The Mechanism of the Selective Antiproliferation Effect of Guanine

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The mechanism of the selective anti-proliferation effect of guanine-based biomolecules and its compensation Junyan Wang, Tao Bing, Nan Zhang, Luyao Shen, Junqing He, Xiangjun Liu, Linlin Wang, and Dihua Shangguan ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00062 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

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ACS Chemical Biology

The mechanism of the selective anti-proliferation effect of guanine-based biomolecules and its compensation Junyan Wang1, 2, Tao Bing1, 2,*, Nan Zhang1, 2, Luyao Shen1, 2, Junqing He1, Xiangjun Liu1, 2, Linlin Wang1, 2, Dihua Shangguan1, 2*

1

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical

Chemistry for Living Biosystems, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China 2

University of the Chinese Academy of Sciences, Beijing 100049, China

* To whom correspondence should be addressed: *e-mail: [email protected], [email protected]

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Abstract As endogenous biomolecules, guanine, guanine-based nucleosides and nucleotides are essential for cellular DNA/RNA synthesis, energy metabolism and signal transduction. However, these biomolecules have been found to have cell-specific anti-proliferation effect at higher concentrations, and the mechanism is unclear. In this study, we demonstrate that guanine deaminase (GDA) is a major factor in determining the cell-type selectivity to the anti-proliferation effect of guanine-based biomolecules. GDA catalyzes the deamination of guanine to xanthine, which is an essential part of guanine degradation pathway. GDA deficient cells could not efficiently remove the excess guanine-based biomolecules. These excess molecules disturb the metabolism of adenine-, cytosine- and thymine- based nucleotides, subsequently inhibit the DNA synthesis and cell growth, and eventually results in the apoptosis/death of GDA deficient cells. The inhibition of DNA synthesis could be relieved by simultaneous addition of adenine- and cytosine-based nucleosides, and the inhibited DNA synthesis could be restarted by post addition of them; which subsequently reduce the anti-proliferation effect of guanine-based biomolecules or even totally restore the cell proliferation. These results provide important information for the development of guanine-based drugs or guanine-rich oligonucleotide drugs, as well as for the safety evaluation of food with high level of guanine-based compounds.


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Introduction Guanine, guanine-based nucleosides and nucleotides are essential biomolecules (Scheme S1) in living organisms.1 They are building blocks of nucleic acid biosynthesis and also the products of nucleic acid catabolism; some of them, such as GTP and cGMP, participate in the energy metabolism and signal transduction. However, several early studies reported that deoxyguanosine (dGuo) and deoxyguanosine triphosphate (dGTP) had strong anti-proliferation effect on T-Lymphoid and B-Lymphoid Cells.2-7 The anti-proliferation effect of dGuo on T cell lymphoma cell lines was reported to involve in de novo ATP synthesis interference and inhibition of cytidine diphosphate reduction.3, 5-7 However, the real mechanism of the dGuo anti-proliferation effect are still unclear. Actually, little attention is paid to dGuo anti-proliferation effect. Recently, guanine-rich oligonucleotides (GROs) have attracted considerable interest as therapeutic agents.8 Many GROs have been reported to have cancer-selective cytotoxicity.9-11 The most representative GRO, AS1411 has been reported to have cytotoxicity in almost 80 tumor cell lines, and had reached phase II clinical trials for acute myeloid leukemia and renal cell carcinoma,12 The biological effects of AS1411 include inhibition of NF-kB activation, S-phase cell cycle arrest, derepression of PRMT5 target genes, reduction of bcl-2 expression, interference with the functions of nucleolin and other unknown proteins.13-16 However, our previous study demonstrated that the selective cytotoxicity of GROs was mainly contributed by the anti-proliferation effect of their guanine-based degradation products, such as



monophosphate deoxyguanosine (dGMP), dGuo and guanine. Guanine and guanine-based nucleotides/nucleosides rather than other nucleobase derivatives showed selective anti-proliferation effect on several kinds of cancer cells besides a T-lymphoblastic leukemia cell line. The IC50 values of guanine-based biomolecules to Jurkat E6-1 cells were 14~18 M; but at concentration lower than 10 M, these compounds did not show any anti-proliferation effect.17 In the phase II clinical studies, AS1411 was administered at a high dosage (40 mg/kg/day) by continuous intravenous infusion for 4 or 7 days.12 AS1411 was found to be rapidly degraded in 10% serum in 6 h, and continuously generate dGMP, dGuo and guanine in 96 h.17 The IC50 values of AS1411 for different cell lines are in the range of 1-10 M, corresponding to 17-170 M dGuo because each AS1411 (26-mer) contains 17 dGuo units, which is much higher than the normal concentrations of guanine, guanosine (Guo), dGuo, dGMP, dGDP, dGTP and GMP in blood.18-21 Above evidences suggest that the anti-proliferation effect of guanine-based biomolecules should not be ignored when discussing the action mechanism and side effects of GRO therapeutic agents. Nucleic acid drugs, such as antisense oligonucleotides, small interfering RNA, steric-blocking oligonucleotides, aptamers, immunostimulatory CpG and molecular decoys, have shown considerable therapeutic promise and attracted continuous scientific attention in the past three decades.22-27 These nucleic acid drugs can be eventually degraded in body to generate the guanine-based compounds. In addition, some foods contain high amount of nucleic acids, nucleotides, nucleosides or nucleobases. Nucleic acid or nucleotide healthy products are consumed all over the


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world. Nucleic acids in food are digested in small intestine to form nucleotides, nucleosides and nucleobases; most of these compounds are absorbed in gut.28 Based on the anti-proliferation of guanine-based biomolecules to certain cells, it is necessary to reconsider the safety of taking high amounts of nucleic acids from drugs or food. However, it is unclear that what kind of cells is sensitive to guanine-based biomolecules and what is the mechanism of the anti-proliferation effect. Therefore, revealing the mechanism of selective anti-proliferation effect of guanine-based biomolecules will offer useful information for the development of nucleic acid drugs and for the safety evaluation on the consumption of food and healthy products containing high level of nucleic acids, nucleotides, nucleosides or nucleobases. In this paper, we further confirmed the selective anti-proliferation effect of guanine-based biomolecules, and compared the overall expression level of proteins in Jurkat E6-1 cells treated with dGuo and deoxyadenosine (dAdo) respectively using quantitative proteomic analysis. By gene transfection and knock out experiments, we confirmed that guanine deaminase (GDA) deficiency was a major factor in determining the cell-type selectivity to dGuo anti-proliferation effect. DNA synthesis assay and cell cycle assay showed that excess dGuo inhibited DNA synthesis, and mixture of dAdo and deoxycytidine (dCyd) could relieve the DNA synthesis inhibition and restore DNA synthesis back to normal. The mechanism of the selective anti-proliferation effect of guanine-based biomolecules was discussed.

Results and Discussion



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The selective anti-proliferation of guanine-based biomolecules

Figure 1. The cell viability of Jurkat E6-1, HEK293, HeLa and A549 cells under 48 h treatment with guanine, Guo, dGuo, GMP, dGMP. Each data represents the mean and S.D. of results from three independent drug-treatment experiments. In previous study, we have reported that guanine, Guo, dGuo and dGTP have strong anti-proliferative effect on Jurkat E6-1 cells and eventually resulting in the cell apoptosis/death, and other nucleobase-related biomolecules did not exhibit significant anti-proliferative effect. dGuo also has found to have anti-proliferative effect on Du145, A549T, PC-3 and K562 cells but no effect on A549 and MCF-7 cells

17.

Garozzo et al. also reported the anti-proliferative effect of guanine, Guo and guanosine monophosphate (GMP) on some human cancer cell lines, but no effect on A549 cells29. Here we further confirmed that the guanine-based biomolecules (guanine, Guo, dGuo, GMP, dGMP) showed strong anti-proliferative effects on Jurkat E6-1 cells (IC50 < 25 μM) and HEK293 cells (IC50 = 40~90 μM), but no effect on HeLa and A549 cells (Figure 1). Other types of nucleoside-based biomolecules (dAdo, dCyd, adenosine (Ado), Cytidine (Cyd) did not show anti-proliferative effect on


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Jurkat E6-1 cells even at the concentration of 200 μM (Figure S1), except that thymidine (dThd) showed weak anti-proliferative effect at the concentrations of 100 and 200 μM. SILAC-based quantitative proteomic analysis of dGuo treated cells In order to reveal the anti-proliferative mechanism of guanine-based biomolecules, we compared the proteomic difference of Jurkat E6-1 cells after treated with dGuo and dAdo for 24 h using SILAC (Stable isotope labeling with amino acids in cell culture)-based quantitative proteomic analysis (Figure S2A).30-34 Jurkat E6-1 cells treated with dAdo was used as a reference, because dAdo did not show anti-proliferative effect on Jurkat E6-1 cells. Through LC-MS/MS analysis, we identified a total of 4068 proteins (Table S1) from three sets of SILAC samples. Among these identified proteins, 428 proteins were up- or down-regulated in dGuo treated cells based on the SILAC ratio (heavy/light) threshold of > 1.2 or < 0.73;32, 35 and 165 proteins were significantly up- or down-regulated based on a stringent threshold of > 1.5 or < 0.67 as (Figure S2B), in which 105 proteins (Table S2) were detected in at least two sets of SILAC samples. Cellular pathway alteration by dGuo Treatment In order to explore the cellular pathways that are altered by dGuo treatment, we subjected the 105 proteins to Ingenuity Pathway Analysis (IPA) using bioinformatics signal pathway analysis software.36-37 The IPA figured out 111 pathways, in which three canonical pathways exhibited the smallest p value (-log p = 4.17, 3.96, 3.7), including Base Excision Repair (BER) pathway, DNA Double-Strand Break Repair



by Non Homologous End Joining (DBRNHEJ) pathway and RAN Signaling pathway (Table S3). In RAN Signaling pathway, the expression ratio (dGuo/dAdo treatment) of RNA transportation related proteins, RanGTPase activating protein 1 (RANGAP1), E3 SUMO-protein ligase RanBP2 (RANBP2), Importin subunit alpha-1 (KPNA2) were significantly decreased to 0.59 ± 0.05, 0.66 ± 0.03 and 0.59 ± 0.04 respectively (Table S5). The down-regulation of RANGAP1 in Jurkat cells was confirmed by western blot assay (Figure S4A). According to previous report, RNAGAP1 and RANBP2 down-regulation would result in inhibition of RNA transportation which induces cell apoptosis.38-39 However, our confocal experiments showed no significant RNA transportation inhibition (Figure S5), which suggests that the alteration of RAN Signaling pathway may not be the major reason of dGuo induced cell apoptosis and death. In the BER and DBRNHEJ pathways (Figure S3), dGuo treatment significantly up-regulated the expression of poly ADP-ribose polymerase 1 (PARP1) (dGuo/dAdo treatment, 2.68 ± 0.55), DNA repair protein (XRCC1) (1.55 ± 0.31) and Double-strand break repair protein MRE11A (MRE11A) (1.62 ± 0.3) (Table S4). The significantly high expression of PARP1 was further confirmed by Western blot analysis (Figure S4A). Since both pathways are related to DNA damage repair,40-41 the high expression of these proteins suggests that dGuo treatment induces DNA damage response. Besides the alteration of the above three cellular pathways, we also noticed that


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dGuo treatment altered the expression of some proteins related to nucleotides metabolism (Table S4). For example, GMP reductase 2 (GMPR2) was down-regulated (dGuo/dAdo treatment, 0.68 ± 0.05), which could catalyze the irreversible NADPH-dependent deamination of GMP to IMP, and transfer the guanine derivatives to adenine derivatives; could maintain the intracellular balance of guanine and adenine nucleotides,42 and reduce intracellular GTP pools partially.43 Uridine-Cytidine kinase 2 (UCK2) was also down-regulated (0.64 ± 0.01). UCK2 could catalyze the phosphorylation of uridine and cytidine to uridine monophosphate (UMP) and cytidine monophosphate (CMP) respectively, which is the first step in the production of the pyrimidine nucleoside triphosphates required for RNA and DNA synthesis.44 The Down regulation of GMPR2 and UCK2 was verified by western blot analysis (Figure S4A). The alteration of these proteins suggests that the dGuo treatment may disturb the nucleotides metabolism Addition of dAdo and dCyd antagonized the anti-proliferation effect of dGuo Above results showed that dGuo treatment induced DNA damage response and altered the expression of GMPR (transformation of GMP to AMP), UCK2 (phosphorylation of uridine and cytidine). Previous studies have reported that dGuo treatment resulted in GTP and dGTP accumulation and dATP depletion in Jurkat cells.45 These implies that dGuo treatment might cause the depletion of other kinds of nucleosides/nucleotides and subsequently results in the anti-proliferation effect. Therefore, we performed a set of experiments to investigate whether the replenishment of other nucleosides can reduce the anti-proliferation effect of dGuo



(Figure 2).

Figure 2. (A) The viability of Jurkat E6-1 cells treated with 50 M dGuo and different concentrations of Ado, dAdo, Cyd, dCyd or dThd for 48 h. (B) The viability of Jurkat E6-1 cells treated with 50 M dGuo and different concentrations of dAdo + dCyd, dAdo + Cyd, dAdo + dThd, dAdo + dCyd + dThd, dAdo + Cyd or Ado + dCyd for 48 h. (C) The viability of Jurkat E6-1 cells treated with different concentrations of mixture of dGuo + dAdo + dCyd + dThd (same concentration of each). Each data represents the mean and S.D. of results from three independent drug-treatment experiments. (D) The viability of Jurkat E6-1 cells treated with 50 M dGuo + 50 M dCyd and different concentrations of dAdo (blue); and the viability of Jurkat E6-1 cells treated with 50 M dGuo + 50 M dAdo and different concentrations of dCyd (red). As shown in Figure 2A, supplementation with different concentrations (0-500 μM) of Ado, dAdo, Cyd, dCyd or dThd, could not reduce the anti-proliferation effect of 50 μM dGuo on Jurkat E6-1 cells at all. This result did not agree with the results reported by Garozzo et al.29, i.e. the single addition of adenine (or Ado) antagonized the anti-proliferation effect of guanine and Guo in U87 glioma cell culture, which may be due to cell-type differences. Surprisingly, supplementation with dAdo and dCyd together gradually reduced the anti-proliferation effect of 50 μM dGuo on Jurkat E6-1 cells, and recovered the cell viability to ~80% with the increase of dAdo and dCyd to


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100 μM (Figure 2B). Similar results were also obtained by supplementation with different concentrations of Ado + Cyd or dAdo + dCyd + dThd. Moreover, the supplementation with dCyd + dAdo was also found to restore the up-regulated protein (PARP1) and down-regulated protein (RanGAP1) by dGuo back to normal, which further confirmed the antagonistic effect on the anti-proliferation of dGuo (Figure S4A). Additionally, the addition of dAdo + dCyd was also found to reduce the anti-proliferation effect of guanine-rich oligonucleotide, AS1411 (Figure S9). However, supplementation with different concentrations of dAdo + dThd could not reduce the anti-proliferation effect of 50 μM dGuo at all (Figure 2B) suggesting that Ado/dAdo and Cyd/dCyd are indispensable for antagonizing the anti-proliferation effect of dGuo. It is well known that cytosine-based nucleosides/nucleotides can transform to thymine/uracil-based nucleoside/nucleotides in cells, thus, the supplementation with dAdo + dCyd could also make up the insufficient dThd. Interestingly, when treated Jurkat E6-1 cells with 50 μM dGuo supplemented with different concentrations of dAdo + Cyd or Ado + dCyd, we obtained two unexpected results. Supplementation with dAdo + Cyd just slightly reduced the anti-proliferation effect of dGuo; in contrary, supplementation with Ado + dCyd not only totally recovered the cell viability, but also slight promoted cell growth in the presence of 50 μM dGuo (Figure 2B). This might due to the complex allosteric effect of ribonucleotide related enzymes, for example, ribonucleotide reductase that catalyzes the reduction of ribonucleotides to deoxyribonucleotides. Ribonucleotide reductase has two allosteric sites: activity site and specificity site, both on the RRM encoded



subunit. Activity site binds either ATP (activating) or dATP (inhibitory).46-47 A high ratio of ATP/dATP favors a higher reductive flux and DNA synthesis, whereas a low ratio signals low demand for dNDP. The specific site binding of (d)ATP induces reduction of CDP or UDP, dGTP induces reduction of ADP, and dTTP induces reduction of GDP.46-48 In this case, the low antagonistic effect of dAdo + Cyd on the anti-proliferation of dGuo might due to the inhibition by dATP (came from the added dAdo) on the ribonucleotide reductase activity, which led to the insufficient of dCyd-based nucleiotides. In contrast, the higher antagonistic effect of Ado + dCyd might because of the elevated ATP (came from Ado) concentration, which could serve as the energy material and ribonucleotide reductase activator. Above results also showed that supplemented with 50 μM mixture of dCyd + dAdo or other mixtures only partly reduced the anti-proliferation effect of 50 μM dGuo, the higher concentration of supplemented mixtures (such as 100 μM) caused a better effect. In addition, treating Jurkat E6-1 cells with increased concentrations of the mixture of dGuo + dAdo + dCyd + dThd (10-200 μM, same concentration of each) gradually reduced the cell viability to 50% (Figure 2C). Since the total concentration of adenine-based nucleotides in cells is much higher than other kinds of nucleotides,49-51 more adenine-based molecules might be required to completely antagonize the anti-proliferation effect of dGuo. Therefore, we further treated Jurkat E6-1 cells with 50 μM dGuo + 50 μM dCyd + different concentration of dAdo, and 50 μM dGuo + 50 μM dAdo + different concentration of dCyd respectively. As shown in Figure 2D, the anti-proliferation effect of 50 μM dGuo was gradually reduced by


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supplementation with 50 μM dCyd + increased concentration of dAdo, and a totally recovery of cell proliferation was observed at 200 μM dAdo. But supplementation with 50 μM dAdo + increased concentration of dCyd only partly recovered the cell proliferation until ~70%, further increasing dCyd above 50 μM could not further reduced the anti-proliferation effect of 50 μM dGuo. This set of results suggests that a higher concentration of adenine-based molecules is needed to maintain the ratio of adenine-, cytosine- and guanine-based molecules in Jurkat E6-1 cells, and also imply a competitive relationship between dGuo and other deoxyribonucleotides. All above results demonstrate that the dGuo treatment resulted in the unbalance of nucleosides and nucleotides in Jurkat E6-1 cells, which may cause the anti-proliferation effect and subsequent cell apoptosis and death. Guanine Deaminase related to the selective anti-proliferation effect of dGuo As an endogenous small molecule in human cells, dGuo has two metabolic directions (Figure S10): converts to dGMP by deoxyguanosine kinase (DGUOK) or deoxycytidine kinase (DCK), and converts to guanine by purine nucleoside phosphorylase (PNP). Guanine is the final product of catabolism of all guanine-based biomolecules and also the starting material of their anabolism. Our previous study17 and the anti-proliferation assay herein showed that guanine, guanine-based biomolecules have the similar anti-proliferation effect on the tested cell lines, which suggest that the guanine metabolism might be related to the selective anti-proliferation effect of the guanine-based biomolecules. Guanine also has two metabolic directions (Figure S10): reversibly converted to



guanine-based biomolecules, such as dGuo, dGMP, dGDP, dGTP, Guo, GTP, GDP and GMP by a serious of enzymes, and degraded by guanine deaminase (GDA) irreversibly. Garozzo et al. reported that anti-proliferation effects of guanine-based purines require hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity.29 HPRT belongs to the salvage pathway of GMP synthesis, can convert guanine to GMP. Therefore, we examined the HPRT expression within dGuo sensitive and insensitive cell lines using western blot analysis. However, the expression of HPRT within all the tested cell lines did not show significant difference (Figure S4B). Thus, the pathway of guanine degradation took our notice. GDA catalyzes the deamination of guanine to xanthine, eliminating the guanine base from reutilization and exhibit variable expression among different tissues.52 Re-examining our quantitative proteomic analysis data, we did not found this protein in the found 4068 proteins. Hence, we investigated the GDA expression in dGuo sensitive cells (Jurkat E6-1, PC-3, HEK293) and insensitive cells (A549, HeLa, MCF-7) by western blot analysis. As we expected, GDA was not detected in all dGuo sensitive cells, but highly expressed in all dGuo insensitive cell lines (Figure 3A). These results suggest that the GDA deficiency might be the cause of the selective anti-proliferation effect of dGuo. To further confirm this results, we performed the GDA gene transfection and knock out experiments. Due to Jurkat E6-1 cells were hard to be transfected, we used HEK293 and HeLa cells to perform these experiments. After transfecting pCMV-Myc-GDA plasmid into dGuo-sensitive cell line, HEK293 (Figure S4C), the anti-proliferation effect of dGuo was significantly reduced (Figure 3D). After


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knocking out GDA from HeLa cells (dGuo insensitive cell) using CRISPR/Cas9 gene editing technology (Figure 3B and Figure S4D), the GDA knocked out HeLa cells became more sensitive to dGuo than original cells (Figure 3C). Further transfecting pCMV-Myc-GDA plasmid to the GDA knocked out HeLa cells, the cells recovered resistance to dGuo anti-proliferation (Figure 3D). These results confirm that GDA deficiency is the main reason of selective anti-proliferation effect of dGuo.

Figure 3. (A) Western blot analysis of GDA expression in dGuo-insensitive cell lines (A549, MCF7, HeLa) and dGuo-sensitive cell lines (PC3, HEK293, Jurkat E6-1). (B) Western blot analysis of GDA knockout in HeLa cells. (C) Cell viability of GDA+ or GDA- HeLa cells treated with 50 M dGuo or 50 M dGuo + 100 M different nucleoside mixtures respectively. (D) Cell viability of pCMV-Myc or pCMV-Myc-GDA overexpressed HEK293 cells and GDA- HeLa cells after dGuo treatment. HeLa GDA- means GDA knocked out HeLa cells. Each data represents the mean and S.D. of results from three independent drug-treatment experiments. dGuo inhibits the DNA Synthesis of GDA Deficient Cells Above results have shown that dGuo treatment might induce DNA damage response and cause the unbalance of nucleosides and nucleotides in GDA deficient cells, which implies that dGuo treatment might inhibit the DNA/RNA synthesis of



GDA deficient cells. Therefore, we further investigated the DNA/RNA synthesis by incorporation of 5-ethynyl-2’-deoxyuridine/5-ethynyluridine (EdU/EU) and its subsequently fluorescent detection of Alex488 linked through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction (‘‘click’’ chemistry).53-54 For the DNA synthesis analysis, all the cells without treatment with dGuo showed two peaks, the peak with lower fluorescence intensity represented background fluorescence, and the peak with higher fluorescence intensity represented the newly synthesized DNA with EdU incorporation (Figure. 4A). After treating with 50 μM dGuo for 24 h, the GDA positive cells (A549 and HeLa) showed two peaks similar with untreated cells; however, the GDA deficient cells (Jurkat E6-1, GDA knocked out HeLa or HEK 293) only showed a main peak between normal EdU incorporation peak and background fluorescence peak, which suggests that the DNA synthesis of GDA deficient cells was stopped when exhaust of the existed some deoxynucleosides/nucleotides after treated by dGuo (Figure 4A). When treated cells with the mixture of 50 μM dGuo +100 μM dAdo + 100 μM dCyd for 24 h, the DNA synthesis of GDA deficient cells was almost not affected (Figure 4A), which suggests that the inhibition of DNA synthesis was caused by the depletion of dAdo, dCyd, and their nucleotides. For the GDA positive cells, the deoxynucleoside mixture treatment did not affect the DNA synthesis of HeLa cells, but promoted the DNA synthesis of A549 cells (Figure 4A), which was confirmed by enhanced proliferation of A549 cells after treated with the deoxynucleoside mixture (Figure S6). The results collected by flow cytometry were further confirmed by confocal imaging (Figure 4B). Compared to the DNA synthesis,


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the RNA synthesis experiment showed that dGuo treatment did not cause significant fluorescence change in both GDA deficient cells and GDA positive cells (Figure S7 and S8), thus the RNA synthesis was not further investigated in this paper. The DNA synthesis inhibition was further confirmed by cell cycle assay of GDA knockout HeLa cells. The dGuo treatment for 24 h caused the cell cycle to arrest at S phase, and no cells were observed in G2/M phase (Figure 4C), which corresponded well to the main peak with medial fluorescence intensity in above EdU experiment (Figure 4A). The treatment with mixture of dGuo + dAdo + dCyd did not affect the cell cycle.

Figure 4. (A) Flow cytometry analysis of EdU incorporation (DNA synthesis) in A549, HeLa, HEK293, Jurkat E6-1 and GDA knockout HeLa cells that pre-treated with control, 50 μM dGuo or deoxynucleoside mixture for 24 h. (B) Confocal imaging of EdU incorporation in HeLa and GDA knockout HeLa cells pre-treated with control, 50 μM dGuo or deoxynucleoside mixture for 24 h. Nucleus stained with Hoechst 33342 as control; the scale bar = 30 µm. (C) Cell cycle analysis of GDA knockout HeLa cells pre-treated with control, 50 μM dGuo or deoxynucleoside mixture for 24 h. Deoxynucleoside mixture is composed of 50 μM dGuo + 100 μM dCyd + 100 μM dAdo. To figure out whether dCyd + dAdo could restart the inhibited DNA synthesis by dGuo, we firstly treated GDA knockout HeLa cells with dGuo for 24 h, then further



incubated with 100 μM dCyd + 100 μM dAdo and performed the cell cycle assay at different incubation times. As shown in Figure 5, the dGuo treatment caused 76.6% cell to accumulate in S phase, and no cells were observed in G2/M phase; after further incubation with dCyd + dAdo for 6 h, most of cells moved to S and G2/M phases and almost no cells were detected in G1 phase, indicating that the inhibited DNA synthesis by dGuo was restarted. When further incubation for 12-24 h, G1 phase cells reappeared and then dominated the cell population, and no G2/M phase cells were observed at 24 hour, suggesting that cells gradually completed division, and was synchronized in G1 phase. When incubation for 24-48 h, cells restarted another cell cycle and the population in each phase restored back to normal (Figure 5A). The EdU incorporation experiment further confirmed that dCyd + dAdo could restart the inhibited DNA synthesis by dGuo within 6 hour (Figure 5B). All these results also suggest that dGuo could serve as a cell synchronization reagent for arresting cells in S phase, and dCyd-dAdo mixture could serve as an antidote to restart the cell growth cycle.

Figure 5. (A) Cell cycle assay of dGuo (50 μM) pre-treated (24 h) GDA knockout HeLa cells after incubation with 100 μM dCyd + 100 μM dAdo for different times. (B) EdU incorporation assay of dGuo (50 μM) pre-treated (24 h) GDA knockout HeLa cells after incubation with 100 μM dCyd + 100 μM dAdo for different times. Normal cells represent GDA knockout HeLa cells without any treatment.


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Discussion As endogenous biomolecules, guanine, guanine-based nucleosides and nucleotides should be maintained at a correct level in body through the subtle regulation and coordination of biosynthesis, catabolism, transportation and uptake from food. GDA catalyzes the hydrolytic deamination of guanine to xanthine, which is an essential part of guanine degradation pathway. The selective anti-proliferation effect of guanine-based biomolecules on GDA deficient cells suggests that these cells could not efficiently remove excess guanine-based molecules through the GDA pathway when they were exposed to a high concentration of these biomolecules, resulting in the accumulation of them in cells. GDA lowly expresses in human bone marrow, and immune

system

(http://www.proteinatlas.org/ENSG00000119125-GDA/tissue),

which just explain the strong anti-proliferation effect of guanine-based biomolecules to T-Lymphoid and B-Lymphoid cells as reported by early studies.2-7 Besides, GDA is associated with the development of nervous systems,55 and highly expresses in human brain, liver, pancreas, gastrointestinal tract and kidney. The high expression in digestion and detoxification tissues suggests an important role of GDA in the regulation of guanine nucleotide pool.52 Our results have shown that dGuo inhibited DNA synthesis of the GDA deficient cells and compelled cell cycle to arrest at S phase, and simultaneous addition of dAdo and dCyd together with dGuo could antagonize the DNA synthesis inhibition caused by dGuo. Moreover, addition of dAdo and dCyd after dGuo treatment could restart the DNA synthesis inhibited by dGuo. These results suggest that the accumulation of



guanine-based molecules in GDA deficient cells led the deficiency of other kinds of deoxynucleotides and resulted in the stop of DNA synthesis. This well explains our previous results of that the addition of dGuo mainly inhibited the cell growth rather than directly killed the cells in a short period.17 With increasing the time of dGuo treatment (> 48 h), the population of apoptotic and dead cells increased,17 which might due to the long-time inhibition of DNA synthesis. Our SILAC-based proteomic analysis showed that dGuo treatment induced DNA damage response, which may be caused by the incomplete DNA synthesis or even the wrong synthesis resulted from deficiency of adenine- and cytosine-based nucleotides. The SILAC-based proteomic analysis also showed that dGuo treatment induced the up- or down-regulation of some proteins involved in nucleotide metabolism, such as GMPR2 that is related to transformation of guanine-based molecules to adenine-based molecules;42 and UCK2 that catalyzes the phosphorylation of uridine and cytidine to UMP and CMP.44 dGuo treatment has been reported to cause GTP and dGTP accumulation and dATP depletion in Jurkat cells.45 It is common sense that GTP/dGTP accumulation would inhibit the de novo synthesis of purine nucleosides. It is also well known that guanine-based nucleoside and nucleotides can transform to adenine-based nucleosides and nucleotides through an intermediate, inosine monophosphate (IMP); and cytosine-based nucleosides and nucleotides can transform to thymine/uracil-based nucleoside and nucleotides (Figure S10). All the transformation and synthesis of nucleosides/nucleotides are achieved by complex series of enzymatic reactions. These evidences could explain why dGuo treatment


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caused the deficiency of other kinds of deoxynucleotides; i.e. the excess guanine-based biomolecules in cells occupied some metabolic enzymes of nucleosides/nucleotides in a competitive manner and disturbed the production and transformation of other deoxynucleotides, which resulted in the exhaustion of other kinds of deoxynucleotides during the DNA synthesis. Thus, the simultaneous- or post-addition of other nucleosides/nucleotides could balance the level of four kinds of nucleosides/nucleotides, and recover the production of deoxynucleotides and DNA synthesis. In summary, the mechanism of the selective anti-proliferation effect of guanine-based biomolecules could be proposed as: the excess guanine-based biomolecules cannot be removed timely in GDA deficient cells; the accumulation of them disturbs the formation and transformation of other deoxynucleotides in cells, led the exhaustion of dATP, dCTP and dTTP, and subsequently inhibited the DNA synthesis, induced the DNA damage like response, and ultimately cause the apoptosis and death of cells. Because of the high complexity of the cellular pathways, currently we cannot figure out the detailed mechanism of the anti-proliferation effect of dGuo in GDA deficient cells. Actually, all the changes in the molecular level (e.g. protein expression, DNA synthesis), as well as the apoptosis/death of cells are the effects of dGuo treatment, and the GDA deficiency is the major determinant of the selective anti-proliferation property of guanine-based biomolecules. This finding implies that an excessive intake of guanine-based compounds or guanine-rich nucleic acid drugs might lead the side effects on GDA deficient tissues, but the side effect may be



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reduced or offset by balanced intake of other nucleosides and nucleotides. Therefore, overall and systematic understanding of the GDA expression in different cells and tissues will helpful in the safety evaluation of health products and foods containing large amount of guanine-based biomolecules.

Methods Cell culture Jurkat E6-1 (Acute T lymphoblastic leukemia) was purchased from Cell Culture Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). A549 (non-small cell lung cancer), HEK293 (embryonic kidney) and HeLa (cervical cancer) cell lines were purchased from Cell Resource Center of Shanghai Institute for Biological Sciences (Chinese Academy of Sciences, Shanghai, China). Jurkat E6-1 and A549 cells were cultured in RPMI 1640 medium (Gibco) supplemented

with

10%

fetal

bovine

serum

(FBS,

Gibco),

and

1%

penicillin/streptomycin (Corning). HEK293 and HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) with the same supplements. All cell lines were routinely maintained at 37 oC in humidified atmosphere with 5% CO2. For SILAC experiments, light or heavy lysine ([13C6,15N2]-L-lysine) and arginine ([13C6]-L-arginine) (Silantes GmbH, Andover, Germany), along with dialyzed FBS (PAN-Biotech Gmbh, Germany) were added to lysine/arginine-depleted RPMI 1640 medium for preparing complete light and heavy RPMI 1640 media. For completely stable isotope incorporating, the Jurkat E6-1 cells were cultured in heavy or light


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RPMI 1640 medium for at least 1 week. SILAC-based proteomic analysis Sample preparation for proteomic analysis: Jurkat E6-1 cells, at a density of 1105 cells/mL in light or heavy RPMI 1640 medium, were treated with 100 μM dGuo or dAdo (Sangon, China) for 24 h. After treatment, the cells were harvested via centrifugation at 250 g at 4°C for 3 min and washed three times with ice-cold phosphate buffered saline (PBS) to remove culture medium and FBS. Subsequently, cells were lysed with CelLyticTM M lysis buffer (Sigma) supplemented with 1 mM PMSF (Sigma) and Cocktail (Sigma). The resulted cell lysates were centrifuged at 15,000 g at 4°C for 10 min. The supernatants were collected and the protein concentration in the supernatants was measured with a Quick Start Bradford Protein Assay Kit (Sangon, China). Three biological replicates of SILAC labeling experiments were performed to ensure the minimization of the systematic errors and the credibility of the dGuo-induced alterations in protein expression. In two forward SILAC experiments, the heavy-labeled dGuo-treated lysate and the light-labeled dAdo-treated lysate were combined equally; in one reverse SILAC experiment, the light-labeled dGuo-treated cell lysate was mixed with the Heavy-labeled dAdo-treated lysate at 1:1 ratio (w/w). Proteomic analysis: 10% SDS-PAGE with a 5% stacking gel was used to separate above equi-mass mixture of light and heavy lysates, and Coomassie Blue was used to stain the gel. Subsequently, the gel was cut into 10 bands, and the proteins in each gel slices were digested overnight at 37°C with trypsin (Promega, Madison, WI) after



in-gel reduction with dithiothreitol (J&K Scientific Ltd, China) and alkylation with iodoacetamide (Sigma). Following digestion, peptides were extracted from gels with 5% acetic acid in H2O and 5% acetic acid in CH3CN/H2O (1:1, v/v). The resulted peptide mixtures were dried and analyzed by LC-MS/MS for protein identification and quantification. Date processing and Ingenuity Pathway Analysis: For peptide identification, the raw MS data were processed with MaxQuant search engine (1.5.5.1) against the human Uniprot database. Common contaminants were added to this database. The maximum number of missed cleavages for trypsin was two per peptide. Cysteine carbamidomethylation was set as fixed modifications, and N-terminal acetylation and methionine oxidation were considered as variable modifications. The tolerances in mass accuracy for MS and MS/MS were 20 ppm and 0.5 Da, respectively. The required false positive discovery rate was set to within 1% at both peptide and protein levels, with the minimal required peptide length being seven amino acids. The quantification was based on three independent SILAC and LC-MS/MS experiments as noted above, which included two forward and one reverse SILAC labeling. According to previous reference,32 we considered a protein as significantly altered only if its differential expression ratio (dGuo treatment/dAdo treatment ) was greater than 1.2 or less than 0.73. The proteins that exhibited stringently expression ratio (>1.5 and

The Mechanism of the Selective Antiproliferation Effect of Guanine

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