Decitabine (5-aza-2′-deoxycytidine, aza-dCyd) is an anti-cancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 (dCTP pyrophosphatase 1) and dUTPase are two ‘house-cleaning’ nucleotidohydrolases involved in the elimination of non-canonical nucleotides. In the present study, we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase, thus suggesting their contribution to the cellular response to this anti-cancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2′-deoxycytidine-5′-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy.

INTRODUCTION

Nucleoside analogues are effective anti-metabolites commonly used as anti-cancer agents. The cytidine analogue decitabine (5-aza-2′-deoxycytidine, aza-dCyd) is a DNA-demethylating agent and genotoxic drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia [1,2]. Decitabine is metabolically activated in vivo through consecutive phosphorylations into aza-dCTP (5-aza-2′-deoxycytidine-5′-triphosphate) that is readily incorporated into DNA and extended by the DNA polymerase [3]. Once in DNA, decitabine acts as a suicidal substrate by covalently trapping DNMT (DNA methyltransferase) molecules that attempt to initiate cytosine methylation. The resulting DNA–protein cross-links trigger the proteasomal degradation machinery and lead to the depletion of the DNA methylation activities of the cell. Consequently, the replacement of deoxycytidine by decitabine results in hypomethylation at the promoter DNA regions and the reactivation of epigenetically repressed genes [4]. The transcriptional activation of tumour-suppressor genes which are aberrantly silenced in cancer cells constitutes the basic principle of epigenetic cancer therapy.

The extent to which the in vivo anti-tumour properties of decitabine and the clinical response to decitabine treatment depend on epigenetic activities remains unclear. It has not been possible to establish a definitive correlation between the inhibition of cell proliferation and clinical response with the reversal of methylation and gene re-expression [5]. In fact, it is generally agreed that decitabine has dual effects on neoplastic cells in a dose-dependent manner. At low doses, cells survive, but reactivation of genes that control proliferation may lead to differentiation, cell cycle arrest and increased apoptosis [6]. At high doses, decitabine induces genome-wide DNA damage and cytotoxicity [7]. Most of the DNA damage arises from the DNA–DNMT cross-links which can block DNA synthesis and induce directly or indirectly DNA double-strand breaks, eventually leading to cell death [8]. Decitabine has also been reported to enhance mutagenesis, mainly point mutations and genome rearrangements most likely due to protein–DNA cross-links [9,10]. The biochemical pathway that initiates repair of DNA–DNMT adducts has not yet been described in detail; however, it may involve DNA double-strand break repair factors at its late stages. It is well established that decitabine treatment induces the activation of a specific DNA damage response that includes the phosphorylation of histone H2AX [7]. Additionally, the cytotoxic and mutagenic properties of decitabine may be partially derived from its intrinsic chemical instability once it is incorporated into DNA or by the accumulation of DNA repair intermediates [3]. Recently, it has been proposed that the BER (base excision repair) mechanism might initiate the repair of decitabine-induced DNA base lesions, although the precise nature of the damage or the DNA glycosylases involved remains unknown [11].

The all-α NTP pyrophosphatase DCTPP1 (dCTP pyrophosphatase 1) is a pyrophosphohydrolase that contributes to the homoeostasis of the dNTP pool in human cells by controlling the levels of dCTP, one of its major substrates. DCTPP1 can also hydrolyse C5-modified dNTPs such as 5-halogenated, 5-methyl and 5-formyl deoxycytidines, and therefore may have an additional ‘house-cleaning’ function [12]. The enzyme dUTPase (deoxyuridine triphosphate nucleotidohydrolase) also plays two roles, providing dUMP for de novo biosynthesis of dTTP and sanitizing the deoxynucleotide pool by the specific removal of dUTP [1315].

In the present study, we have investigated the role of DCTPP1 and dUTPase in the metabolic response to decitabine. We found that DCTPP1 or dUTPase down-regulation increased decitabine-induced toxicity in HeLa and MRC-5 cells at a wide range of doses. DCTPP1 can hydrolyse the activated form of decitabine, aza-dCTP, and might constitute a novel route in the detoxification of this DNA-methylation inhibitor. On the other hand, both dUTPase- and DCTPP1-deficient cells exhibited an accumulation of dUTP in the nucleotide pool and uracil in their genomic DNA as a consequence of the treatment, suggesting an alternative mode of action for decitabine in which the metabolism of decitabine may ultimately lead to the genotoxic accumulation of dUTP in the cellular pool.

MATERIALS AND METHODS

Nucleotides, nucleosides and antibodies

Decitabine, ara-Cyd (cytarabine) and dCyd (2′-deoxycytidine) were purchased from Sigma; aza-dCTP, ara-CTP (cytarabine triphosphate), dF-CTP (gemcitabine triphosphate; 2′-difluorocytidine-5′-triphosphate) and ddCTP (zalcitabine triphosphate; 2′,3′-dideoxycytidine-5′-triphosphate) were obtained from Jena Bioscience. Anti-DCTPP1 [12] and anti-dUTPase (the present study) are anti-rabbit polyclonal antibodies generated in our laboratory. DCTD (dCMP deaminase), TMPK (thymidylate kinase), TS (thymidylate synthase) and TK (thymidine kinase) detection was carried out with anti-rabbit polyclonal antibodies from Santa Cruz Biotechnology. CDA (cytidine deaminase) and DCK (deoxycytidine kinase) were detected with rabbit anti-CDA and mouse anti-DCK antibodies respectively (Abcam). Anti-α-tubulin was purchased from Ab Frontier.

Enzymatic assays and kinetic analysis

The pyrophosphohydrolase activity of DCTPP1 was determined using a continuous spectrophotometric assay described previously [12]. In a standard reaction (1 ml final volume), 10–250 μM nucleotide substrate was incubated in reaction buffer (20 mM MgCl2, 100 mM KCl, 0.75 mg/ml BSA and 4 mM DTT) with DCTPP1 concentrations ranging from 0.1 to 1 μM. All reactions were carried out at 25°C. Kinetic parameters resulted from adjusting the data to the Hill equation:

 
formula

where V0 is the initial rate, Vmax is the maximum reaction rate, [S] is the substrate concentration, h is the Hill coefficient, and Km is the Michaelis–Menten constant.

Cell cultures and transfections

Two wild-type p53-expressing cell lines were used, the normal human fibroblast line MRC-5 (ATCC CCL-171) and the tumour epithelial line HeLa (ATCC CCL-2). Both cell lines were obtained from the ATCC (Manassas, VA, U.S.A.) and authenticated by STR (short tandem repeat) DNA profiling. In the present study, cells were not passaged for more than 6 months following purchase. MRC-5 was cultured in Eagle's minimum essential medium (Gibco/Life Technologies) with 2 mM L-glutamine (GlutaMAX™, Gibco/Life Technologies) and HeLa cell line was cultured in Dulbecco's modified Eagle's medium (PAA Laboratories/GE Healthcare) supplemented with 1× MEM Non-Essential Amino Acids Solution (Gibco/Life Technologies) and 2 mM L-glutamine. Media were supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco/Life Technologies). Cell lines were cultured in a humidified atmosphere of 5% CO2 at 37°C.

DCTPP1 and dUTPase silencing were carried out with an siRNA oligonucleotide pool (ON-TARGETplus smart pool, Dharmacon). The DCTPP1-specific siRNAs have been previously validated [12]. For dUTPase silencing, the following mRNA sequences were targeted: 5′-GCUCAUUUGCGAACGGAUU-3′, 5′-UGUAGGAGCUGGUGUCAUA-3′, 5′-UAGAGGAAAUGU-UGGUGUU-3′ and 5′-UGCCUAUGAUUACACAAUA-3. The negative control consisted of four non-targeting siRNA oligonucleotides (ON-TARGETplus non-targeting pool, Dharmacon). Transient transfections were carried out according to the manufacturer's instructions.

Proliferation and clonogenic assays

A total of 103 cells were exposed to increasing doses of decitabine, deoxycytidine or cytarabine at 37°C for 24 h (HeLa) or 72 h (MRC-5). Incubation times with the nucleoside analogues were based on differences in cell proliferation and sensitivity to decitabine. The medium was exchanged and the drug replenished every 24 h. Viable cell number was determined by Resazurin (Sigma) reduction. For the clonogenic assay, cells were seeded in a six-well plate at a density of 300 cells per well and treated with decitabine. After completion of the treatments, fresh growth medium was added to the wells and cells were left to proliferate for 7 (Hela) or 12 (MRC-5) additional days. Colonies were stained with a solution of 0.5% Crystal Violet in methanol.

Quantification of DNA methylation by liquid chromatography–tandem mass spectrometry

DNA was isolated using the Tissue & Cell GenomicPrep MiniSpin Kit (GE Healthcare), quantified and subjected to hydrolysis with formic acid as described previously [12]. UPLC (ultraperformance LC)–MS/MS analysis was carried out using a Waters XEVO TQ-S spectrometer at the Center of Scientific Instrumentation (University of Granada, Granada, Spain).

Intracellular dNTP pool size determination

dNTP levels were measured using a DNA polymerase assay with minor modifications [1618]. A total of 106 cells were extracted with 1 ml of 1:1 (v/v) methanol/water at −20°C and the suspension was vortex-mixed vigorously. Samples were then subjected to two freeze–thaw cycles (10 min each in solid CO2/ethanol and ice), before centrifugation at 16000 g for 20 min and 4°C. The supernatants were collected, dried under vacuum and dissolved in 40 μl of dUTPase buffer (34 mM Tris/HCl, pH 7.8, and 5 mM MgCl2) or dUTPase buffer plus 30 ng of human dUTPase [19] for 20 min at 37°C. To stop the reaction, samples were precipitated with methanol, centrifuged at 16000 g for 20 min, dried and used for the quantification of the dNTP pool size as described in [12].

Quantitative determination of uracil in DNA

Uracil incorporation into DNA was measured using a qPCR (quantitative real-time PCR)-based assay following the protocol described by Horváth and Vértessy [20] with minor variations [21]. Genomic DNA was extracted using MasterPure DNA Purification Kit (Epicentre), digested with SacI-HF (New England Biolabs) and 3–5 kb fragments containing the target template [GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene], isolated and purified from 1% agarose gel. A 2-fold dilution series of the DNA samples was amplified with PfuTurbo Hotstart (Agilent Technologies) or Taq polymerase (Bioline) and primers 5′-CTCCTGCCCTTTGAGTTTGATG-3′ and 5′- CAGCAGAGAAGCAGACAGTTATG-3′. qPCR was performed with a CFX96 Real Time System C1000 Thermal Cycler (Bio-Rad Laboratories). Cq values obtained were used to determine uracil content as described in [20]. All values are referred to non-treated control siRNA-transfected cells.

Immunofluorescence analysis of γH2AX foci

Cells were grown on sterile glass coverslips, fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature and permeabilized in PBS containing 0.1% Triton X-100 for 10 min at room temperature. Cells were then incubated with 1:250 diluted monoclonal anti-γH2AX antibody (clone JBW301, Millipore) for 30 min at 37°C and detected with secondary Alexa Fluor® 488-conjugated goat anti-mouse IgG (Invitrogen/Life Technologies). Coverslips were dehydrated in methanol and mounted in Vectashield with DAPI (Vector Laboratories). Digital images were captured using a Leica TCS SP5 confocal microscopy system and analysed with Fiji software.

Statistics

One or two-way ANOVA followed by Dunnett's post-hoc test (referred to control or non-treated cells) was used to analyse the data. Normality and homogeneity of variance assumptions were checked for each analysis. When the variances were different, a Welch test followed by a Games–Howell post-test were used. Results are expressed as means±S.D. of at least three independent replicates (except data in Figure 6B). A χ2 test was performed for γH2AX-positive cell analysis. A Fisher test was applied when the assumptions were not fulfilled. Data presented in Figure 6(B) were obtained from two independent experiments. Differences were considered significant when P<0.05.

RESULTS

DCTPP1 and dUTPase expression is up-regulated in response to decitabine

We have shown previously that media supplementation with an excess of deoxycytidine induces an increase in the expression of DCTPP1 in HeLa cells [12]. Hence we expected that exposure to decitabine might induce a similar modulation of enzyme levels in response to the accumulation of aza-dCTP. As shown in Figure 1, the amount of DCTPP1 protein increased up to 3-fold at 24 h after addition to the medium of 10 μM decitabine. At the same time, CDA, DCTD, TS and dUTPase expression was also up-regulated, suggesting the generation of metabolic intermediates different from those leading to the formation of aza-dCTP.

Modulation of enzyme expression by decitabine

Figure 1
Modulation of enzyme expression by decitabine

HeLa cells were exposed to 10 μM decitabine (aza-dCyd) and enzyme levels in 10 μg of protein cell extract were determined by Western blotting (upper panel). Expression at 24 h was quantified with ImageQuant software (GE Healthcare) (lower panel).

Figure 1
Modulation of enzyme expression by decitabine

HeLa cells were exposed to 10 μM decitabine (aza-dCyd) and enzyme levels in 10 μg of protein cell extract were determined by Western blotting (upper panel). Expression at 24 h was quantified with ImageQuant software (GE Healthcare) (lower panel).

Depletion of DCTPP1 or dUTPase sensitizes cells to decitabine

The observation that cell exposure to decitabine translates into the overexpression of two house-cleaning nucleotidohydrolases with the capacity to hydrolyse non-canonical nucleotides led us to evaluate their potential protective role against the cytotoxic action of decitabine once activated and incorporated into the nucleotide pool. HeLa and MRC-5 cells were transfected with an siRNA pool against DCTPP1 or dUTPase and then treated with increasing concentrations of the nucleoside analogue. In all of the experiments performed, a non-targeting siRNA pool was used as a negative control. siRNA-mediated depletion of the proteins was checked by Western blotting (Figure 2A). Strong down-regulation of DCTPP1 or dUTPase increased the toxic effect of decitabine in HeLa cells or in the non-tumoral cell line MRC-5 (Figure 2B). In contrast, cells transfected with the non-targeting siRNA pool did not exhibit a decrease in viability even in the presence of 100 μM decitabine. The protective effect was specific for decitabine since the absence of DCTPP1 or dUTPase did not have a significant impact on cell proliferation after exposure to the canonical nucleoside deoxycytidine or to the cytidine analogue cytarabine (Figure 2B).

Down-regulation of DCTPP1 and dUTPase sensitizes cells to decitabine

Figure 2
Down-regulation of DCTPP1 and dUTPase sensitizes cells to decitabine

(A) Western blot analysis of HeLa and MRC-5 cells transfected with DCTPP1-specific siRNAs, dUTPase-specific siRNAs or a non-targeting (Control) siRNA. Cell extracts were prepared at the fourth day post-transfection. Protein loading was normalized with anti-α-tubulin. (B) HeLa (upper row) and MRC-5 cells (lower row) were exposed to decitabine (aza-dCyd), 2′-deoxycytidine (dCyd), and cytarabine (ara-Cyd). Percentage of viability was calculated relative to the number of viable cells in the absence of exogenous nucleosides. All values are mean±SD (n=3). Data concerning the sensitivity of DCTPP1 siRNA-transfected cells to dCyd have been published elsewhere [12] and are included for comparison purposes. (C) Clonogenic survival of HeLa and MRC-5 cells transfected with Control, DCTPP1- or dUTPase-specific siRNAs and treated with increasing doses of decitabine. All values are mean±SD (n=4).

Figure 2
Down-regulation of DCTPP1 and dUTPase sensitizes cells to decitabine

(A) Western blot analysis of HeLa and MRC-5 cells transfected with DCTPP1-specific siRNAs, dUTPase-specific siRNAs or a non-targeting (Control) siRNA. Cell extracts were prepared at the fourth day post-transfection. Protein loading was normalized with anti-α-tubulin. (B) HeLa (upper row) and MRC-5 cells (lower row) were exposed to decitabine (aza-dCyd), 2′-deoxycytidine (dCyd), and cytarabine (ara-Cyd). Percentage of viability was calculated relative to the number of viable cells in the absence of exogenous nucleosides. All values are mean±SD (n=3). Data concerning the sensitivity of DCTPP1 siRNA-transfected cells to dCyd have been published elsewhere [12] and are included for comparison purposes. (C) Clonogenic survival of HeLa and MRC-5 cells transfected with Control, DCTPP1- or dUTPase-specific siRNAs and treated with increasing doses of decitabine. All values are mean±SD (n=4).

DCTPP1 or dUTPase-deficient cells incubated with decitabine also displayed a reduced colony-forming capacity compared with control cells (Figure 2C). To investigate a potential resistance response to decitabine mediated by DCTPP1 and dUTPase, we tested whether the overexpression of these proteins might confer additional protection to decitabine by comparing clonogenic survival of reference HeLa cells with that of cells overproducing DCTPP1 or dUTPase after exposure to a range of decitabine doses (Figure 3). At 1 μM decitabine, the proportion of surviving cells expressing dUTPase was significantly enhanced compared with the number of viable colonies obtained with cells transfected with the empty vector. The overexpression of DCTPP1 had no impact on cell survival at any of the concentrations tested, suggesting that endogenous levels of DCTPP1, but not of dUTPase, are sufficient to deal with potentially cytotoxic metabolites generated from decitabine.

Effect of DCTPP1 and dUTPase overexpression on the survival against decitabine

Figure 3
Effect of DCTPP1 and dUTPase overexpression on the survival against decitabine

Left panel, Western blot showing the levels of dUTPase and DCTPP1 proteins in HeLa cells transfected with pcDNA3-DCTPP1 (DCTPP1 OE), pcDNA3-dUTPase (DUT OE) or the empty pcDNA3 vector (Control). Right panel, Clonogenic survival of HeLa cells expressing normal or increased levels of DCTPP1 or dUTPase and treated with increasing doses of decitabine. All values are mean±SD (n=3).

Figure 3
Effect of DCTPP1 and dUTPase overexpression on the survival against decitabine

Left panel, Western blot showing the levels of dUTPase and DCTPP1 proteins in HeLa cells transfected with pcDNA3-DCTPP1 (DCTPP1 OE), pcDNA3-dUTPase (DUT OE) or the empty pcDNA3 vector (Control). Right panel, Clonogenic survival of HeLa cells expressing normal or increased levels of DCTPP1 or dUTPase and treated with increasing doses of decitabine. All values are mean±SD (n=3).

DCTPP1 modulates DNA hypomethylation induced by decitabine

Since DCTPP1 catalyses the hydrolysis of dCTP to dCMP and pyrophosphate, we investigated its potential role in detoxification of aza-dCTP (the activated form of decitabine). In vitro, DCTPP1 is active on aza-dCTP (Km=54.04±0.34 μM; kcat=7.54±0.03 s−1) (Figure 4A), exhibiting kinetic parameters very similar to those obtained for dCTP (Km=47.63±2.66 μM; kcat=5.69±0.18 s−1) [12]. The sigmoidal character of the aza-dCTP saturation curve and the double-reciprocal plot showing non-linear kinetics indicate that substrate binding is occurring with positive co-operativity (h=2.29±0.03) (Figure 4A). No activity was detected with other nucleotide analogues tested such as ara-CTP, dF-CTP or ddCTP.

DCTPP1 hydrolyses aza-dCTP and prevents DNA demethylation in human cells

Figure 4
DCTPP1 hydrolyses aza-dCTP and prevents DNA demethylation in human cells

(A) Left panel, the enzymatic assay was performed by incubating DCTPP1 (0.5 μM) with increasing concentrations of aza-dCTP (20–250 μM) under optimized reaction conditions as previously described [12]. Kinetic parameters and the Hill's coefficient (nH) resulted from adjusting the data to the Hill's equation. All values are mean±SD (n=3). Right panel, double reciprocal plot showing non-linear kinetics for aza-dCTP. (B) Global DNA methylation in HeLa cells exposed to increasing concentrations of decitabine for 24 or 48 h. All values are mean±SD (n=3).

Figure 4
DCTPP1 hydrolyses aza-dCTP and prevents DNA demethylation in human cells

(A) Left panel, the enzymatic assay was performed by incubating DCTPP1 (0.5 μM) with increasing concentrations of aza-dCTP (20–250 μM) under optimized reaction conditions as previously described [12]. Kinetic parameters and the Hill's coefficient (nH) resulted from adjusting the data to the Hill's equation. All values are mean±SD (n=3). Right panel, double reciprocal plot showing non-linear kinetics for aza-dCTP. (B) Global DNA methylation in HeLa cells exposed to increasing concentrations of decitabine for 24 or 48 h. All values are mean±SD (n=3).

The ability of DCTPP1 to act upon aza-dCTP suggests that, under decitabine exposure, this enzyme may be preventing the accumulation of azacytosine in DNA and consequently keeps the level of DNA methylation constant. To address this question, we determined changes in global methylation by measuring the ratio of 5-methylcytosine to cytosine by LC–MS/MS (Figure 4B). Under our experimental conditions, we observed a moderate decrease in global genome methylation in control cells only after exposure to the highest dose of decitabine for 48 h (P=0.026). Depletion of DCTPP1 further increased the DNA hypomethylating effect of decitabine, which, in the absence of the nucleotidohydrolase, caused significant reductions in the level of methylcytosine at 10 and 100 μM. These data support a potential role for DCTPP1 in preventing incorporation of azacytosine into DNA upon treatment of cells with decitabine.

Decitabine treatment alters the nucleotide pool composition

It has been suggested that deaminated derivatives of decitabine could interfere with the pyrimidine biosynthesis pathway by inhibiting TS [22]. To investigate whether exposure to decitabine promotes the expansion of the dUTP pool in a similar way to other TS inhibitors such as 5-fluorouracil [23], we measured perturbations in the nucleotide pool upon treatment with decitabine and the role of the nucleotidohydrolases DCTPP1 and dUTPase in these potential perturbations (Figure 5).

Effect of decitabine on the intracellular dNTP pool

Figure 5
Effect of decitabine on the intracellular dNTP pool

Nucleotide concentration was measured in HeLa cells transfected with non-targeting, DCTPP1 or dUTPase-specific siRNA pools. Treatments were carried out with 0, 10 and 100 μM of decitabine for 24 h. All values are mean±SD (n=4).

Figure 5
Effect of decitabine on the intracellular dNTP pool

Nucleotide concentration was measured in HeLa cells transfected with non-targeting, DCTPP1 or dUTPase-specific siRNA pools. Treatments were carried out with 0, 10 and 100 μM of decitabine for 24 h. All values are mean±SD (n=4).

The DNA polymerase-based assay used to quantify dNTPs does not allow discrimination between dCTP and aza-dCTP or dUTP and aza-dUTP so we will refer to them as (aza-)dCTP or (aza-)dUTP. In control cells, only the treatment with the highest dose of decitabine induced a substantial increase in the concentration of intracellular (aza-)dCTP (from 3.9 to 4.1 and 8.2 pmol/106 cells at 10 μM or 100 μM respectively) probably due to the conversion of decitabine into aza-dCTP and the saturation of catabolic activities. On the other hand, the depletion of DCTPP1 promoted a substantial accumulation of aza-(dCTP) by treatment with decitabine (from 5.6 to 13.8 and 17.3 pmol/106 cells) suggesting that DCTPP1 is likely the main catabolic activity of the activated form of decitabine. Unexpectedly, in the absence of decitabine, DCTPP1-silenced cells also exhibited a significant pool of dUTP (0.9 pmol/106 cells) which cannot be detected in control cells. This pool increased 2-fold after exposure to 100 μM decitabine (1.9 pmol/106 cells). We hypothesize that dUTP might be generated as a result of the allosteric activation of dCMP deaminase by the abnormally high levels of dCTP that accumulate in these cells [12] providing an excess of dUMP that is subsequently converted into dUTP.

In the case of cells subjected to siRNA-mediated depletion of dUTPase and exposed to decitabine, important alterations in the nucleotide pool were observed. These alterations consisted in a strong increase in the intracellular amount of pyrimidine nucleotides: (aza-)dCTP (from 4.5 to 7.3 and 9.6 pmol/106 cells), dTTP (from 8.6 to 12.1 and 11.5 pmol/106 cells) and (aza-)dUTP (from 0.4 to 1.6 and 2.2 pmol/106 cells). Although it is true that the expansion of the dCTP pool induced by decitabine can be mostly attributed to the formation of aza-dCTP, the increase of dTTP pools might be the consequence of an up-regulation of dNTP synthesis in response to DNA damage as reported for other genotoxic agents [24]. Indeed, in DNA-damage-stressed cells, the synthesis of dTTP needed for recovery from DNA damage has been reported to be essentially mediated by TK [24]. In spite of such an expanded dTTP pool, decitabine exposure caused a 2-fold increase in the dUTP/[dUTP+dTTP] ratio that elevates substantially the risk of uracil misincorporation into DNA.

Assessment of uracil and DNA damage induced by decitabine in DCTPP1- and dUTPase-deficient backgrounds

An expected consequence of the accumulation of dUTP in the nucleotide pool as a result of the treatment with decitabine would be the misincorporation of uracil into DNA, especially in the absence of dUTPase. To monitor changes in the uracil genome content in the genome, we have used a qPCR assay which utilizes the B-type DNA polymerase of Pyrococcus furiosus (Pfu) and the Taq DNA polymerase for amplifications [20]. The Pfu/Taq PCR method is based on the fact that the DNA polymerase Pfu is strongly inhibited by uracil-containing DNA, whereas Taq DNA polymerase can replicate through the deaminated base. The differences in product formation by the two enzymes provides a means to quantify the amount of uracil in the DNA sample [25]. Genomic DNA was obtained from HeLa cells transfected with control, DCTPP1 or dUTPase siRNAs and treated with 0, 1, 10 and 100 μM decitabine for 24 or 48 h. Treatment of control cells with decitabine for 24 h produced a significant increase in the DNA uracil content which was enhanced further after exposure to the nucleoside analogue for 48 h (P<0.00001) (Figure 6A). We next analysed the presence of uracil in DCTPP1- or dUTPase-silenced cells. Consistent with the existence of an endogenous dUTP pool, DCTPP1-deficient cells contain higher constitutive levels of uracil in their genomic DNA and accumulate higher amounts of uracil after exposure to decitabine than control cells.

Decitabine induces uracil accumulation and DNA strand breaks specially in the absence of DCTPP1 or dUTPase

Figure 6
Decitabine induces uracil accumulation and DNA strand breaks specially in the absence of DCTPP1 or dUTPase

(A) Uracil content in genomic DNA from HeLa cells transfected with Control, DCTPP1 or dUTPase siRNAs and treated with increasing concentrations of decitabine for 24 or 48 h. Values resulting from genomic DNA isolated from non-treated Control siRNA-transfected cells were used as reference. All values are mean±SD (n=3). P-values underlined with dotted line are referred to treated Control siRNA cells. (B) Left panel, representative microscopy images showing γH2AX staining in non-treated or decitabine-treated HeLa cells. All chosen fields contain a similar number of cells as determined by DAPI staining (not shown). Right panel, percentage plots of γH2AX-positive cells in Control, DCTPP1, and dUTPase siRNA cells treated with decitabine. γH2AX-positive cells (> 5 foci) were scored from a total of two hundred cells per cell line and dose.

Figure 6
Decitabine induces uracil accumulation and DNA strand breaks specially in the absence of DCTPP1 or dUTPase

(A) Uracil content in genomic DNA from HeLa cells transfected with Control, DCTPP1 or dUTPase siRNAs and treated with increasing concentrations of decitabine for 24 or 48 h. Values resulting from genomic DNA isolated from non-treated Control siRNA-transfected cells were used as reference. All values are mean±SD (n=3). P-values underlined with dotted line are referred to treated Control siRNA cells. (B) Left panel, representative microscopy images showing γH2AX staining in non-treated or decitabine-treated HeLa cells. All chosen fields contain a similar number of cells as determined by DAPI staining (not shown). Right panel, percentage plots of γH2AX-positive cells in Control, DCTPP1, and dUTPase siRNA cells treated with decitabine. γH2AX-positive cells (> 5 foci) were scored from a total of two hundred cells per cell line and dose.

Similarly to DCTPP1, the lack of dUTPase provoked an increase in the basal levels of uracil in DNA, supporting the notion that dUTP is being constantly generated by the normal metabolism of the cell and removed by dUTPase from the nucleotide pool. Upon decitabine treatment, uracil incorporation into DNA increased further in dUTPase-silenced cells, suggesting a major role for this enzyme in counteracting the formation of aza-dUTP.

Previous studies have reported that cell treatment with decitabine leads to the formation of DNA double-strand breaks in a direct or indirect manner and also promotes the accumulation of γH2AX foci (H2AX pSer139), a hallmark of the cellular response to this type of DNA damage [7]. We proceeded to monitor γH2AX foci by immunofluorescence microscopy in DCTPP1- and dUTPase-depleted HeLa cells after incubation with decitabine (Figure 6B). Control cells exposed to 10 μM decitabine exhibited γH2AX foci in only 7% of the cell population, but this increased up to 10% and 12% in the absence of DCTPP1 and dUTPase respectively. At 100 μM decitabine, the proportion of γH2AX-positive cells increased from 21% in control cells to 33% and 38% in DCTPP1- and dUTPase-depleted cells respectively, an observation that supports the notion that at least some of the DNA breaks in these genetic backgrounds can arise as a consequence of the incorporation of uracil or aza-uracil during replication.

DISCUSSION

Catabolic activities may influence metabolism and thereby the pharmacological efficacy of anti-cancer nucleoside analogues. Mammalian cells can take up decitabine and, upon entering the cellular nucleotide pool, the pyrimidine analogue can be potentially channeled into different biochemical pathways (Figure 7). Decitabine can be deaminated by CDA to aza-deoxyuridine, which is poorly phosphorylated by TK [26] or, alternatively, decitabine can be converted into its active form via a phosphorylation pathway that includes DCK, UMP/CMP kinase and NDPK (nucleoside diphosphate kinase). The ability of DCTPP1 to hydrolyse the activated form of decitabine and the hypersensitivity of DCTPP1-deficient cells exposed to this nucleoside analogue suggest that this enzyme may be involved in the catabolism of aza-dCTP. Indeed, our data showing increased cytosine hypomethylation in DCTPP1-silenced cells support the notion that this nucleotidohydrolase is interfering with the primary mode of action of decitabine and prevents the incorporation of aza-dCTP into DNA. Since the action of DCTPP1 entails the reversion of the phosphorylation steps involved in decitabine activation, it is plausible that the relative activity ratio of the kinases UMP/CMP kinase and NDPK with regard to DCTPP1 may have predictive clinical value in treatments with this nucleoside analogue.

Role of DCTPP1 and dUTPase in the response to decitabine

Figure 7
Role of DCTPP1 and dUTPase in the response to decitabine

Decitabine is converted into its active form via a phosphorylation pathway initiated by deoxycytidine kinase (DCK). DCTPP1 can hydrolyse aza-dCTP and could prevent its incorporation into DNA and the formation of DNMT-DNA cross-links. Alternatively, decitabine can be deactivated by cytidine deaminase (CDA) to aza-deoxyuridine (aza-dUrd) which is poorly phosphorylated by thymidine kinase (TK). The aza-dCMP intermediate can also be deaminated by dCMP deaminase (DCTD) to produce aza-dUMP, which might potentially inhibit thymidylate synthase (TS). The accumulation of genotoxic (aza-)dUTP would promote uracil misincorporation and DNA damage.

Figure 7
Role of DCTPP1 and dUTPase in the response to decitabine

Decitabine is converted into its active form via a phosphorylation pathway initiated by deoxycytidine kinase (DCK). DCTPP1 can hydrolyse aza-dCTP and could prevent its incorporation into DNA and the formation of DNMT-DNA cross-links. Alternatively, decitabine can be deactivated by cytidine deaminase (CDA) to aza-deoxyuridine (aza-dUrd) which is poorly phosphorylated by thymidine kinase (TK). The aza-dCMP intermediate can also be deaminated by dCMP deaminase (DCTD) to produce aza-dUMP, which might potentially inhibit thymidylate synthase (TS). The accumulation of genotoxic (aza-)dUTP would promote uracil misincorporation and DNA damage.

Importantly, a number of pieces of evidence of the present study suggest that aza-dCMP can also be deaminated by dCMP deaminase to produce aza-dUMP. Early reports have demonstrated the formation of aza-dUMP in vivo after exposure to [3H]decitabine [27]. The increase in dCMP deaminase expression (Figure 1) would certainly contribute to the generation of aza-dUMP. Moreover, the presence of elevated levels of aza-dCTP resulting from decitabine metabolic activation would further favour deamination due to the allosteric activation of dCMP deaminase [28]. In addition to the potential formation of aza-dUTP, it is likely that aza-dUMP itself acts as a competitive TS inhibitor. In agreement with this notion, a recent screening of TS inhibitors using a cellular thermal shift assay led to a product of decitabine metabolism which was later identified as 5-aza-2′-deoxyuridine 5′-monophosphate [29]. On the other hand, the up-regulation of TS is a well-known mechanism of resistance to short-term exposure to TS inhibitors [30]. It has been proposed that TS controls its own translation by binding its own mRNA, whereas the interaction with the inhibitor disrupts this autoregulatory loop, leading to translational derepression and increased TS protein levels [31,32].

In the absence of normal levels of dUTPase or DCTPP1, cells exposed to decitabine accumulated a significant amount of intracellular (aza-)dUTP and increased levels of genomic uracil. Numerous studies have established that the DNA damage associated with misincorporation of uracil in DNA is a significant mechanism of cytotoxicity induced by TS-inhibiting chemotherapeutic agents [23]. Whereas the dUTPase activity preserves genomic integrity by removing (aza-)dUTP from the nucleotide pool, the absence of DCTPP1 might be promoting decitabine cytotoxicity by two different mechanisms: first, it allows the accummulation of aza-dCTP in the cellular pool and, secondly, the excess of (aza-)dCTP stimulates the synthesis of toxic aza-dUMP derivatives through the allosteric activation of dCMP deaminase (Figure 7).

We postulate that decitabine enhances uracil misincorporation into DNA and activates BER mechanisms. Indeed, a recent study has demonstrated that XRCC1 (X-ray repair cross-complementing 1) mutant cells, defective in BER, are hypersensitive to decitabine and this phenotype is associated with an accumulation of abasic sites and DNA strand breaks in genomic DNA [11]. It is therefore possible that some of these breaks are an indication of the formation of DNA repair intermediates induced by the presence of uracil and aza-uracil.

In summary, the data of the present study suggest a novel mode of action for decitabine in which decitabine may ultimately lead to the toxic accumulation of (aza-)dUTP in the nucleotide pool and its incorporation into the genome. Further research will allow us to establish whether inhibition of these nucleotidohydrolases may constitute a novel approach to improve the efficacy of anti-tumour therapy with decitabine and circumvent potential drug resistance phenotypes. Indeed, novel molecules capable of specifically inhibiting the activity of dUTPase and DCTPP1 have been recently identified to enhance the cytotoxic effect of pyrimidine anti-cancer agents [33,34], thus highlighting the relevance of these nucleotidohydrolases in the mode of action of pyrimidine derivatives currently used in anti-tumour therapy.

AUTHOR CONTRIBUTIONS

Cristina Requena, Dolores González-Pacanowska and Antonio Vidal conceived and designed the study. Cristina Requena, András Horváth and Beáta Vértessy developed the methodology. Cristina Requena and Guiomar Pérez-Moreno acquired data. Cristina Requena, Guiomar Pérez-Moreno, Luis Ruíz-Pérez, Dolores González-Pacanowska and Antonio Vidal analysed and interpreted data. Cristina Requena, András Horváth, Beáta Vértessy, Luis Ruíz-Pérez, Dolores González-Pacanowska and Antonio Vidal wrote, reviewed, and/or revised the paper.

We thank Aurora Constán for her technical assistance.

FUNDING

This work was supported by the Ministerio de Economía y Competitividad (Plan Nacional de Investigación) [grant numbers SAF2011-27860 (to A.E.V.) and SAF2013-48999-R (to D.G.-P.)], Junta de Andalucía [grant numbers BIO-199 and P12-BIO-2059 (to D.G.-P.)], Hungarian National Research, Development and Innovation Office [grant numbers NK-84008 and K-109486], and the International Centre for Genetic Engineering and Biotechnology (ICGEB) [grant number CRP HUN14-01 (to B.G.V.)].

Abbreviations

     
  • ara-CTP

    cytarabine triphosphate

  •  
  • ara-Cyd

    cytarabine

  •  
  • aza-dCTP

    5-aza-2′-deoxycytidine-5′-triphosphate

  •  
  • aza-dCyd

    5-aza-2′-deoxycytidine

  •  
  • BER

    base excision repair

  •  
  • CDA

    cytidine deaminase

  •  
  • DCK

    deoxycytidine kinase

  •  
  • DCTD

    dCMP deaminase

  •  
  • DCTPP1

    dCTP pyrophosphatase 1

  •  
  • dCyd

    2′-deoxycytidine

  •  
  • ddCTP

    2′,3′-dideoxycytidine-5′-triphosphate

  •  
  • dF-CTP

    2′-difluorocytidine-5′-triphosphate

  •  
  • DNMT

    DNA methyltransferase

  •  
  • NDPK

    nucleoside diphosphate kinase

  •  
  • qPCR

    quantitative real-time PCR

  •  
  • TK

    thymidine kinase

  •  
  • TMPK

    thymidylate kinase

  •  
  • TS

    thymidylate synthase

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