstrated that sires of a strain of nonmonogamous laboratory mice initiate parental retrieval behavior in response to olfactory and auditory signals from the dam during isolation in a new environment. This behavior is rapidly lost in the absence of such signals when the sires are caged alone. The neural circuitry and hormones that control paternal behavior are not well-understood. CD38, a membrane glycoprotein, catalyzes synthesis of cyclic ADP-ribose and facilitates oxytocin (OT) secretion due to cyclic ADP-ribose-dependent increases in cytosolic free calcium concentrations in oxytocinergic neurons in the hypothalamus. In this paper, we studied CD38 in the nucleus accumbens (NAcc) and the role of OT on paternal pup retrieval behavior using CD38 knockout (CD38−/−) mice of the ICR strain.ResultsCD38−/− sires failed to retrieve when they were reunited with their pups after isolation together with the mate dams, but not with pup, in a novel cage for 10 min. CD38−/− sires treated with a single subcutaneous injection of OT exhibited recovery in the retrieval events when caged with CD38−/− dams treated with OT. We introduced human CD38 in the NAcc of CD38−/− sires using a lentiviral infection technique and examined the effects of local expression of CD38. Pairs of knockout dams treated with OT and sires expressing CD38 in the NAcc showed more retrieval (83% of wild-type sire levels). Complete recovery of retrieval was obtained in sires with the expression of CD38 in the NAcc in combination with OT administration. Other paternal behaviors, including pup grooming, crouching and huddling, were also more common in CD38−/− sires with CD38 expression in the NAcc compared with those in CD38−/− sires without CD38 expression in the NAcc.ConclusionsCD38 in the NAcc and OT are critical in paternal behavior.

A Novel Fluorescent Cell Membrane-permeable Caged Cyclic ADP-ribose Analogue

Cyclic adenosine diphosphate ribose is an endogenous Ca(2+) mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca(2+) increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca(2+) release from endoplasmic reticulum, with concomitant Ca(2+) influx in Jurkat cells. Ca(2+) release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca(2+) influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca(2+) release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca(2+) release from endoplasmic reticulum via RyRs and triggers Ca(2+) influx via TRPM2.

Catalysis-Based Inhibitors of the Calcium Signaling Function of CD38

CD38 is a signaling enzyme responsible for catalyzing the synthesis of cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate; both are universal Ca(2+) messenger molecules. Ablation of the CD38 gene in mice causes multiple physiological defects, including impaired oxytocin release, that result in altered social behavior. A series of catalysis-based inhibitors of CD38 were designed and synthesized, starting with arabinosyl-2'-fluoro-2'-deoxynicotinamide mononucleotide. Structure-function relationships were analyzed to assess the structural determinants important for inhibiting the NADase activity of CD38. X-ray crystallography was used to reveal the covalent intermediates that were formed with the catalytic residue, Glu226. Metabolically stable analogues that were resistant to inactivation by phosphatase and esterase were synthesized and shown to be effective in inhibiting intracellular cADPR production in human HL-60 cells during induction of differentiation by retinoic acid. The inhibition was species-independent, and the analogues were similarly effective in blocking the cyclization reaction of CD38 in rat ventricular tissue extracts, as well as inhibiting the α-agonist-induced constriction in rat mesentery arteries. These compounds thus represent the first generally applicable and catalysis-based inhibitors of the Ca(2+) signaling function of CD38.

Cytosolic CD38 Protein Forms Intact Disulfides and Is Active in Elevating Intracellular Cyclic ADP-ribose

CD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca(2+) messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca(2+) signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254-Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism.

Involvement of phospholipase C-independent calcium-mediated abscisic acid signalling during Arabidopsis response to drought

The present study investigated whether Ca2+ mobilization independent of phosphoinositide-specific phospholipase C (PI-PLC) would delay wilting in Arabidopsis thaliana (L.) Heynh. cv. Columbia through mediating stomatal closure at abscisic acid (ABA) concentrations rising beyond a drought-specific threshold value. In wild type (WT) epidermis, the PI-PLC inhibitor (U73122) affected the stomatal response to 20 µM ABA but not to 30 µM ABA. Disruption in GTP-binding protein ά subunit 1 (GPA1) affected the stomatal response to 30 µM ABA, but not to 20 µM ABA. In the gpa1-4 mutant, the inhibitory effects of the Ca2+ buffer, 1,2-bis(0-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), the inactive mastoparan analogue, mas17 and the antagonist of cyclic ADP-ribose synthesis, nicotinamide, were differentially attenuated on 30 µM ABA-induced stomatal closure. By contrast, the NADPH oxidase atrbohD/F double mutation fully suppressed inhibition of 20 µM ABA-induced stomatal closure by BAPTA or U73122 as well as inhibition of 30 µM ABA-induced stomatal closure by BAPTA, mas17 or nicotinamide. On the contrary, The Al resistant alr-104 mutation modulated ABA-induced stomatal closure by a stimulatory effect of U73122 and an increased sensitivity to mas17, nicotinamide and BAPTA. Compared to WT, the atrbohD/F double mutant was more hypersensitive than the gpa1-4 mutant to wilting under the tested water stress conditions, whereas wilting was delayed in the alr-104 mutant. Since the atrbohD/F mutation breaks down ABA-induced Ca2+ signalling through fully preventing apoplastic Ca2+ to enter into the guard cells, these results showed that a putative guard cell GPA1-dependent ADP-ribosyl cyclase activity should contribute to drought tolerance within PI-PLC-independent-Ca2+-mediated ABA signalling.

Putative primary involvement of Arabidopsis phosphoinositide-specific phospholipase C1 within abscisic acid-induced stomatal closing

Stomatal closing to abscisic acid (ABA) was studied in leaf epidermal peels of a dexamethasone (Dex)-inducible transgenic line expressing the phospholipase C AtPLC1 antisense in the Columbia genetic background. In the absence of Dex, the Ca2+ buffer, ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and the phopholipase C inhibitor, 1-[6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl]-1H-pyrrole-2,5-dione (U73122) specifically inhibited the response to 20 µM ABA, whereas the Ca2+ buffer, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) inhibited the response to 20 or 30 µM ABA. Neither EGTA nor BAPTA increased the U73122 effect. Applying 30 µM Dex specifically affected 20 µM ABA-induced stomatal closing through reducing its magnitude as well as suppressing the EGTA, BAPTA and U73122 inhibitory effects. Neither Dex nor U73122 changed the specific inhibitory effects of both the antagonist of cyclic ADP-ribose synthesis, nicotinamide and the GTP-binding protein (G protein) modulators, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2 (GP Ant-2) and mas17 on 30 µM ABA-induced stomatal closing. When tested in combination, substituting nicotinamide for mas17, but not for GP Ant-2, enhanced their inhibitory effect to an extent that BAPTA did not increase. These results supported that AtPLC1 primarily mediates the Ca2+-dependent stomatal closing response to 20 µM ABA as much as 30 µM Dex did not affect 20 µM ABA-induced stomatal closing when tested on the wild type Columbia-4 ecotype. Furthermore, the present study suggested that Ca2+ mobilization did not involve any dependency between AtPLC1 and a putative G protein-coupled ADP-ribosyl cyclase at the tested ABA concentrations.

The role of dietary niacin intake and the adenosine-5′-diphosphate-ribosyl cyclase enzyme CD38 in spatial learning ability: is cyclic adenosine diphosphate ribose the link between diet and behaviour?

The pyridine nucleotide NAD+ is derived from dietary niacin and serves as the substrate for the synthesis of cyclic ADP-ribose (cADPR), an intracellular Ca signalling molecule that plays an important role in synaptic plasticity in the hippocampus, a region of the brain involved in spatial learning. cADPR is formed in part via the activity of the ADP-ribosyl cyclase enzyme CD38, which is widespread throughout the brain. In the present review, current evidence of the relationship between dietary niacin and behaviour is presented following investigations of the effect of niacin deficiency, pharmacological nicotinamide supplementation and CD38 gene deletion on brain nucleotides and spatial learning ability in mice and rats. In young male rats, both niacin deficiency and nicotinamide supplementation significantly altered brain NAD+ and cADPR, both of which were inversely correlated with spatial learning ability. These results were consistent across three different models of niacin deficiency (pair feeding, partially restricted feeding and niacin recovery). Similar changes in spatial learning ability were observed in Cd38- / - mice, which also showed decreases in brain cADPR. These findings suggest an inverse relationship between spatial learning ability, dietary niacin intake and cADPR, although a direct link between cADPR and spatial learning ability is still missing. Dietary niacin may therefore play a role in the molecular events regulating learning performance, and further investigations of niacin intake, CD38 and cADPR may help identify potential molecular targets for clinical intervention to enhance learning and prevent or reverse cognitive decline.

Design, synthesis, and biological activity of carbocyclic analogues of cyclic ADP-ribose, a Ca2+-mobilizing second messenger

An efficient method for the total synthesis of cyclic ADP-ribose (cADPR, 1) analogues was established. In this procedure, formation of the characteristic 18membered ring was key step, which was achieved by the AgNO3-or I2-promoted condensation with the phenylthiophosphate-type substrate forming an intramolecular pyrophosphate linkage. Using this method, a variety of carbocyclic analogues of cADPR have been synthesized to investigate the structure-activity-relationship, where cyclic ADP-carbocyclic-ribose (2) was identified as a stable and cell-type selective cADPR agonist.

Identification and Functional Characterization of the Hepatic Stellate Cell CD38 Cell Surface Molecule

The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, because these cells are the main producers of extracellular matrix proteins in the liver and contribute to the modulation of inflammatory responses via the secretion of several cytokines and the expression of adhesion molecules. The goal of the present study was to characterize cell surface proteins that regulate HSC activation. To this end, a panel of monoclonal antibodies (mAbs) was generated. mAb 14.27 recognized a protein of 45 kd that was highly expressed on HSCs. Affinity purification of this protein followed by sequencing revealed that protein to be CD38. We subsequently demonstrated that CD38 was constitutively expressed by HSCs and that its expression increased after in vitro and in vivo activation. mAb 14.27 induced an increase in cytosolic Ca2+ levels in HSCs, showing that it functions as an agonistic antibody. Moreover, the effects mediated by the CD38 mAb included induction of the proinflammatory cytokine interleukin-6 and up-regulation of the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecule. Collectively, our data suggest that CD38 can act as a regulator of HSC activation and effector functions.

Antagonists to Retinoid Receptors Down-Regulate CD38 Expression and Inhibit In Vitro Differentiation of Cord Blood Derived CD34+ Cells.

CD38, originally described as a differentiation marker, has emerged as an important multifunctional protein. Its most well-characterized function is the ability to catalyze the synthesis of cyclic ADP-ribose (cADPR) from NAD. However, its major enzymatic activity is the hydrolysis of NAD (NADase) implicating it as the major regulator of cellular NAD levels. CD38 expression increases with commitment and differentiation. It is not clear, however, whether such changes in CD38 are merely phenotypic, or reflect an active role for CD38 in the regulation of cell differentiation. The regulation of CD38 gene expression is under the direct control of retinoid receptors (RAR). Antagonists to RAR abolish up-regulation of CD38 gene expression as well as RA induction of granulocytic differentiation down-stream of the myeloid compartment.In the present study we evaluated the involvement of CD38 in the regulation of HPC differentiation by treatment of ex-vivo cultures with LMW antagonists, targeted to either CD38 expression or to its biological activities.CB derived CD34+ cells were cultured with cytokines (S,T,F,6). Treatment of these cultures with an RAR-antagonist (AGN194310) abolished the expression of surface CD38. After 3 weeks in culture, the content of CFC was 3 ±1.1-fold higher, the content of CD34+ cells was 2.4 ± 0.24-fold higher and percentage CD34+ cells displaying CD34+Lin− phenotype was by 35 ± 10-fold higher (p<0.05, n= 14) in RAR-antagonist (10−6M) compared to cytokines-only treated cultures. Colonies derived from RAR-antagonist treated cultures sustained high re-plating capacity, a property that was lost during the first 3-weeks of expansion with cytokines only. In long-term cultures, the peak of CFUc and CD34+ cell expansion of RAR-antagonist treated cultures was 6–10 weeks later than control cultures. At the peak of expansion, cumulative numbers of CD34+ and CFUc were by 130- and 512-fold higher (p<0.05, n=4), respectively, in treated than in control cultures. CFU-MIX colonies were exclusively observed in RAR-antagonist treated cultures (between weeks 7–10). Interestingly, limited (1 week) exposure to the RAR-antagonist was sufficient for this long-term effect. Similarly, we tested the effect of an RXR antagonist (LGN 100754) (10−9 – 10−5 M) on short- and long-term cultures. Treatment with the RXR-antagonist did not down-regulate CD38 expression and only slightly improved ex-vivo expansion parameters over cytokines-only treated cultures.We next evaluated whether inhibition of CD38 enzymatic activities will also modulate in-vitro differentiation of cultured cells. To this end, CD34+ cell cultures were treated with nicotinamide (NA), a non-competitive inhibitor of CD38 NADase, previously demonstrated to abolish its enzymatic activities. 3-week treatment with NA (5mM) resulted in a marked decrease in CD38 expression and a marked increase in the fraction of CD34+Lin− cells as compared to cytokines-only treated cultures (48.0 ± 3.7% vs. 2.8 ± 0.7% and 18.6 ± 3% vs. 0.7 ± 0.06%, n=6, p<0.05, respectively). As with the RAR-antagonist, long-term expansion potential, as determined by CFC and CD34+ cell content, was significantly higher in cultures treated with NA relative to cytokines-only treated cultures.These results demonstrate that both a pan-RAR antagonist and NA inhibit differentiation and promote ex-vivo expansion of progenitor cells, suggesting the possible involvement of CD38 protein in these processes.

Nicotinamide, a Potent SIRT2 Inhibitor, Delays Differentiation of Hematopoietic Progenitor Cells.

CD38, originally described as a differentiation marker, has emerged as an important multifunctional transmembrane protein. Its most intriguing and well-characterized function is its ability to catalyze the synthesis of cyclic ADP-ribose (cADPR) from NAD. Of particular interest is its presence on the inner membrane of the nucleus, suggesting that CD38/cADPR may play a direct role in mediating nuclear activation and gene expression. Our studies on ex vivo expansion of Hematopoietic Stem Cells (HSCs) have led us to test whether alteration of CD38 function carries the potential of affecting cell fate decisions of HSCs. Inhibition of CD38 enzymatic activity was achieved by treating CD34+ cell cultures with nicotinamide (NA), a well-known base-exchange inhibitor demonstrated to inhibit the synthesis of cADPR from NAD.We report here that exogenously added nicotinamide (5–10 mM) to CD34+ cell cultures supplemented with cytokines (SCF, TPO, IL-6, FLt3, +/− IL-3) resulted in significant enrichment of CD34+CD38− (79±9.3%, n=9) and CD34+CD38−Lin− (19±3%, n=8) cells, as compared with control cultures treated only with cytokines (6.3±1.8%, n=9, and 0.7±0.06%, n=8, respectively, p<0.01). The functionality of these early progenitor subsets was demonstrated using the extended LTC-CFC assay, performed in the absence of NA. These results raised the intriguing possibility that cADPR production may have a pivotal role in regulation of CD34+ cell fate. However, inhibition of cADPR downstream signal transduction pathways by its specific antagonist, 8-amino-cADPR did not yield any effect on CD34+ cell cultures, excluding the possibility that nicotinamide modulates CD34+ cell fate solely by inhibition of cADPR synthesis.Nicotinamide is also a well-known potent inhibitor of SIRT2, a unique NAD(+)-dependent type III histone deacetylase (HDAC) with mono-ADP-ribosyltransferase activity involved in gene silencing, metabolism, apoptosis and aging. NA blocks NAD(+) hydrolysis by binding to an adjacent conserved pocket, and is therefore suggested as the physiologically relevant regulator of SIRT2 enzymes. This additional function of nicotinamide raises the intriguing possibility that HSC enrichment achieved by nicotinamide treatment may be related to specific inhibition of SIRT2 deacetylase activity and modulation of chromatin architecture leading to re-activation of previously silenced genes.In line with this hypothesis, Milhem et all. recently reported that addition of trichostatin A, a specific HDAC (type I and II) inhibitor, along with a DNA hypomethylating agent, modulated HSC fate ex vivo resulting in the retention of stem cell phenotype, number, and function (Blood , 2004; 103; 4102).Ongoing work is aimed at elucidating whether inhibition of SIRT2 is specifically involved in NA mechanism of activity leading to modulation of hematopoietic stem cell fate in ex vivo conditions.

Nuclear CD38 in retinoic acid-induced HL-60 cells

The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD + and hydrolysis of either NAD + or cADPR to ADP-ribose. All- trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD + glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. With Western blotting, we detected in the nuclear protein fraction from RA-treated cells a ∼43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the ∼43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.

CD38 is expressed on human mature monocyte-derived dendritic cells and is functionally involved in CD83 expression and IL-12 induction.

Dendritic cell (DC) maturation is characterized by the gain or loss of immunological functions and by expression of distinctive surface receptors. CD38 is an ectoenzyme that catalyzes the synthesis of cyclic ADP ribose (a potent second messenger for Ca(2+) release), as well as a receptor that initiates transmembrane signaling upon engagement with its counter-receptor CD31 or with agonistic monoclonal antibodies. Since CD38 is expressed by resting monocytes, we aimed to monitor CD38 expression during the differentiation of human monocyte-derived DC (MDDC) and to investigate the possibility that CD38 plays a functional role during DC maturation. CD38 is down-modulated during differentiation into immature MDDC and expressed again upon maturation. The extent of CD38 expression is dependent on the stimulus adopted (LPS > IFN-gamma > CD40 cross-linking). Although weak, IFN-gamma consistently induces DC maturation. De novo-synthesized CD38 is enzymatically active, and its expression in mature (m) MDDC is dependent on NF-kappa B activity. However, CD38 is not merely a maturation marker but also mediates signaling in mMDDC, where it maintains its functions as a receptor. Activation via agonistic anti-CD38 mAb induces up-regulation of CD83 expression and IL-12 secretion, whereas disruption of CD38/CD31 interaction inhibits CD83 expression, IL-12 secretion and MDDC-induced allogeneic T cell proliferation.

Niacin and Carcinogenesis

The dietary status of niacin (vitamin B3) has the potential to influence DNA repair, genomic stability, and the immune system, eventually having an impact on cancer risk, as well as the side effects of chemotherapy in the cancer patient. In addition to its well-known redox functions in energy metabolism, niacin, in the form of NAD, participates in a wide variety of ADP-ribosylation reactions.Poly(ADP-ribose) is a negatively charged polymer synthesized, predominantly on nuclear proteins, by at least seven different enzymes. Poly(ADP-ribose) polymerase-1 (PARP-1) is responsible for the majority of polymer synthesis and plays important roles in DNA damage responses, including repair, maintenance of genomic stability, and signaling events for stress responses such as apoptosis. NAD is also used in the synthesis of mono(ADP-ribose), often on G proteins, with poorly understood roles in signal transduction. Last, NAD and NADP are required for the synthesis of cyclic ADP-ribose and nicotinic acid adenine dinucleotide (NAADP), two mediators of intracellular calcium signaling pathways. Disruption of any of these processes has the potential to impairgenomic stability and deregulate cell division, leading to enhanced cancer risk. There are various sources of evidence that niacin status does have an impact on cancer risk, including animal models of leukemogenesis and skin cancer, as well as epidemiological data from human populations.

Expression of CD38 in human promyelocytic leukemia HL-60 cell line during differentiation by niacin-related compounds.

It was found that three niacin-related compounds, isonicotinic acid, nicotinamide, and nicotinamide N-oxide, induced granulocytic differentiation in HL-60 cells. We investigated the expression of CD38, which catalyzes the synthesis of cyclic ADP-ribose, a Ca2+ mobilizer, during differentiation by niacin-related compounds. It was found that CD38 was induced by isonicotinic acid, whereas nicotinamide and nicotinamide N-oxide containing an amino group did not induce it. The difference in expression of CD38 may provide some useful information for the elucidation of the mechanisms of cell differentiation.

NAD(P) +-glycohydrolase from human spleen: a multicatalytic enzyme

NAD(P) +-glycohydrolase (NADase, EC 3.2.2.6) was partially purified from microsomal membranes of human spleen after solubilization with Triton X-100. In addition to NAD + and NADP +, the enzyme catalyzed the hydrolysis of several NAD + analogues and the pyridine base exchange reaction with conversion of NAD + into 3-acetylpyridine adenine dinucleotide. The enzyme also catalyzed the synthesis of cyclic ADP-ribose (cADPR) from NAD + and the hydrolysis of cADPR to adenosine diphosphoribose (ADPR). Therefore, this enzyme is a new member of multicatalytic NADases recently identified from mammals, involved in the regulation of intracellular cADPR concentration. Human spleen NADase showed a subunit molecular mass of 45 kDa, a p I of 4.9 and a K m value for NAD + of 26 μM. High activation of ADPR cyclase activity was observed in the presence of Ag + ions, corresponding to NADase inhibition.

Hydrolysis of NADP + by platelet CD38 in the absence of synthesis and degradation of cyclic ADP-ribose 2′-phosphate

CD38 is a multifunctional cell surface ectoenzyme that catalyzes both the synthesis of cyclic ADP-ribose from NAD + and its hydrolysis to ADP-ribose. In this work, we investigated the metabolism of NADP + by CD38 expressed on human platelets. Incubation of either platelet membranes or intact cells with NADP + resulted in the rapid and time-dependent accumulation of ADP-ribose 2′-phosphate that paralleled the consumption of the substrate. However, under the same conditions, synthesis of cyclic ADP-ribose 2′-phosphate was not observed. By immunoprecipitation experiments, we identified CD38 as the enzyme responsible for the observed NADP + glycohydrolase activity. The lack of detection of cyclic ADP-ribose 2′-phosphate was not due to its rapid hydrolysis, since direct incubation of platelet membranes with cyclic ADP-ribose 2′-phosphate did not result in the formation of ADP-ribose 2′-phosphate. By contrast, the same membrane samples expressed a significant ability to hydrolyze cyclic ADP-ribose to ADP-ribose. The absence of cyclic ADP-ribose 2′-phosphate hydrolase activity was also confirmed using high concentrations of substrate and by analysing both intact Jurkat T-lymphocytes and immunoprecipitated CD38. These results indicate that CD38, which is a multifunctional enzyme towards NAD +, displays exclusively a NADP + glycohydrolase activity and is unable to catalyze both the synthesis and the hydrolysis of cyclic ADP-ribose 2′-phosphate.

NADP+-Dependent Internalization of Recombinant CD38 in CHO Cells

CD38 is a 46-kDa type II transmembrane glycoprotein that catalyses the synthesis of cyclic ADP-ribose (cADPR) from NAD+. cADPR is a second messenger known to regulate intracellular Ca2+-induced Ca2+-release (CICR). A recent study has revealed that CD38 in Namalwa B cells undergoes internalization upon exposure to external NAD+. In this study, recombinant rat CD38 was expressed in Chinese hamster ovary (CHO) cells and the possibility of the protein to undergo internalization upon exposure to a substrate analog NADP+was examined. It was found that such treatment of CHO cells resulted in a decrease of ADP-ribosyl cyclase activity, as well as immunofluorescence of CD38 on the cell surface. The same treatment of CHO cells also resulted in intracellular clustering of CD38 molecules as revealed by confocal microscopic analysis. The internalized CD38 was purified using a streptavidin/biotin-based method and was found to exhibit both ADP-ribosyl cyclase and cADPR hydrolase activities. On immunoblot, the internalized CD38 appeared as a monomer of 46 kDa under reducing condition of SDS–PAGE. Our data demonstrate that NADP+can efficiently induce internalization of CD38, a process that may be important in the production of cADPR intracellularly to regulate CICR.