Laura Formentini

Flavio Moroni

MD Alberto Chiarugi
email: alberto.chiarugi@unifi.it

Detection and Pharmacological Modulation of Nicotinamide Mononucleotide (NMN) in vitro and in vivo

Keywords: NAD, PARP, mitochondria, FK866, NMNAT, NaPRT

The emerging key role of NAD consuming enzymes in cell biology has renewed the interest in NAD resynthesis through the rescue pathways. The first step of the nicotinamide-dependent NAD rescue pathway is operated by nicotinamide phosphoribosyl transferase (NaPRT) forming nicotinamide mononucleotide (NMN). Because of the difficulties in measuring NMN, numerous open questions exist about the pathophysiological relevance of NaPRT and NMN itself. Here, we describe a new method of fluorimetric NMN detection upon derivatization of its alkylpyridinium group with acetophenone. By adopting this method, we analyzed the kinetics of nicotinamidedependent NAD recycling in HeLa and U937 cells. Measurement of NMN contents in subcellular fractions revealed that the nucleotide is highly enriched in mitochondria, suggesting intramitochondrial NAD synthesis. NMN increases in cells undergoing hyperactivation of the NADconsuming enzyme poly(ADP-ribose) polymerase (PARP)-1, or exposed to gallotannin, a putative inhibitor of NMN-adenylyl transferases. Evidence that the inhibitor of NAD resynthesis FK866 selectively inhibits NaPRT having no effect on NMNAT activity is also provided. Importantly, NMN reduces NAD and ATP depletion in cells undergoing PARP-1 hyperactivation, significantly delaying cell death. Finally, we show that a single injection of FK866 in the mouse induces long-lasting (up to 16 h) but mild (20%) reduction of NMN contents in different organs, suggesting slow rate of basal NAD consumption in vivo. Data provide new information on the biochemistry and pharmacology of NAD biosynthesis, allowing a better understanding of pyridine nucleotide metabolism.

Pyridine nucleotides have long been considered hydride ion carriers exclusively involved in oxidoreduction reactions. However, the recent identification of different enzyme families having nicotinamide adenine dinucleotide (NAD) as substrate has significantly renewed the interest in the biochemistry and pharmacology of pyridine nucleotides [START_REF] Berger | The new life of a centenarian: signalling functions of NAD(P)[END_REF][START_REF] Belenky | NAD+ metabolism in health and disease[END_REF][START_REF] Magni | Enzymology of mammalian NAD metabolism in health and disease[END_REF]. In particular, being these enzyme families responsible for the irreversible transformation of NAD into different products, attention has been focused on the mechanisms of NAD resynthesis into eukaryotic cells. Classically, two metabolic pathways regulate the formation of NAD in mammalian cells; i.e. the de novo (also called the "kynurenine") pathway, leading to NAD from tryptophan, and the rescue pathway using the nicotinamide moiety produced as a by-product by the NAD-consuming enzymes. The Preiss-Handler route also contributes to pyridine nucleotide formation, transforming nicotinic acid absorbed from the gut into NAD [1;4;5]. Notably, a recently identified metabolic route leading to NAD formation from nicotinamide riboside also exists [6;7]. Conversely, vertebrates are unable to convert nicotinamide into nicotinic acid [START_REF] Brenner | Evolution of NAD biosynthetic enzymes[END_REF].

A central metabolite in NAD resynthesis is nicotinamide mononucleotide (NMN, Fig. 1). It is produced by nicotinamide phosphoribosyl transferase (NaPRT) and by nicotinamide riboside kinase (NRK). NMN is then converted into NAD by three isoforms of NMN-adenilyl transferase (NMNAT). Recently, NaPRT received much attention by the scientific community because of its pleiotypic functions. Specifically, besides being an intracellular enzyme involved in NAD resynthesis, the protein has been also identified as a released factor behaving as an adipokine and as an inflammatory cytokine [START_REF] Yang | Nampt/PBEF/Visfatin: a regulator of mammalian health and longevity?[END_REF]. Adipokines are fat tissue-derived hormones with central roles in metabolism and disease pathogenesis. A protein with the same amino acid structure of NaPRT has been identified as a 52 kDa release product of visceral fat and therefore called visfatin [9;10].

Controversies exist as for the endocrine properties of visfatin/NaPRT. Although originally identified as an insulin receptor-interacting protein able to improve glucose-stimulated insulin secretion, the protein is now thought to be devoid of insulin receptor-interacting properties but still able to regulate glucose homeostasis by promoting pancreatic islet insulin secretion. Of note, these properties are entirely dependent on NMN production by secreted visfatin/NaPRT [START_REF] Revollo | Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme[END_REF]. The NaPRT/visfatin/PBEF is released by various immune cells, promotes TNF, IL1 and IL-6 production, and is increased in the sera of patients affected by sepsis, acute lung injury, myocardial infarction, rheumatoid arthritis and inflammatory bowel disease. The protein is also able to inhibit neutrophil apoptosis, thereby promoting the immune response [START_REF] Luk | Pre-B cell colony-enhancing factor (PBEF)/visfatin: a novel mediator of innate immunity[END_REF]. Whether NMN production contributes to the immunoregulatory functions of NaPRT/visfatin/PBEF is still unknown but a specific receptor has not been identified. Also, the permeability of NMN through the plasma membrane is not clear and, again, a possible NMN-interacting receptor waits to be identified.

Given the potential relevance of NaPRT/visfatin/PBEF to pathophysiology, several ELISA kit able to detect the protein have been recently made commercially available. Yet, these kits have different sensitivities and are heterogeneous in nature. These inconsistencies are probably responsible for the apparent contrasting properties of secreted NaPRT/visfatin/PBEF present in the literature. Also, since the functional properties of NaPRT/visfatin/PBEF may in part depend on its enzymatic activity [11;13], evaluation of intra-as well as extracellular NMN production can certainly help understanding the pathophysiological role of the protein. Unfortunately, analytical determination of NMN is difficult because of its physicochemical features. Indeed, very few studies have measured NaPRT activity and/or NMN concentrations in biological fluids or tissue extracts using radioactive or complex HPLC/MS techniques [11;13;14]. In the present study we provide new information on NMN metabolism and pharmacological modulation by using an original method of NMN measurement as well as the newly identified inhibitor of NaPRT (E)-N-[4-(1-benzoylpiperidin-4-yl) butyl]-3-(pyridin-3-yl) acrilamide (FK866) [15;16].

Materials and methods

Cell culture and treatment

Unless otherwise stated, all chemicals and cell culture products were from Sigma (Milan, Italy).

HeLa or U937 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2mM glutamine, 10% fetal bovine serum and antibiotics. Cultures were brought to 50-70% confluence and exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 100 µM) , nicotinamide 3-yl) acrilamide (FK866, 1-100 µM) was obtained from NIH, Bethesda, USA. Cell viability was evaluated by measuring lactate dehydrogenase (LDH) release in the incubating media or reduction of methylthiazolyl tetrazolium (MTT) and described [START_REF] Cipriani | Nuclear poly(ADPribose) polymerase-1 rapidly triggers mitochondrial dysfunction[END_REF]. An inverted Nikon TE-2000U microscope equipped with a CDD camera was used for cell visualization.

Cell fractionation

As previously described [START_REF] Cipriani | Nuclear poly(ADPribose) polymerase-1 rapidly triggers mitochondrial dysfunction[END_REF], cells were disrupted using a glass/glass homogenizer in 500 µl of buffer A (Tris HCl 50 mM, pH 7.4, mannitol 225 mM, saccarose 75 mM, 1mM PMSF, 10 µl of protease inhibitor cocktail), and centrifuged at 600 g to obtain the nuclear pellet. Supernatants were centrifuged at 12000 g to obtain the mitochondrial pellet and the cytosolic fraction. Cell fractions were then processed for NMN determination as described above.

NMN, NAD and ATP measurement

Ultra pure NMN standards (Sigma, Milan, Italy) were dissolved in water and solutions analyzed by HPLC with UV or fluorimetric detection. For UV detection, cells grown in a 48 well plate were scraped with 100 µl of HCl 0.6. The cell extract was centrifuged at 14.000 g/5 min and 25 µL of the supernatant injected in an HPLC system consisting in a mobile phase of 0.1M buffer phosphate pH 6.5, 1% acetonitrile, 10 mM tetra butyl-ammonium bromide (TBAB), a Supelco 25 cm column (5µm) and an UV detector (Perkin Elmer) set at 260 nm. For fluorimetric detection, we modified a method previously described [START_REF] Putt | An enzymatic assay for poly(ADP-ribose) polymerase-1 (PARP-1) via the chemical quantitation of NAD(+): application to the high-throughput screening of small molecules as potential inhibitors[END_REF]. Briefly, cells grown in a 48 well plate were lysed with 100 µl of HClO 4 1N, whereas mouse organs were sonicated in HCLO 4 1N (1:4 w/v). Then, 100 µl of the extract were neutralized with KOH 1N and, after 5 min, additional 100 µl of 0.1 M bicine pH 7.4 were added. The cell extract was centrifuged at 14.000 g/5 min and 10 µL of the supernatant were mixed with 100 µl of KOH 1N and 50 µl of acetophenone. The solution was incubated for 15 min at 4°C, then 100 µl formic acid were added and the solution incubated 5 min at 100 °C. By means of this derivatization procedure, NMN is converted into a highly fluorescent compound as shown in NAD contents were quantified by means of an enzymatic cycling procedure as described [START_REF] Shah | Methods for biochemical study of poly(ADP-ribose) metabolism in vitro and in vivo[END_REF]. ATP was measured by the ATPlight kit (Perkin Elmer, Milan).

In vivo experiments

CD1 male albino mice (20-25 g) (n=4 per group) were injected i.p. with FK866 (100 mg/kg) and sacrificed 4, 8 and 16 h later. Organs were rapidly collected and processed as described above for NMN content determination. Procedures involving animals and their care were conducted in compliance with the Italian guidelines for animal care (DL 116/92) in application of the European Communities Council Directive (86/609/EEC) and was formally approved by the Animal Care Committee of the Department of Pharmacology of the University of Florence.

Results

Spectrofluorometric HPLC analysis of NMN

Isocratic HPLC analysis of NMN with UV absorbance detection is virtually impossible because of its extremely short elution time. Indeed, under our experimental conditions (see Methods), standard NMN had a retention time of 1'54''(Fig. 2B). Addition of tetra butyl-ammonium bromide (up to 10 mM) to the mobile phase to increase NMN retention only minimally influenced this parameter (not shown). We were unable to identify an unequivocal peak with a retention time corresponding to NMN in a whole cellular extract from different cell lines (Fig. 2C and not shown).

It is well known that N-alkylpyridinium compounds are transformed into fluorescent compounds through reactions with ketone moieties followed by heating in acidic environment. Accordingly, methyl-nicotinamide and NAD have been quantified spectrofluorometrically for different purposes [18;20]. We therefore attempted to adapt this method to NMN measurement (see Methods). Upon derivatization, NMN became fluorescent with excitation and emission maxima at 332 and 454 nm, A c c e p t e d M a n u s c r i p t 7 respectively (Fig. 2D). Of note, elution time of derivatized NMN standard was increased up to 4'18''(Fig. 2E), and a single peak with the identical elution time was present in a whole cellular extract derivatized as described above (Fig. 2F). The peak area linearly increased by spiking the extract with different amounts on NMN (not shown). Optimization of the derivatization procedure showed that NMN fluorescence increased linearly by augmenting the temperature of step 2 up to 100°C (Fig. 2G). At this temperature, incubations longer than 5 min reduced fluorescence (Fig. 2H). Maximal efficiency of step 1 was reached with derivatization times of 15 min or longer. NMN fluorescence increased linearly over the range of 8 to 2500 pmoles with a threshold sensitivity of 750 fmoles (Fig. 2I).

Modulation of NMN content in HeLa and U937 cells

Basal contents of NMN in U937 cells were 3,7 ± 0.1 nmol/mg prot, and about 10-fold lower in HeLa cells (0,48 ± 0.02 nmol/mg prot). These findings suggested low basal activity of NaPRT in HeLa cells. We therefore attempted to evaluate in intact cells the contribution of NaPRT to NMN contents by adding nicotinamide to the cell medium. As shown in Fig. 3A, nicotinamide increased NMN contents in a concentration-dependent manner in U937 but not in HeLa cells, again suggesting low expression levels/activity of NaPRT in this cell type. In contrast with this interpretation, however, addition of nicotinamide to the culture medium similarly increased NAD contents in both cell types.

Taken together, these findings suggested that NaPRT is active in both U937 and HeLa cells, and that NMN does not accumulate in HeLa because of its rapid transformation into NAD by NMNAT.

Reportedly, NaPRT is the rate-limiting enzyme of the mammalian NAD rescue pathway [START_REF] Sauve | NAD+ and vitamin B3: from metabolism to therapies[END_REF]. When NMN was added to the culture medium for 1 h, its intracellular contents increased in both cells types (Fig. 1C), indicating that the nucleotide readily permeates the plasma membrane.

Accordingly, NAD contents increased in both HeLa and U937 cells exposed to NMN (Fig. 3D). We also measured the subcellular distribution of NMN in these cell lines. As shown in Fig. 2E, NMN was significantly more concentrated in the mitochondrial fraction than in the cytosolic or nuclear ones. However, when subcellular NMN levels were expressed as % of total cellular content, the nucleotide amount appeared higher in the nuclear fraction (Fig. 3F).

A c c e p t e d M a n u s c r i p t 8

Pharmacological modulation of intracellular NMN contents

It has been repeatedly reported that, in condition of massive DNA damage, hyperactivation of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 leads to depletion of the nucleotide pool and cell death [START_REF] Chiarugi | PARP-1: killer or conspirator? The suicide hypothesis revisited[END_REF]. The cell's capability to face PARP-1 dependent NAD depletion by activating the NAD rescue pathway is therefore central to cell survival. Yet, how NMN concentrations vary when PARP-1 hyperactivates is still unknown. As shown in Fig. 4A andB, exposure of U937 cells to the prototypical PARP-1 activator MNNG [START_REF] D'amours | Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions[END_REF][START_REF] Ha | Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion[END_REF][START_REF] Yu | Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor[END_REF] only marginally increased NMN contents despite massive NAD depletion. The increase of NMN and depletion of NAD were prevented by the PARP-1 inhibitor phenanthridinone (PHE). These findings suggested that the rate of PARP-1-dependent NAD utilization exceeded that of NMNAT-mediated NAD resynthesis. However, the possibility of a compartmentalized pool of NMN not readily convertible into NAD must also be considered. Additionally, massive ATP depletion that typically follows PARP-1 activation could differently affect the activity of the ATP-dependent enzymes NaPRT and NMNAT. Gallotannin has been reported to inhibit NMNAT [START_REF] Berger | Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms[END_REF]. In keeping with this finding, we found that NMN contents increased in cells exposed to gallotannin (Fig. 4C). Evaluating the effect of the latter in cells undergoing PARP-1 hyperactivation indicated that the gallotannin-and PARP-1 activation-dependent increases of NMN were additive (Fig. 4C). FK866 is a recently identified inhibitor of NaPRT [15;16]. It is unknown, however, whether FK866 also inhibits NMNAT. We report here that FK866 similarly reduced both NAD and NMN contents in U937 cells (Fig. 4D andE), confirming its ability to inhibit NaPRT activity. However, increases in NAD contents prompted by the addition of NMN to the culture medium were proportionally similar in the presence or absence of FK866 (Fig. 4F), indicating that the drug does not affect NMNAT activity.

Effect of NMN or NAD on PARP-1-dependent cell death

It has been reported that strategies aimed at increasing intracellular NAD contents provide cytoprotection under different pathological conditions [see [2;3;21;27] for reviews]. Hence, to evaluate the potential cytoprotective properties of NMN, we analyzed various cell death 5C). Accordingly, MTT reduction and LDH release assays showed that both NMN and NAD partially counteracted cell death 24 h after PARP-1-hyperactivation (Fig. 5D andE). NMN-or NAD-treated cells died 36 h after MNNG exposure (not shown), indicating that nucleotide cytoprotection was only transient.

NMN content in mouse organs and effect of FK866

To our knowledge, the impact of FK866 on NMN contents in vivo has not been reported. We therefore injected the drug in mice (100 mg/kg, i.p.) and measured NMN contents in various organs at different times after injection. Reportedly, mouse NaPRT activity is high in liver, intermediate in heart, and under detectable levels in brain [START_REF] Revollo | Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme[END_REF]. Accordingly, we found that NMN contents were higher in the liver than in the heart (6.7±0.3 and 4.4±0.2 nmol/mg tissue, respectively). However, the brain contents of NMN (3.86±0.1 nmol/mg tissue) were similar to those of the heart.

Importantly, injection of FK866 reduced NMN contents in the three organs after 4 and 8 hrs. At 16 hrs from the injection, NMN contents in the organs were still lower than control with a tendency to return to basal values (Fig. 6).

Discussion

In the last several years we have witnessed a renewed interest in NAD biology and basic biochemistry of pyridine nucleotide metabolism [START_REF] Berger | The new life of a centenarian: signalling functions of NAD(P)[END_REF]. Among the biochemical routes leading to NAD formation, the rescue pathway operated by NaPRT and NMNATs is of pivotal importance to NAD homeostasis in mammals. Indeed, this pathway rescues nicotinamide which is a by-product of several enzymes such as poly and mono ADP-ribose transferases, sirtuins as well as ADP-ribosyl cyclase, which are responsible for substantial consumption of NAD under constitutive and pathological conditions. However, the intracellular contents as well as subcellular A c c e p t e d M a n u s c r i p t 10 compartmentalization of NMN, the first metabolite of the nicotinamide-dependent NAD-rescue pathway, are in large part unknown. Furthermore, whether NMN formation mediates the signalling properties of extracellular NaPRT/visfatin/PBEF waits to be clearly understood. Current lack of knowledge about the biological relevance of NMN can be ascribed, at least in part, to difficulties in nucleotide measurements in biological samples. Previous studies have quantified NMN by means of complex HPLC systems coupled to UV detection and subsequent mass spectrometry analysis [11;13;28]. Indirect methods or radioactive precursors have been also adopted to follow NMN formation and degradation [START_REF] Bieganowski | Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans[END_REF]. In the present study, we report a new method to quantify NMN in biological samples, based on the ability of its N-alkylpyridinium moiety to be transformed into fluorescent compounds.

By monitoring the effect of nicotinamide addition to the incubating media of HeLa or U937 cells on their NMN and NAD content, we confirm that, at least in HeLa cells, NaPRT is the rate-limiting step of the rescue pathway [29;30]. Also, evidence that intracellular NMN contents promptly increase when the nucleotide is added to the culture media indicates that plasma membrane is permeable to this nucleotide. These findings suggest that the pharmacologic effects of exogenous NMN in cultured cells and mice [11;13] are due to cellular uptake and changes in NAD contents. Whether NMN permeates the plasma membrane through the putative NAD uptake mechanisms [START_REF] Billington | Characterization of NAD uptake in mammalian cells[END_REF] remains to be clarified. We also report that NMN was more concentrated in the mitochondrial fraction than in the nuclear or cytosolic ones. Although we adopted a crude cell fractionation technique with possible subcellular fraction contamination, data suggest high mitochondrial NMN contents. This finding is of particular relevance if we consider that it is still debated whether NaPRT activity is present in the mitochondria. NaPRT can be detected by Western blotting in mitochondria extracted from cultured cells or mouse liver, and the NaPRT inhibitor FK866 reduces mitochondrial NAD content when added to a pure organelle preparation [START_REF] Yang | Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival[END_REF]. Overall, these findings suggest that NMN is synthesized in mitochondria by NaPRT. Although the presence of a mitochondrial NaPRT is conceivable if we consider that mitochondria contain a specific NMNAT isoform [START_REF] Berger | Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms[END_REF], how NaPRT enters the organelles is unknown given that a mitochondrial-targeting sequence has not been reported [START_REF] Khan | Molecular basis for the inhibition of human NMPRTase, a novel target for anticancer agents[END_REF]. In light of the recent identification of mitochondrial NAD carriers [28;32], the Regardless, evidence that mitochondria contain significant amounts of NMN (Fig. 3E), as well as NMNAT3 [START_REF] Berger | Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms[END_REF], clearly indicates that at least the last step of the NAD rescue pathway occurs in these organelles. This makes sense if one consider that NAD consuming enzymes such as sirtuins and mono-ADP-ribose transferases are present in mitochondria [21;33]. Conversely, the presence of a mitochondrial PARP-1 is still debated [17;34-36].

A large body of evidence identifies PARP-1 as a key player of cell death. It has been repeatedly

proposed that the molecular pathway responsible for PARP-1-dependent cell death stems from an excessive consumption and ensuing depletion of NAD pools [START_REF] Chiarugi | PARP-1: killer or conspirator? The suicide hypothesis revisited[END_REF]. It is unknown, however, whether/how PARP-1 hyperactivation affects the kinetics of the NAD rescue pathway. We show here that the massive depletion of intracellular NAD that follows PARP-1 activation is not accompanied by a concomitant depletion of NMN. Rather, a small but significant increase in NMN content occurred upon PARP-1 activation (Fig. 4A). This finding suggests that NMNAT becomes limiting the rate of the NAD rescue pathway when the dinucleotide is massively consumed by PARP-1. Also, it indicates that strategies aimed at circumventing the "NMNAT bottleneck" could be of cytoprotective relevance in conditions of hyperactivation of PARP-1. In good agreement with this hypothesis and previous findings [START_REF] Ying | NAD+ repletion prevents PARP-1-induced glycolytic blockade and cell death in cultured mouse astrocytes[END_REF], we report that exogenous NAD reduced early PARP-1dependent bioenergetic derangement and cell death. On the one hand this result further corroborates the hypotheses that NAD depletion is causative in this type of cell demise [START_REF] Berger | Poly (ADP-ribose) in the cellular response to DNA damage[END_REF], and on the other that NAD permeates through the plasma membrane [START_REF] Billington | Characterization of NAD uptake in mammalian cells[END_REF]. Of note, NMN also reduced PARP-1-dependent energy failure and cell death, despite to a lower extent when compared to NAD. This is in keeping with the NMNAT rate-limiting activity for NAD re-synthesis during overactivation of PARP-1. We report, however, that protection afforded by NMN or NAD on cells undergoing PARP-1 hyperactivation is only transient. This indicates that the partial prevention of NAD/ATP depletion is not compatible with cell survival, and/or that delayed events in addition to nucleotide depletion [i.e. poly(ADP-ribose)-dependent mitochondrial AIF release [START_REF] Yu | Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death[END_REF]] contribute to cell demise prompted by PARP-1. Indeed, NMN can improve energy dynamics working as a A c c e p t e d M a n u s c r i p t 12 precursor of NAD but also favour poly(ADP-ribose) formation by the detrimental NMNAT-PARP-1 complex [START_REF] Berger | Regulation of poly(ADP-ribose) polymerase 1 activity by the phosphorylation state of the nuclear NAD biosynthetic enzyme NMN adenylyl transferase 1[END_REF].

Despite the emerging complexity and pathophysiological relevance of NAD metabolism, pharmacology of NAD re-synthesis is extremely poor, with FK866 being the most studied chemical.

Evidence that gallotannin increases intracellular NMN contents (Fig. 4C) is in keeping with prior work showing that gallotannin inhibits NMNAT activity [START_REF] Berger | Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms[END_REF]. Although gallotannin is a complex mixture of tannins extracted from oak gall [START_REF] Formentini | Mono-galloyl glucose derivatives are potent poly(ADP-ribose) glycohydrolase (PARG) inhibitors and partially reduce PARP-1-dependent cell death[END_REF], these findings could be of relevance for the identification of the pharmacophore of more potent and specific NMNAT inhibitors. Further, the demonstration that FK866 reduced basal NMN and NAD contents in cultured cells, but did not prevent the increase of NAD in cells exposed to NMN (Fig. 4D-F), indicates for the first time that the drug selectively inhibits NaPRT, having no effects on NMNAT activity. This is of significance if one considers that the chemical is currently in clinical trial for cancer treatment [START_REF] Holen | The pharmacokinetics, toxicities, and biologic effects of FK866, a nicotinamide adenine dinucleotide biosynthesis inhibitor[END_REF].

In keeping with data on tissue distribution of NaPRT expression [START_REF] Revollo | Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme[END_REF] and activity [START_REF] Dietrich | Nicotinamide mononucleotide pyrophosphorylase activity in animal tissues[END_REF], we report that NMN is more concentrated in the mouse liver than in the heart or brain. Notably, NMN contents in the brain are comparable to those of the heart, despite evidence that NaPRT expression is almost undetectable in the brain [START_REF] Revollo | Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme[END_REF]. Although we do not know the cell type(s) in which NMN is contained, data suggest active NMN uptake from neural cells. Treatment of mice with FK866 induced long-lasting but mild reduction of NAD contents in liver, heart and brain.

Obviously, mild reduction can be primarily ascribed to the pharmacodynamic and pharmacokinetic features of FK866. Indeed, metabolism of FK866 is still to be clearly understood. Data, however, hint that under constitutive conditions NAD consumption and/or recycling through nicotinamide occurs at a slow rate. This hypothesis is consistent with knowledge that NAD half life in rat liver is about 10 hrs [START_REF] Ijichi | Studies on the biosynthesis of nicotinamide adenine dinucleotide. 3. Comparative in vivo studies on nicotinic acid, nicotinamide, and quinolinic acid as precursors of nicotinamide adenine dinucleotide[END_REF].

In conclusion, the development of a new method for NMM measurement allowed us to gather new information on the biochemistry and pharmacology of NAD biosynthesis. Given that the pathophysiological relevance of NaPRT and NMN is still in large part obscure, the present study helps addressing some of the numerous unanswered questions existing in the field of pyridine nucleotide metabolism and NAD signaling.

A c c e p t e d M a n u s c r i p t 4 secreted

 4 protein can also behave as a cytokine called Pre-B cell colony-enhancing factor (PBEF).

  µM, NMN 10-1000 µM, NAD 0.1-1 mM. (E)-N-[4-(1-benzoylpiperidin-4-yl) butyl]-3-(pyridin-

Fig. 1 .

 1 Fig. 1. Excitation and emission spectra of the fluorescent compound were determined by means of

A c c e p t e d M a n u s c r i p t 6 a

 6 spectro fluorophotometer RF5000 (Shimadzu, Milan, Italy). Samples were injected into the HPLC system consisting in a mobile phase of 0.1M buffer phosphate pH 6.5, 10% acetonitrile, a Supelco 25 cm column (5µm) and a fluorimetric detector (Perkin Elmer) with excitation and emission wavelength of 332 and 454 nm, respectively.

A c c e p t e d M a n u s c r i p t 9 parameters

 9 of HeLa and U937 cells undergoing NAD and ATP depletion because of PARP-1 activation. As shown in Fig.5A and B, both NMN and NAD were able to counteract the reduction in NAD and ATP contents in HeLa and U937 cells exposed 1 h to the PARP-1 activating agent MNNG. Twenty three hrs later, MNNG-challenged cells appeared shrunken and joined in clusters of dead cells. Conversely, those exposed to MNNG plus NMN or NAD in part conserved their healthy morphology (Fig.

A

  c c e p t e d M a n u s c r i p t 11 possibility exists that part of the mitochondrial NMN pool is uptaken from the cytoplasm.

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Optimization on HPLC analysis of NMN. (A) NAD biosynthetic pathways in mammals. NAD is synthesized de novo through the kynurenine pathway originating from tryptophan. Intracellular NAD degrading enzymes such as PARPs, mono(ADP-ribosyl)transferases (MARTs) and Sirtuins hydrolyze NAD forming different products and nicotinamide (NAM). The later is utilized to re-synthesize NAD through the NAD rescue pathway including NaPRT and NMNAT1-3. Nicotinic acid (NA) originating from food is transformed into NAD through the Preiss-Handler pathway composed by NA phosphoribosyl transferase (NAPRT), NMNAT1-3 and NAD deamidase (NADASE). Nicotinamide riboside kinase (NRK) converts nicotinamide riboside (NR) into NMN. The site of action of the NaPRT inhibitor FK866 is shown. (B), Chromatogram of NMN (250 pmol) obtained with an HPLC apparatus coupled to an UV detector (see Methods). (C), Chromatogram of a whole cell extract (U937 cells) injected into the same apparatus described in (B). (D), Absorption and emission spectra of NMN derivatized as described in Methods. (E), Chromatogram of

A

  c c e p t e d M a n u s c r i p t 18 derivatized NMN (250 pmol) obtained with an HPLC apparatus coupled to a fluorimetric detector (see Methods). (F) Chromatogram of a whole cell extract (U937 cells) derivatized as described in Methods and injected into the same apparatus described in (E). (G) Effect of the temperature of the step 2 of the derivatization procedure on efficiency of NMN derivatization. (H) Effect of duration of step 1 or 2 of the derivatization procedure on efficiency of NMN derivatization. (I) NMN calibration curve. Fluorescence of NMN is linear up to 2500 pmol injected. In G-I, bars/points represent the mean ± SEM of three experiments conducted in duplicate.

Figure 3 .

 3 Figure 3. NMN and NAD contents in HeLa and U937 cells. NMN and NAD contents were measured in HeLa and U937 cells under control conditions or after exposure for 1 h to different concentrations of nicotinamide (Nam) (A and B) or NMN (C and D). (E, F) Contents of NMN in cytosol, nuclei and mitochondria of HeLa or U937 cells. *p<0.05, **p<0.01 vs Cytosol (ANOVA + Tukey's post hoc test). In A-E, each point/bar represents the mean ± SEM of three experiments conducted in duplicate.

Figure 4 .

 4 Figure 4. Effect of PARP-1 activation, gallotannin or FK866 on NMN or NAD contents in U937 cells. The NMN (A) or NAD (B) contents were measured in cells under control conditions and at different times after exposure to the PARP-1-activating agent MNNG (100 µM) in the presence or absence of the PARP-1 inhibitor phenanthridinone (PHE, 30 µM). (C) The contents of NMN were measured in cells exposed for 1 h to different concentrations of gallotannin (GLT) in the presence or absence of MNNG 100 µM/1h. Effect of a 4 hrs exposure to FK866 on intracellular NMN (D) or NAD (E) contents. (F) NAD contents in cells exposed 1 h to different concentrations of NMN and preincubated 4 hrs with FK866 (10 µM). Each bar represents the mean ± SEM of at least three experiments conducted in duplicate. *p<0.05, **p<0.01 vs Crl (ANOVA + Tukey's post hoc test). In (F) § p<0.05 vs Crl in the presence of FK866 (ANOVA + Tukey's post hoc test).

Figure 5 .

 5 Figure 5. Cytoprotection form PARP-1-dependent cell death by NMN or NAD. Effect of NMN or NAD added to the incubating media of HeLa and U937 cells (1 h preincubation) on reduction of

Figure 6 .

 6 Figure 6. Effect of FK866 on NMN contents in different mouse organs. NMN was measured in different mouse organs under control conditions (C) and at different times after i.p. injection of FK866 (100 mg/kg). Each bar represents the mean of 2 experiments (n = 4 per group). *p<0.05 vs C (ANOVA + Tukey's post hoc test).

  Figure 1

Figure

  Figure

  Figure

Acknowledgments

This study was supported by grants from the University of Florence, the Italian Ministry of University and Scientific and Technological Research, Associazione Italiana Sclerosi Multipla and Ente Cassa di Risparmio di Firenze.


Luke R E Harrison

Christopher J Ottley

D Graham Pearson

Roger J Griffin

Stephen R Wedge

M Eileen Dolan

David R Newell

Michael J Tilby

The kinase inhibitor O 6 -cyclohexylmethylguanine (NU2058) potentiates the cytotoxicity of cisplatin by mechanisms that are independent of its effect upon CDK2

Keywords: cisplatin, melphalan, potentiation, kinase inhibitor, NU2058 ICP-MS, inductively coupled plasma mass spectroscopy, IR, ionising radiation, Rb, retinoblastoma protein

Abstract

O 6 -Cyclohexylmethylguanine (NU2058) was developed as an inhibitor of CDK2 and was previously shown to potentiate cisplatin cytotoxicity in vitro. The aim of this study was to investigate the mechanism of cisplatin potentiation by NU2058.

SQ20b, head and neck cancer cells were treated for 2 hours with NU2058 (100 μM) and then for a further 2 hours with cisplatin and NU2058. NU2058 increased cisplatin cytotoxicity, by clonogenic assay, with a dose modification factor (DMF) of 3.1.

NU2058 increased total intracellular platinum levels 1.5-fold, and platinum-DNA adduct levels 2-fold. Furthermore, the cisplatin-DNA adducts formed were more toxic in the presence of NU2058. To investigate whether the effects of NU2058 on cisplatin adduct levels and toxicity were dependent on CDK2 activity, additional CDK2 inhibitors were tested. NU6230 (CDK2 IC 50 18 μM) was equipotent to NU2058 (CDK2 IC 50 17 μM) as a CDK2 inhibitor in cell-free and cell-based assays, yet did not potentiate cisplatin cytotoxicity. Furthermore, NU6102 was >1000-fold more potent than NU2058 as a CDK2 inhibitor (CDK2 IC 50 5 nM) yet was no more active than NU2058 in potentiating cisplatin.

NU2058 also potentiated melphalan (DMF 2.3), and monohydroxymelphalan (1.7), but not temozolomide or ionising radiation. While NU2058 increased melphalan cytotoxicity, it did not increase melphalan-DNA adduct formation. These studies demonstrate that NU2058 alters the transport of cisplatin, causing more Pt-DNA adducts, as well as sensitizing cells to cisplatin-and melphalan-induced DNA damage. However, the effects of NU2058 are independent of CDK2 inhibition.

A c c e p t e d M a n u s c r i p t

Introduction

Platinum compounds such as cisplatin and carboplatin remain amongst the most widely used chemotherapeutic drugs in the treatment many tumours types, including head and neck, testicular, lung, and ovarian carcinomas. For example, the combination of bleomycin, etoposide and cisplatin is considered curative therapy for testicular cancer, with survival rates of over 95% [START_REF] Einhorn | Curing metastatic testicular cancer[END_REF]. Like most cytotoxic anticancer drugs, cisplatin acts via the induction of DNA damage involving a number of types of DNA adducts [START_REF] Wang | Cellular processing of platinum anticancer drugs[END_REF].

Although cisplatin has clinical utility against some tumour types it has little activity against others. Furthermore, initially sensitive tumours can become resistant to platinum complexes. Acquired as well as intrinsic cisplatin resistance can be caused by several mechanisms, including altered drug uptake, increased activity of cellular detoxification systems and DNA repair [START_REF] Safaei | The role of copper transporters in the development of resistance to Pt drugs[END_REF][START_REF] Safaei | Copper transporters regulate the cellular pharmacology and sensitivity to Pt drugs[END_REF][START_REF] Siddik | Cisplatin: mode of cytotoxic action and molecular basis of resistance[END_REF][START_REF] Rabik | Molecular mechanisms of resistance and toxicity associated with platinating agents[END_REF][START_REF] Boulikas | Cisplatin and platinum drugs at the molecular level. (Review)[END_REF][START_REF] Fink | The role of DNA mismatch repair in platinum drug resistance[END_REF]. Modulation of cellular responses to established drugs such as cisplatin have the potential to both improve efficacy and attenuate toxicity.

Protein kinases are involved in a wide range of cellular events including cell growth, DNA repair, replication and cell survival. Many protein kinases are deregulated in the development and progression of cancer, and protein kinase inhibitors are currently being used therapeutically to exploit the molecular pathology of neoplasms.

Trastuzumab and imatinib are two clinically successful anti-cancer drugs which act via inhibition of kinase signalling pathways [START_REF] Piccart-Gebhart | Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer[END_REF][START_REF] Druker | Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia[END_REF]. Encouraged by the success of imatinib and trastuzumab, the identification of protein kinases that are deregulated in Cyclin dependent kinases (CDK), and their partner cyclins, are key proteins in the regulation of the cell cycle and are frequently deregulated in cancer cells [START_REF] Shapiro | Cyclin-dependent kinase pathways as targets for cancer treatment[END_REF]. The CDKs have been investigated as targets for cancer therapy and the antitumour activity of CDK inhibitors has been demonstrated in preclinical models, both in vitro and in vivo [START_REF] Drees | Flavopiridol (L86-8275): selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells[END_REF]. However, CDK inhibitors have so far shown little or no single agent clinical activity, with the possible exception of flavopiridol for the treatment of chronic lymphocytic leukaemia [START_REF] Byrd | Flavopiridol administered using a pharmacologically derived schedule is associated with marked clinical efficacy in refractory, genetically high-risk chronic lymphocytic leukemia[END_REF]. These latter data are consistent with preclinical studies, where inhibition of CDKs by flavopiridol was shown to induce cell death [START_REF] Shapiro | Flavopiridol induces cell cycle arrest and p53-independent apoptosis in non-small cell lung cancer cell lines[END_REF]. It has also been suggested that CDK inhibitors could be used to increase the efficacy of cytotoxic therapies by modulating cellular responses, possibly by recruiting or holding cells in a drug sensitive phase of the cell cycle [START_REF] Bible | Cytotoxic synergy between flavopiridol (NSC 649890, L86-8275) and various antineoplastic agents: the importance of sequence of administration[END_REF].

O 6 -Cyclohexylmethylguanine (NU2058, Fig. 1A) is a compound that was identified in a programme to develop potent and selective inhibitors of CDK2. NU2058 showed modest inhibition of CDK2 with an IC 50 (concentration required to give 50% inhibition of CDK2 in an isolated enzyme assay) value of 17 µM [START_REF] Arris | Identification of novel purine and pyrimidine cyclin-dependent kinase inhibitors with distinct molecular interactions and tumor cell growth inhibition profiles[END_REF]. NU2058 has previously been shown to potentiate the cytotoxicity of cisplatin in vitro [START_REF] Fishel | Effect of cell cycle inhibition on Cisplatin-induced cytotoxicity[END_REF] and it was hypothesised that inhibition of CDK2 was responsible for this effect.

In the present work, we tested the hypothesis that CDK2 inhibition was responsible for the potentiation of cisplatin cytotoxicity by NU2058 through the use of two structurally-related compounds; NU6102 and NU6230 (Fig. 1A). Furthermore, to

Methods

Materials

All materials were purchased from Sigma-Aldrich (UK) unless otherwise stated.

Monohydroxymelphalan was synthesised according to Tilby et al [START_REF] Tilby | Application of a sensitive immunoassay to the study of DNA adducts formed in peripheral blood mononuclear cells of patients undergoing high-dose melphalan therapy[END_REF].

Temozolomide was provided by the Cancer Research UK Formulation Unit, Strathclyde University, UK. Cells were irradiated using X-rays (310 kV, dose rate 3 Gy per min.). NU2058, NU6230, NU6102 were synthesised as described previously [START_REF] Arris | Identification of novel purine and pyrimidine cyclin-dependent kinase inhibitors with distinct molecular interactions and tumor cell growth inhibition profiles[END_REF][START_REF] Davies | Structure-based design of a potent purine-based cyclin-dependent kinase inhibitor[END_REF] and stored as 100 mM stock solutions in DMSO at -20°C.

Cell lines

The human head and neck cancer cell line, SQ20b, was kindly provided by Dr.

Michael Beckett (Department of Radiation and Cellular Oncology, University of Chicago, USA) and maintained in DMEM: Ham's F-12 medium, supplemented with 20% (v/v) foetal calf serum, and 0.4 μg/ml hydrocortisone, as previously described [START_REF] Fishel | Enhancement of platinum-induced cytotoxicity by O6-benzylguanine[END_REF]. Cells were grown in the presence of penicillin (100 units/ml) and streptomycin (100 μg/ml) at 37 °C and 5% CO 2 .

Colony formation assay

Cells were seeded into 6 well plates (350,000 cells/well) and allowed to attach overnight. On the day of the experiment, growth media were replaced with media containing 100 μM of either NU2058, NU6102, NU6230 or DMSO (0.1% (v/v) final concentration) for 2 hours followed by a further 2 hours in the additional presence of cytotoxic drugs, unless otherwise indicated. For the radiation experiments, cells were treated for a total of 4 hours with NU2058, and irradiated after the first 2 hours. After treatment, the cells were washed twice with PBS, trypsinised, and replated into 100 mm Petri dishes at various cell densities (300 -50,000 cells per plate). After approximately 12 days, media was removed, and cells were fixed with Carnoy's reagent (75% (v/v) methanol, 25% (v/v) acetic acid), stained with crystal violet (0.4% (w/v) in water) and colonies were counted using a ColCount™ image analyser (Oxford Optronix, UK). Percentage survival was calculated relative to the survival of control cells, treated with the compound vehicle alone (DMSO).

Western blotting

Phosphorylation of retinoblastoma protein (Rb) was measured in cells after incubation with each compound or DMSO. Exponentially growing cells were treated in 15 cm dishes with 100 μM of either NU2058, NU6230, NU6102 or vehicle (0.1 % (v/v) DMSO) for a period of 4 or 24 hours. After treatment, cells were washed twice with ice cold PBS, and lysed with Laemmli buffer [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. Samples were sonicated and protein concentration quantified using BCA protein assay (Pierce, USA). Equal amounts of protein (15 μg) were separated on a 4-20% (w/v) tris-glycine SDS polyacrylamide gel (Invitrogen, UK) and transferred to a PVDF blotting membrane (BioRad, USA). The membrane was blocked in 5% (w/v) milk in tris buffered saline containing 0.2% (v/v) tween (TBST), and the primary monoclonal phosphospecific (pRb-T 821 ) antibody (Biosource, CA, Cat. No. 44-582G) was added at a dilution factor of 1:1000 for two hours, washed with TBST, and incubated with a rabbit secondary antibody (DakoCytomation, Denmark, Cat. No. P0217) in TBST containing 5% (w/v) milk for a further two hours. The ECL system (Pierce, USA) was used for detection of phospho-Rb. The image was captured with photographic film and developed. For measurement of actin, membranes were incubated as above but with a primary anti-

Measurement of total cellular platinum and platinum-DNA adducts

Actively growing cells were treated with NU2058 (100 μM) or DMSO control for 2 hours and then a further 2 hours in the additional presence of cisplatin (0-50 μM).

After treatment, cells were either washed twice with ice-cold PBS, trypsinised and collected by centrifugation, or washed twice with warm growth medium and further incubated at 37ºC for periods up to 24 hours. For platinum-DNA adduct analysis, DNA was extracted from cells using the Qiagen DNeasy kit according to the manufacturer's instructions (Qiagen, UK). Isolated DNA was quantified by measuring OD 260 using a ND-1000 Spectrophotometer (NanoDrop, USA). Extracted DNA was hydrolysed with 3.5% nitric acid (v/v) Spectrosol grade (Merck, UK) overnight at 70°C, prior to analysis by inductively coupled plasma mass spectroscopy (ICP-MS).

For measurement of total cellular platinum, cell pellets were sonicated and protein measured as described above. Samples were then incubated with 1 M NaOH for 1 hour at 60°C, followed by an overnight acid hydrolysis with 1 M nitric acid at 70°C. Platinum concentrations in samples were determined by ICP-MS as described previously [START_REF] Meczes | Specific adducts recognised by a monoclonal antibody against cisplatin-modified DNA[END_REF], and compared to a standard curve analysed on the same day to correct for any instrument drift.

Measurement of melphalan-DNA adducts

For measurement of melphalan-DNA adducts, cells were pre-treated with 100 μM NU2058 or vehicle (0.1% (v/v) DMSO) for two hours followed by a further two hours in the additional presence of melphalan (0 -50 μM). After drug treatment, cells were washed twice with ice-cold PBS and released using trypsin. Cells were then centrifuged and washed once more with PBS, and pellets were stored at -20°C until required. DNA was extracted using the phenol hydroxyapatite method as described by Tilby et al [START_REF] Tilby | Application of a sensitive immunoassay to the study of DNA adducts formed in peripheral blood mononuclear cells of patients undergoing high-dose melphalan therapy[END_REF]. DNA concentration was determined by measuring OD 260 using a ND-1000 Spectrophotometer (NanoDrop, USA). Melphalan-DNA adducts were quantified using a competitive ELISA as described previously [START_REF] Tilby | Immunological detection of DNA damage caused by melphalan using monoclonal antibodies[END_REF]. Briefly, several dilutions of DNA from treated cells were mixed with a constant amount of MP5/73 antibody, which was specific for melphalan-guanine adducts on DNA, and incubated in wells coated with a standard amount of melphalan-treated DNA. The amount of antibody that bound to the wells was determined by a fluorogenic enzyme assay. The quantities of adducts per assay well that caused a 50% reduction of the assay signal were determined by a curve fitting procedure.

NU2058 did not markedly inhibit CDK2 activity in vitro

The hypothesis that NU2058-enhanced cisplatin cytotoxicity is due to CDK2 inhibition was tested through a comparative study with the structurally-related compounds NU6230 and NU6102 (Fig. 1A). NU6230 (CDK2 IC 50 = 18 μM) is an equipotent CDK to inhibitor to NU2058 (CDK2 IC 50 = 17 µM), whilst NU6102 (CDK2 IC 50 = 5 nM) is >1000-fold [START_REF] Davies | Structure-based design of a potent purine-based cyclin-dependent kinase inhibitor[END_REF] more potent as an inhibitor of this enzyme. The effects of NU2058, NU6230, and NU6102 on cisplatin cytotoxicity were evaluated by treating SQ20b cells for 2 hours with each compound alone at a concentration of 100 μM, followed by a further 2 hours with each compound in the presence of cisplatin. All compounds were non-toxic when used alone in the absence of cisplatin, using the schedule described. The previously reported pronounced potentiation of cisplatin cytotoxicity by NU2058 in SQ20b cells [START_REF] Fishel | Effect of cell cycle inhibition on Cisplatin-induced cytotoxicity[END_REF] was confirmed (Fig. 1B). In these experiments the LC 50 values (concentration of cytotoxic drug that reduces plating efficiency by 50%) were determined. NU2058 enhanced cisplatininduced cell death with a dose modification factor (DMF) of 3.1 fold (p < 0.001, twoway ANOVA). DMF is defined as the ratio of the LC 50 for cytotoxic drug alone to the LC 50 for combined treatment with cytotoxic drug and NU2058 (or NU6102 / NU6230).

NU6102 significantly enhanced the cytotoxicity of cisplatin (Fig. 1B), with a DMF of 2.3 (p < 0.05, two-way ANOVA). In contrast, NU6230 caused no significant enhancement of cisplatin mediated cell death (Fig. 1B, p < 0.05, two-way ANOVA).

To evaluate whether the failure of NU6230 to enhance the cytotoxicity of cisplatin was due to inadequate intracellular accumulation of the compound, and hence failure Attempts to re-probe the membranes for total Rb failed to give reproducible results, and so actin was used as a loading control. Cells incubated with NU6102 for 4 and 24 hours showed a clear reduction in levels of phospho-Rb (Fig. 1C). NU2058 and NU6230 caused a slight decrease in Rb phosphorylation after four hours and a clear decrease after 24 hours. Thus NU6230 was at least as effective as NU2058 at decreasing the phosphorylation of Rb in a cell-based assay, and at inhibiting CDK2 in a cell-free enzyme assay, but did not potentiate cisplatin cytotoxicity. Conversely, NU6102 was markedly more effective than NU2058 in both cell-free and cell-based CDK2 assays, but was no more effective than NU2058 at potentiating cisplatin cytotoxicity. Together, these data do not support the hypothesis that the potentiation of cisplatin cytotoxicity by NU2058 is due to CDK2 inhibition.

NU2058 increased cellular levels of cisplatin and Pt-DNA adducts without affecting DNA repair

To investigate whether the increased cytotoxicity of cisplatin in the presence of NU2058 was due to effects on drug-DNA interaction, cells were harvested immediately after drug treatment, DNA extracted, and the amount of platinum bound to DNA measured using ICP-MS. Platinum-DNA adducts were linearly related to extracellular cisplatin concentration, and NU2058 increased the number of platinum-DNA adducts by 2-fold (Fig. 2A, p < 0.001). The formation and loss of DNA-adducts over time was examined in cells treated with NU2058 or vehicle (0.1% (v/v) DMSO) for 2 hours followed by a further 2 hours in combination with 25 μM cisplatin. Cells were then washed with, and further incubated in, drug-free medium and harvested at To investigate whether the increase in cisplatin-DNA adduct formation induced by NU2058 was due to increased intracellular cisplatin levels, the total concentration of platinum in cells after treatment with cisplatin in the presence or absence of NU2058 was evaluated. Total cellular platinum was also linearly related to extracellular cisplatin concentration, and for any given concentration of cisplatin, NU2058 caused a 1.5 fold increase in the levels of cellular platinum (Fig. 2C, p < 0.001). To examine whether the increased cytotoxicity of cisplatin in the presence of NU2058 was due solely to increased cisplatin-DNA adduct formation, the relationship between cisplatin-DNA adduct levels and survival was studied by combining data from Figure 2D shows that for equal levels of cisplatin-DNA adducts, cell survival was lower in the presence of NU2058.

Potentiation of cisplatin cytotoxicity by NU2058 was highly schedule dependent

To determine whether the sequence of drug administration was important for the enhancement of cisplatin cytotoxicity by NU2058, various treatment schedules were studied. Exposure to cisplatin for all schedules was for two hours, and the sequence of cisplatin and NU2058 treatments was as described in the figure legend (Fig. 3).

Schedules that involved no overlap of NU2058 and cisplatin exposure produced no significant potentiation of cisplatin cytotoxicity (Fig. 3A and3D), and the A c c e p t e d M a n u s c r i p t 14 Linking powered by eXtyles enhancement of cisplatin cytotoxicity by NU2058 was therefore dependent on both drugs being present simultaneously (Fig. 3B andC).

NU2058 potentiated melphalan and monohydroxymelphalan but not temozolomide or ionising radiation

The ability of NU2058 to potentiate other DNA damaging agents was investigated.

NU2058 potentiated the cytotoxic effects of melphalan (DMF of 2.2 (p < 0.05, twoway ANOVA)) and monohydroxymelphalan (DMF of 2.1 (p < 0.05, two-way ANOVA)), but not temozolomide (p = 0.22, two-way ANOVA) or ionising radiation (p = 0.99, two-way ANOVA) (Fig. 4A-D). Since NU2058 increased cisplatin-DNA adduct levels (Fig. 2A), the effect of NU2058 on melphalan-DNA adduct levels was studied using an immunoassay based on an antibody that is specific for melphalanguanine adducts. Although, in contrast to cisplatin, NU2058 did not affect melphalan-DNA adduct levels (p > 0.05, two-way ANOVA, Fig. 5A), NU2058 did increase the cytotoxicty of the adducts formed (Fig. 5B).

A c c e p t e d

M a n u s c r i p t

Discussion

The primary aim of this study was to investigate the mechanism by which NU2058 potentiates cisplatin cytotoxicity. NU2058 was developed as an inhibitor of CDK2, and to determine whether its effect on cisplatin cytotoxicity was mediated via CDK2 inhibition, two structurally-related compounds, NU6102 and NU6230, were also studied. NU6230 showed no potentiation of cisplatin (Fig. 1B) despite being similar to NU2058 in its ability to inhibit purified CDK2 (NU2058 CDK2 IC 50 = 17 µM; NU6230 CDK2 IC 50 = 18 μM). The lack of potentiation of cisplatin cytotoxicity was not due to poor access of NU6230 to CDK2 in cells, because NU6230 caused similar inhibition of Rb phosphorylation to NU2058, a widely used marker of CDK2 activity (Fig. 1C). In contrast to NU6230 and NU2058, NU6102 was a much more potent inhibitor of purified CDK2 (NU6102 CDK2 IC 50 5 nM) and, consistent with the greater potency of the compound, exerted a much greater effect on Rb phosphorylation in SQ20b cells than did NU2058 (Fig. 1C). However, despite its greater potency as a CDK2 inhibitor, NU6102 was no more effective than NU2058 at potentiating cisplatin cytotoxicity. These data demonstrate that inhibition of CDK2 was not the mechanism by which NU2058 potentiated cisplatin cytotoxicity. The effects reported here are not specific to SQ20b cells because comparable potentiation of cisplatin has been observed in other human cell lines representing other types of cancer (for example LoVo and SW620, data not shown).

Since NU2058 and NU6102 inhibit CDK2 by interaction at the ATP binding site [START_REF] Arris | Identification of novel purine and pyrimidine cyclin-dependent kinase inhibitors with distinct molecular interactions and tumor cell growth inhibition profiles[END_REF][START_REF] Davies | Structure-based design of a potent purine-based cyclin-dependent kinase inhibitor[END_REF], and ATP binding sites are found on multiple proteins, we propose that the potentiation of cisplatin and certain other cytotoxic drugs by NU2058, was due to the interaction with another, as yet unknown ATP-binding protein, possibly a protein kinase. The lack of efficacy of NU6230 is presumably due to the large substituent at the C-8 position (Fig. 1A), and NU6230 represents an important negative control compound for further mechanistic studies. Since NU2058 is an analogue of guanine it could, in principle, become converted to a nucleotide and even incorporated into DNA. However, the large substituent at the O6-position is likely to compromise the ability of NU2058 to act as a substrate for hypoxanthine/guanine phosphoribosyl transferase, and subsequently nucleotide kinases and DNA polymerase. Nevertheless, we cannot exclude the possibility that the biological effects of NU2058 are related to its activation via the purine salvage pathway.

NU2058 also potentiated the cytotoxicity of carboplatin, oxaliplatin (data not shown) and the nitrogen mustard, melphalan. However, it did not have any effect on the sensitivity of cells to ionising radiation or temozolomide. NU2058 also potentiated the monofunctional derivative of melphalan, monohydroxymelphalan; however, as expected from previous studies with ML-1 cells [START_REF] Gould | p53 elevation in relation to levels and cytotoxicity of mono-and bifunctional melphalan-DNA adducts[END_REF] monohydroxymelphalan was approximately 20-fold less toxic than melphalan towards SQ20b cells (Fig. 4F). The cytotoxicity of monohydroxymelphalan, and the associated potentiation by NU2058, could not be attributed to low levels of intact bifunctional melphalan being present because HPLC analysis of the monohydroxymelphalan used showed that any such contamination was <0.2% (H. Thomas, personal communication), a level too low to account for the observed cytotoxicity. Therefore, the potentiation of monohydroxymelphalan by NU2058 demonstrates that the potentiation of melphalan by NU2058 was not solely dependent on the bifunctional properties of the latter molecule. The lack of potentiation by NU2058 of both temozolomide (a DNA methylating agent) and ionising radiation (which involves DNA single and double strand breaks) demonstrates that the type of DNA damage is an important determinant of the ability of NU2058 to enhance cytotoxicity. Since cisplatin-DNA adduct levels were based on total platinum bound to DNA, they reflect mainly the levels of intra-strand 1,2 crosslinks as these account for the majority of adducts present on DNA [START_REF] Fichtinger-Schepman | Detection and quantification of adducts formed upon interaction of diamminedichloroplatinum (II) with DNA, by anion-exchange chromatography after enzymatic degradation[END_REF]. DNA interstrand cross-links could be particularly important for cytotoxicity but [START_REF] Knox | Mechanism of cytotoxicity of anticancer platinum drugs: evidence that cis-diamminedichloroplatinum(II) and cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II) differ only in the kinetics of their interaction with DNA[END_REF], since these only constitute about 1% of the total adducts [START_REF] Roberts | Quantitative estimation of cisplatin-induced DNA interstrand cross-links and their repair in mammalian cells: relationship to toxicity[END_REF], the present data cannot rule out an effect of NU2058 on repair of these specific lesions despite the lack of any effects of NU2058 on overall cisplatin adduct removal (Fig. 2B). However, as discussed above, the potentiation observed with monohydroxymelphalan unequivocally demonstrates that the effect of NU2058 does not involve a mechanism specific for DNA crosslinks.

Total cellular platinum content following cisplatin exposure was increased 1.5 fold by NU2058 and this could be responsible, fully or in part, for the increase in platinum-DNA adduct formation induced by NU2058 (Fig. 2C). NU2058 could affect cellular platinum levels either by causing an increase in cisplatin influx or a decrease in efflux, effects which in turn could cause an increase in drug-DNA interactions. The transport of cisplatin has recently been shown to be related to the transport of copper [START_REF] Safaei | The role of copper transporters in the development of resistance to Pt drugs[END_REF][START_REF] Safaei | Copper transporters regulate the cellular pharmacology and sensitivity to Pt drugs[END_REF], and three copper transporters have been identified which can transport cisplatin; Ctr1, the copper influx carrier protein which passively transports copper across the cell membrane, and ATP7A and ATP7B, two ATP-dependent copper efflux transporters. However, when cell survival was related to level of platinum-DNA adducts it was shown that for equivalent adduct levels, cells treated with In contrast to the effect on cisplatin-DNA adducts, NU2058 had no effect on melphalan-DNA adduct levels (Fig. 5), and potentiation was therefore solely due to the increased cytotoxicity of the adducts that were formed. We hypothesise that the mechanism of potentiation of melphalan is the same as the second mechanism proposed above for the potentiation of cisplatin; namely, that NU2058 alters the cellular response to DNA adducts. To investigate whether the altered cellular response induced by NU2508 was related to effects on DNA repair processes, cisplatin-DNA adduct formation and loss were studied. NU2058 did not alter the rate of DNA adduct loss after removal of cisplatin. Furthermore, consistent with the lack of involvement of DNA repair in the effects of NU2058, it is particularly noteworthy that for potentiation of cisplatin by NU2058 it was necessary for both drugs be present simultaneously (Fig. 3), exposure to NU2058 directly after cisplatin, when repair would be expected to be active, having no effect. Further experiments are required to verify this hypothesis.

As shown here, the potentiation of cisplatin was independent of CDK2 inhibition, and hence it seems likely that potentiation by NU2058 was due to effects involving other protein kinases or ATP binding proteins. Although part of the cisplatin potentiation observed was due to an effect on cisplatin transport, the major effect appears to be an The effect of NU2058 on DNA-cisplatin adduct levels and cisplatin uptake. A) Cisplatin-DNA adduct levels after treatment of SQ20b cells with 100 µM NU2058 or vehicle (0.1% (v/v) DMSO) for 2 hours followed by a further 2 hours in the presence of cisplatin. Total platinum-DNA adduct levels were measured using ICP-MS. B) Cisplatin-DNA adduct formation and loss over time. Cells were treated with 100 µM NU2058 or vehicle (0.1% (v/v) DMSO) between -4 to 0 hours, with cisplatin (25 µM) present between -2 and 0 hours. Cells were then incubated in drug free media for up to 24 hours. C) Cellular cisplatin uptake. Cells were treated as described in A), pellets were sonicated and total cellular platinum determined as described in methods. D) Survival of cells in relation to DNA-cisplatin adduct levels. Values were generated using data from Figures 1B) and

A c c e p t e d M a n u s c r i p t 5

 5 Linking powered by eXtyles cancer and the development of kinase inhibitors is currently a major drug discovery priority.

A

  c c e p t e d M a n u s c r i p t improve our understanding of the mechanism of potentiation of cisplatin by NU2058 we also investigated: 1) whether NU2058 affected cisplatin-DNA interactions and cellular drug uptake; 2) if NU2058 potentiated the cytotoxicity of other DNA damaging agents; 3) whether the effect of NU2058 on potentiation of cisplatin was schedule dependent.

  , UK. Cat. No. A4700) and with a rabbit anti-mouse secondary antibody (DakoCytomation, Denmark, Cat. No. P0260).

  A c c e p t e d M a n u s c r i p t 10 Linking powered by eXtyles

  A c c e p t e d M a n u s c r i p t 11 Linking powered by eXtyles

A

  c c e p t e d M a n u s c r i p t 12 Linking powered by eXtyles to inhibit CDK2, the effects of the compounds on the phosphorylation of Rb at threonine 821 were measured, a well characterised CDK2 phosphorylation site[START_REF] Zarkowska | Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases[END_REF].

A

  c c e p t e d M a n u s c r i p t 13 Linking powered by eXtyles the indicated times. As shown in Figure 2B, NU2058 again increased the formation of platinum-DNA adducts, but did not alter their subsequent rate of removal.

Figures

  Figures 1B and 2A to evaluate the cytotoxicity as a function of DNA adduct levels.

  eXtyles NU2058 showed an approximately 2-fold increase in cytotoxicity in relation to cells treated with cisplatin alone. This latter result suggests that there are two mechanisms involved in the potentiation of cisplatin cytotoxicity by NU2058, 1) that NU2058 affects the transport of cisplatin, resulting in increased DNA adduct formation, and 2) that NU2058 alters the cellular response to the drug-DNA adducts, such that adducts are more toxic.

  eXtyles alteration to the way that cells respond to DNA damage, a response that is common to cisplatin and melphalan. The lack of an effect on temozolomide and IR cytotoxicity could reflect the different cellular response mechanisms to DNA damage caused by these latter two treatments, and since the available data are not consistent with an effect on DNA damage repair, it seems likely that NU2058 is affecting early cellular responses to DNA damage. Phosphorylation events on chromatin have been shown to occur rapidly after cisplatin treatment[START_REF] Wang | Cisplatin-induced post-translational modification of histones H3 and H4[END_REF], and could have a role to play in the cellular response to cisplatin. An alternative explanation is that NU2058 is altering the nature or distribution of the DNA damage across the genome, although not necessarily the overall levels. Ongoing in vitro and in vivo experiments are aimed at identifying kinases that could be the targets by which NU2058 and related compounds potentiate the toxicity of cytotoxic drugs, and the therapeutic potential of cisplatin-NU2058 combinations.

Figure 1 A

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Figure 2

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  2A).

  -C points are the mean of 3 independent experiments, and bars the standard deviation. In Figures 2A and 2C the lines were fitted by linear regression analysis.

Figure 3 Effect

 3 Figure 3

Figure 4 Effect

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Figure 5 The

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Acknowledgement

The authors would like to acknowledge AstraZeneca, the Medical Research Council, and Cancer Research UK for funding this project.