Regulation of apoptosis by cyclic nucleotides in human erythroleukemia (HEL) cells and human myelogenous leukemia (K-562) cells

Biochemical Pharmacology 112 (2016) 13–23

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Regulation of apoptosis by cyclic nucleotides in human erythroleukemia (HEL) cells and human myelogenous leukemia (K-562) cells Fanni Dittmar, Sabine Wolter, Roland Seifert ⇑ Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany

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Article history: Received 9 March 2016 Accepted 29 April 2016 Available online 6 May 2016 Chemical compounds studied in this article: cAMP (PubChem CID: 6076) cAMP-AM (PubChem CID: 71312192) cCMP (PubChem CID: 19236) cGMP (PubChem CID: 24316) cUMP (PubChem CID: 3081385) brefeldin A (PubChem CID: 5287620) probenecid (PubChem CID: 4911) Z-VAD-FMK (PubChem CID: 5497174) Keywords: Apoptosis Cyclic CMP Mitochondrial membrane potential

a b s t r a c t The cyclic pyrimidine nucleotides cCMP and cUMP have been recently identified in numerous mammalian cell lines, in primary cells and in intact organs, but very little is still known about their biological function. A recent study of our group revealed that the membrane-permeable cCMP analog cCMPacetoxymethylester (cCMP-AM) induces apoptosis in mouse lymphoma cells independent of protein kinase A via an intrinsic and mitochondria-dependent pathway. In our present study, we examined the effects of various cNMP-AMs in human tumor cell lines. In HEL cells, a human erythroleukemia cell line, cCMP-AM effectively reduced the number of viable cells, effectively induced apoptosis by altering the mitochondrial membrane potential and thereby caused changes in the cell cycle. cCMP itself was biologically inactive, indicating that membrane penetration is required to trigger intracellular effects. cCMP-AM did not induce apoptosis in K-562 cells, a human chronic myelogenous leukemia cell line, due to rapid export via multidrug resistance-associated proteins. The biological effects of cCMP-AM differed from those of other cNMP-AMs. In conclusion, cCMP effectively induces apoptosis in HEL cells, cCMP export prevents apoptosis of K-562 cells and cNMPs differentially regulate various aspects of apoptosis, cell growth and mitochondrial function. In a broader perspective, our data support the concept of distinct second messenger roles of cAMP, cGMP, cCMP and cUMP. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction Although research on the non-canonical pyrimidine nucleotide cytidine 30 ,50 -cyclic monophosphate (cCMP) started many years ago, little is known about its biological function. In the beginning, there were several failures due to insufficient specificity and sensitivity of the available detection methods [1–3]. Meanwhile, cCMP was detected in several cell lines, primary cells and in intact organs using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) [4–7]. cCMP is produced by soluble adenylyl cyclase (sAC) [8] and soluble guanylyl cyclase (sGC) [9], degraded by phosphodiesterase (PDE) 7A [10] and exported via multidrug resistance-associated

protein (MRP) 5 [11]. Protein kinase G (PKG)-dependent vasodilation as well as PKG-dependent inhibition of platelet aggregation was observed using the dibutyrylated analog of cCMP (DB-cCMP) [12]. The current knowledge on cCMP and another cyclic pyrimidine nucleotide, cUMP, as signaling molecules has been recently reviewed [13,14]. Moreover, dynamic mass restribution (DMR) measurements revealed that the acetoxymethylester analog of cCMP (cCMP-AM) induced cellular responses independent of protein kinase A (PKA) and PKG in HEK293 and B103 cells and increased c-Fos expression in HEK293 cells [15]. These data show that the AM-analog is an appropriate tool to examine the cellular functions of cCMP. After membrane penetration, the AM-group is cleaved by esterases inside the cell and cCMP is released [13,15].

Abbreviations: AM, acetoxymethylester; APC, allophycocyanin; cAMP, adenosine 30 ,50 -cyclic monophosphate; BFA, brefeldin A; cCMP, cytidine 30 ,50 -cyclic monophosphate; Cq, quantification cycle; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; cGMP, guanosine 30 ,50 -cyclic monophosphate; HEL, human erythroleukemia cell line; JC-1, 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide; K-562, human chronic myelogenous leukemia cell line; MRP, multidrug resistanceassociated protein; cNMP, nucleoside 30 ,50 -cyclic monophosphate; PDE, phosphodiesterase; PE, phycoerythrin; PI, propidium iodide; PKA, protein kinase A; PKG, protein kinase G; PO4-AM3, phosphate tris(acetoxymethyl)ester; PROB, probenecid; cUMP, uridine 30 ,50 -cyclic monophosphate. ⇑ Corresponding author. E-mail addresses: [email protected] (F. Dittmar), [email protected] (S. Wolter), [email protected] (R. Seifert). http://dx.doi.org/10.1016/j.bcp.2016.04.018 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.

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PO4-AM3 is used as control reagent to test for side effects of the delivery mechanism, such as the formation of acetic acid and formaldehyde [16]. PO4-AM3 contains three AM groups and is therefore applied at a threefold lower concentration than cNMPAMs in experiments [15]. Using these tools, our group has recently shown that cCMP-AM induces apoptosis in S49 mouse lymphoma cells independent of PKA via an intrinsic, mitochondriadependent pathway whereas cAMP-AM, cGMP-AM and cUMPAM do not induce apoptosis [6]. Based on these findings, in our present study, we addressed the question whether cCMP induces apoptosis not only in mouse but also in human tumor cell lines. We studied HEL cells [17], which were obtained from a patient with erythroleukemia and K-562 cells [18], which were established from a patient with chronic myelogenous leukemia. Both cell lines exhibit low cCMP concentrations under basal conditions [5]. In our study, we used brefeldin A (BFA) as positive control for apoptosis. BFA is a fungal metabolite which blocks protein transport from the endoplasmic reticulum to the Golgi apparatus and thereby induces apoptosis within 48 h, depending on the cell type [19].

2. Materials and methods 2.1. Reagents The cyclic 30 ,50 -nucleotides cAMP, cCMP, cGMP and cUMP as well as their acetoxymethylester-analogs (cNMP-AMs) and PO4AM3 were purchased from BioLog Life Science Institute (Bremen, Germany). Stock solutions (100 mM cNMP-AMs, 33 mM PO4AM3) were prepared in dimethyl sulfoxide (DMSO) water-free. Annexin-V-APC was obtained from MabTag (Friesoythe, Germany), protease and phosphatase inhibitors from Biotool (Munich, Germany), BD MitoScreen kit from BD Biosciences Pharmingen (San Diego, CA, USA). Brefeldin A (BFA) and probenecid (PROB) were purchased from Sigma-Aldrich (Seelze, Germany). BFA stock solutions were prepared in ethanol (18 mM) and 1 PBS (1 mM), PROB stock solution (100 mM) was prepared in DMSO. RNase A was obtained from Macherey-Nagel (Dueren, Germany) and stock solution (50 mg/ml) was prepared in dest. H2O. Z-VAD-FMK was purchased from R&D Systems (Minneapolis, MN, USA) and stock solution (20 mM) was prepared in DMSO. All other reagents were of analytical grade and from standard suppliers.

2.4. Detection of apoptosis by flow cytometry Cells (1  105 cells/ml) were incubated in a 24-well plate with 1 ml of media and 150 lM cNMPs or cNMP-AMs, 50 lM PO4-AM3 as negative control and 5 lM BFA as positive control for 24 h (HEL) or 48 h (K-562). In a second approach, cells were preincubated for 1 h with the pan-caspase inhibitor Z-VAD-FMK (20 lM) [20]. In a third approach, cells were pre-incubated for 5 min with the MRP inhibitor probenecid (500 lM) [21]. After incubation, cells were centrifuged at 300g for 10 min at room temperature and resuspended in 100 ll binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2 and dest. H2O; pH 7.4). Annexin-V-APC was added and samples were incubated for 20 min in the dark at room temperature. To stop the reaction, another 200 ll binding buffer was added to each sample. Propidium iodide (PI) (1.67 lg/ml) was added immediately before apoptosis was detected using a MACSQuant Analyzer from Miltenyi Biotech (Bergisch Gladbach, Germany). Cells appear according to their size and granularity in a forward scatter/sideward scatter (FSC/SSC) dot plot. In a fluorescence dot plot (APC/PI), different populations were distinguished as follows: the lower left (LL) quadrant (annexin-V-APC/PI) represents the population of viable cells, the lower right (LR) quadrant (annexin-V-APC+/PI) is regarded as the cell population in the early apoptotic state and the upper right (UR) quadrant (annexin-V-APC+/PI+) is considered as the population at late apoptotic state and necrotic cells. Data were analyzed with MACSQuantify software. 2.5. Sample preparation for western blot analysis HEL cells (2  105 cells/ml) were incubated in a 6-well plate with 5 ml of media and 150 lM cNMP-AMs, 50 lM PO4-AM3 as negative control and 5 lM BFA as positive control for 18 h. Cells were transferred to 15 ml falcon tubes, centrifuged at 300g for 10 min at room temperature, resuspended in 1 PBS and transferred to 1.5 ml tubes. After recentrifugation, supernatant fluids were discarded and cell pellets were stored at 20 °C. Pellets were thawed, resuspended in 150 ll 1 PBS with protease and phosphatase inhibitors (1:1000) from Biotool (Munich, Germany) and lysed by sonication. Based on the protein concentrations determined by bicinchoninic acid (BCA) protein assay, samples were mixed with Pierce Lane Marker Reducing Sample Buffer from Thermo Scientific (Schwerte, Germany), heat denatured at 96 °C for 10 min and stored at 20 °C until use.

2.2. Cell culture 2.6. Western blot analysis The human erythroleukemia cell line HEL 92.1.7 was obtained from Prof. Dr. Armin Buschauer (University of Regensburg, Germany), the human chronic myelogenous leukemia cell line K-562 was obtained from American Type Culture Collection (ATCC, Manassas, Virginia, USA). Cells were cultured and maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin, 1% (v/v) MEM non-essential amino acids (100) and 1 mM sodium pyruvate solution (100 mM), both from PAA Laboratories (Pasching, Austria) and incubated at 37 °C in a humidified atmosphere containing 5% (v/v) CO2.

2.3. Determination of the number of viable cells Cells (1  105 cells/ml) were incubated in a 48-well plate with 300 ll of media together with 100 lM cNMP-AMs and 33 lM PO4-AM3 as negative control. After 24, 48 and 72 h, viable cells were distinguished from dead cells using the trypan blue dye exclusion method and counted using a hemocytometer.

Equal protein quantities (50 lg) were separated by SDS-PAGE using gels containing 10% (m/v) acrylamide for detection of PKG and gels containing 12.5% (m/v) acrylamide for detection of caspase 3 and cleaved caspase 3. Proteins were transferred to nitrocellulose membranes and analyzed with specific antibodies: anticaspase-3, #9662 and anti-cleaved caspase-3, #9661 from Cell Signaling Technology (Frankfurt/Main, Germany); and anti-alpha tubulin, sc-8035 from Santa Cruz Biotechnology (Heidelberg, Germany). Secondary antibodies were purchased from Cell Signaling Technology. Proteins were detected using WesternSure Chemiluminescent Substrates and a C-DiGit Blot Scanner from Li-Cor Biotechnology (Bad Homburg, Germany). Images were analyzed with Image Studio Lite software, values were referred to a tubulin expression and normalized to untreated cells. 2.7. Caspase 3 assay Cells (2  105 cells/ml) were incubated in a 6-well plate with 4 ml of media and 150 lM cNMP-AMs, 50 lM PO4-AM3 as negative

F. Dittmar et al. / Biochemical Pharmacology 112 (2016) 13–23

control and 5 lM BFA as positive control for 20 h. The assay was performed using the Caspase-3/CPP32 Fluorometric Assay Kit from Enzo Life Sciences (Loerrach, Germany) according to its protocol with the following modifications: cell suspension was centrifuged at 1200g for 10 min at 4 °C in two 2 ml tubes, cells were transferred to a black 96 well plate before substrate was added and then incubated for 2 h. Fluorescence was measured using a Synergy 4 microplate reader from BioTek (Bad Friedrichshall, Germany). 2.8. Measurement of mitochondrial membrane potential Dw Experiments were performed using the BD MitoScreen kit from BD Biosciences Pharmingen (San Diego, CA, USA). HEL cells at a density of 2  105 cells/ml were incubated in a 6-well plate with 3 ml of media and 150 lM cNMP-AMs, 50 lM PO4-AM3 as negative control and 5 lM BFA as positive control for 24 h. The protocol of the staining procedure was modified as follows: Three ml cell suspension was transferred to 1.5 ml tubes in two steps. After each step, cells were centrifuged at 300g for 10 min at room temperature, resuspended in 0.5 ml JC-1 staining solution and incubated for 12.5 min. Afterward, 1 ml of 1 assay buffer was added to each sample and another centrifugation step (see above) followed. Cells were resuspended in 0.5 ml 1 assay buffer and mitochondrial membrane potential Dw was analyzed by flow cytometry with a MACSQuant Analyzer. In a fluorescence dot plot (PE/FITC), both populations can be distinguished as follows: Cells with a polarized Dw (PE"/FITC") appear diagonally above cells with a depolarized Dw (PE;/FITC"). 2.9. Cell cycle analysis HEL cells (2  105 cells/ml) were treated with 150 lM cNMPAMs and 50 lM PO4-AM3 for 24 h in a 12-well plate with 2.5 ml of media. Cells were transferred to 15 ml falcon tubes, centrifuged at 300g for 10 min at 4 °C and resuspended in 1 ml sample buffer (1 PBS with 1 g/l glucose). 10 ml sample buffer was added, cells were sedimented by centrifugation again (see above) and resuspended in approximately 100 ll supernatant fluid. This washing step was repeated. Afterward, cells were mixed and 1 ml ice-cold 70% (v/v) ethanol was added dropwise. Samples were fixed overnight at 4 °C. Then, 10 ml sample buffer was added to each sample, cells were centrifuged at 500g for 10 min at 4 °C; and resuspended in approximately 100 ll supernatant fluid. One ml staining solution containing 50 lg/ml PI and 100 U/ml RNase A was added. After incubation for 35 min in the dark at room temperature, samples were analyzed by flow cytometry with a MACSQuant Analyzer and MACSQuantify software.

2.10. Gene expression analysis Cells (4  105) were centrifuged at 650g for 10 min at room temperature, pellets were stored at 20 °C. RNA was isolated using the NucleoSpin RNA II kit from Macherey-Nagel (Dueren, Germany) according to the manufacturer’s protocol. Afterward, cDNA synthesis was performed and samples were stored at 20 °C. mRNA expression was analyzed with a StepOnePlus Real-Time PCR System from Life Technologies (Darmstadt, Germany) using hydrolysis probes for MRP4 (ABCC4, Hs00988717_m1), MRP5 (ABCC5, Hs00981087_m1) and b-glucuronidase (GUSB, Hs00939627_m1) as well as the TaqMan Gene Expression Master Mix from Life Technologies according to the manufacturer’s protocol. Data were analyzed using the DCq method [22].

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2.11. Statistical analysis All results are expressed as mean ± SD, and are based on at least three independent experiments. GraphPad Prism 6.07 software (San Diego, CA, USA) was used for calculation of p-values by means of unpaired t-test with Welch’s correction or a one-way ANOVA followed by Dunnett’s multiple comparison test with n.s., not significant; ⁄, p < 0.05; ⁄⁄/##, p < 0.01; ⁄⁄⁄/$$$, p < 0.001 and ⁄⁄⁄⁄/### #/$$$$, p < 0.0001. Bonferroni’s multiple comparison test was additionally used to compare selected pairs of columns. 3. Results 3.1. cNMP-AMs reduce number of viable cells The number of viable HEL cells was determined by using the trypan blue dye exclusion method. Viable cells were significantly reduced after 24 h of incubation with every cNMP-AM in comparison to untreated cells (p < 0.0001 for cAMP-AM and cCMP-AM, p < 0.01 for cGMP-AM and cUMP-AM). Surprisingly, this reduction was no longer significant for cUMP-AM after 48 and 72 h of incubation (p > 0.05) (Fig. 1A). The number of viable K-562 cells was significantly reduced by cCMP-AM and cGMP-AM after incubation for 48 h only (p < 0.05, each) (Fig. 1B). 3.2. Induction of apoptosis by cNMP-AMs is time- and concentrationdependent Detection of apoptosis was performed by flow cytometry using annexinV/PI-staining [23]. A time curve showed that, in HEL cells, treatment with cNMP-AMs as well as with BFA led to a decrease of viable cells and a simultaneous increase of early apoptotic cells over the period of time. Late apoptotic cells were only slightly increased and untreated cells as well as PO4-AM3-treated cells were almost steady over time in these populations (Fig. 2A–C). cNMP-AMs induced apoptosis in the following order: cCMPAM > cAMP-AM > cGMP-AM > cUMP-AM. For cCMP-AM, the percentage of early apoptotic cells increased from 33.5% after 18 h treatment to 83.2% after 72 h. In K-562 cells, only BFA caused a decrease of viable cells accompanied by an increase of early and late apoptotic cells (Fig. 2D–F); untreated cells as well as cNMPAM- and PO4-AM3-treated cells were in the similar range over time regarding the three populations. cNMP-AMs and BFA induced apoptosis in a concentrationdependent manner in HEL cells (Fig. 3A–C). The negative control, PO4-AM3, also induced apoptosis in a concentration-dependent manner with 34.7% early apoptosic cells at 66 lM (relative to 200 lM cNMP-AMs) compared to 15.6% at 50 lM (relative to 150 lM cNMP-AMs). These results lead to the following EC50 values in HEL cells: 110 lM for cAMP-AM, 90 lM for cCMP-AM, 80 lM for cGMP-AM, 140 lM for cUMP-AM and 55 lM for PO4-AM3. At a concentration of 200 lM (or 66 lM PO4-AM3 or 10 lM BFA), early apoptotic cells amount to 36.8% for cAMP-AM, 41.7% for cCMP-AM, 32.2% for cGMP-AM, 40.5% for cUMP-AM, 34.7% for PO4-AM3 and 59.5% for BFA (Fig. 3B); whereas late apoptotic cells amount to 24.7% for cAMP-AM, 20.2% for cCMP-AM, 23.8% for cGMP-AM, 18.7% for cUMP-AM, 12.8% for PO4-AM3 and 14.3% for BFA (Fig. 3C). In K-562 cells (Fig. 3D–F), only BFA caused a concentration-dependent increase in apoptosis, whereas cCMPAM and cGMP-AM induced apoptosis only at 200 lM. 3.3. cNMP-AMs induce caspase-dependent apoptosis After incubation of HEL cells with 150 lM cNMP-AMs, the population of early and late apoptotic cells in each sample was

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Fig. 1. Absolute number of viable cells after incubation with cNMP-AMs. HEL and K-562 cells at a density of 1  105 cells/ml were incubated with 100 lM cNMP-AMs and 33 lM PO4-AM3 for 72 h. Viable cells were counted after 24, 48 and 72 h using a hemocytometer and trypan blue dye exclusion. Data shown are mean ± SD of three independent experiments and were assessed by a one-way ANOVA with Dunnett’s post-test (n.s., not significant, p > 0.05; ⁄, p < 0.05; ⁄⁄, p < 0.01; ⁄⁄⁄, p < 0.001; ⁄⁄⁄⁄ , p < 0.0001). Bonferroni’s post-test was additionally used to compare cAMP-AM and cCMP-AM. (A) Absolute number of viable HEL cells. (B) Absolute number of viable K-562 cells.

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Fig. 2. Time dependency of cNMP-AM-induced apoptosis. HEL and K-562 cells were incubated with 150 lM cNMP-AMs, 50 lM PO4-AM3 and 5 lM BFA for indicated points of time. Data shown are mean ± SD of three independent annexinV/PI-staining experiments. (A) Viable HEL cells. (B) Early apoptotic HEL cells. (C) Late apoptotic HEL cells. (D) Viable K-562 cells. (E) Early apoptotic K-562 cells. (F) Late apoptotic K-562 cells.

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Fig. 3. Concentration dependency of cNMP-AM-induced apoptosis. HEL cells were incubated for 24 h (A–C) and K-562 cells for 48 h (D–F) with indicated concentrations of cNMP-AMs. Corresponding concentrations of (i) PO4-AM3 were 33, 50 and 66 lM; (ii) BFA were 1, 5 and 10 lM. Data shown are mean ± SD of three independent annexinV/PIstaining experiments. (A) Viable HEL cells. (B) Early apoptotic HEL cells. (C) Late apoptotic HEL cells. (D) Viable K-562 cells. (E) Early apoptotic K-562 cells. (F) Late apoptotic K-562 cells.

significantly increased compared to untreated cells (p < 0.0001) (Fig. 4A). The effects for cCMP-AM and cUMP-AM were in the same range as these of cAMP-AM and cGMP-AM. In the case of K-562 cells, increase of apoptotic cells was not significant compared to untreated cells (p > 0.05), but the increase of cells treated with 5 lM BFA was significant (p < 0.0001) (Fig. 4B). To assess whether apoptosis is caspase-dependent, we preincubated HEL cells with the pan-caspase inhibitor Z-VAD-FMK [20] (Fig. 4A). For all cNMP-AMs, Z-VAD-FMK caused a significant reduction of apoptotic cells (Fig. 4A) compared to the previous approach (p < 0.001). Furthermore, we performed western blots with HEL cell lysates to examine potential changes in caspase 3 and cleaved caspase 3 protein amount after treatment with cNMP-AMs (Fig. 5A–D). Surprisingly, differences in both approaches were not significant (p > 0.05). Hence, we additionally performed a caspase 3 assay to determine caspase 3 activity (Fig. 5E). Cells treated with cAMP-AM, cCMP-AM, cGMP-AM and BFA showed significant higher caspase 3 activity than untreated cells (p < 0.01, p < 0.001, p < 0.05 and p < 0.0001, respectively).

[24,25]. Conversely, JC-1 does not accumulate in mitochondria with depolarized Dw, which is associated with apoptosis, but remains in the cytoplasm as monomers, resulting in a decrease of red fluorescence [14,15]. Changes in the fluorescence signal intensity were measured by flow cytometry. After incubation of HEL cells, Dw was significantly depolarized for cAMP-AM and cGMP-AM treated cells (p < 0.05 each) as well as for cCMP-AM and for BFA treated cells (p < 0.001 and p < 0.0001, respectively) compared to untreated cells (Fig. 6A). cUMP-AM had no effect on the mitochondrial membrane potential (p > 0.05). To investigate if depolarization of the mitochondrial membrane potential caused by cCMP-AM is due to caspasedependent apoptosis and not just a general sign of mitochondrial dysfunction or toxicity, we pre-incubated HEL cells with the general caspase inhibitor Z-VAD-FMK [20] (Fig. 6B). For cCMP-AM treated cells, Z-VAD-FMK caused a significant reduction of cells with depolarized Dw (p < 0.05).

3.6. cCMP-AM increases subG1 population 3.4. Incubation with unmodified cNMPs does not lead to induction of apoptosis Apoptosis was detected by flow cytometry using annexinV/PIstaining. Incubation of HEL cells with unmodified cNMPs did not result in a significant increase of apoptotic cells (p > 0.05) (Fig. 4C) but for cells incubated with cCMP-AM and BFA (p < 0.0001, each). 3.5. cCMP-AM reduces the mitochondrial membrane potential Dw The fluorescent dye JC-1, which was used in this assay, is rapidly taken up by mitochondria of viable cells whose mitochondrial membrane potential (Dw) is polarized, leading to the formation of JC-1 aggregates with high red fluorescence signal intensity

The cell cycle can be subdivided into interphase, which encompasses G1, S, and G2; and stages of mitosis (M phase). G1 and G2 represent the ‘‘gaps” between the landmarks of the cell cycle, DNA synthesis and mitosis. In the first gap (G1), the cell is preparing for DNA synthesis. S phase cells are synthesizing DNA and therefore contain varying amounts of DNA (between 2n and 4n). During the second gap (G2), the cell prepares for M phase. G0 cells are not in the cycle but have the potential for division [26]. G0/G1 phase cells are diploid (2n) and have half of the DNA content of tetraploid G2/M phase cells (4n), whereas cells in subG1 phase contain less DNA due to fragmentation induced by apoptosis. The fluorescent dye PI binds stoichiometrically to DNA, so that changes in DNA quantity are directly correlated to fluorescence intensity [27].

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P

30

20

µM

30

****

50

cA

40

****

60

0

50

70

15

****

60

apoptotic cells [%]

90

70

+

HEL cells

100

apoptotic cells [%]

+

c

Z-VAD-FMK 20 µM

untreated

15 GM 0 P µM

0

Fig. 4. Analysis of apoptosis of HEL and K-562 cells. (A) HEL cells were treated with 150 lM cNMP-AMs, 50 lM PO4-AM3 and 5 lM BFA for 24 h and pre-incubated with 20 lM Z-VAD-FMK for 1 h. Data shown are mean ± SD of three independent annexinV/PI-staining experiments and were assessed by a one-way ANOVA with Dunnett’s posttest (n.s., not significant, p > 0.05; ⁄⁄/##, p < 0.01; ⁄⁄⁄⁄/####, p < 0.0001). Statistical analysis of the approach without Z-VAD-FMK is compared to untreated cells and marked with ⁄, whereas statistical analysis of the approach with Z-VAD-FMK is compared to untreated cells + Z-VAD-FMK and marked with #. Bonferroni’s post-test was additionally used to compare both approaches (marked with $; $$$, p < 0.001). (B) K-562 cells were incubated for 48 h with 150 lM cNMP-AMs, 50 lM PO4-AM3 and 5 lM BFA. (C) HEL cells were incubated with 150 lM unmodified cNMPs, 150 lM cCMP-AM and 5 lM BFA for 24 h. (B, C) Data shown are mean ± SD of three independent annexinV/PI-staining experiments and were assessed by a one-way ANOVA with Dunnett’s post-test (⁄⁄⁄⁄, p < 0.0001).

Cell cycle analysis was performed with HEL cells using flow cytometry where results are displayed as a fluorescence histogram with intervals for the different phases according to the DNA content. These sections were used to calculate statistics (Fig. 7). SubG1 population was significantly increased by cCMP-AM and cGMP-AM (p < 0.001 and p < 0.01, respectively) (Fig. 7A) while G2/M population was simultaneously decreased (p < 0.01, each) (Fig. 7D), suggesting a cell cycle block at the G2/M checkpoint. cAMP-AM and cUMP-AM were inactive in this assay (p > 0.05). 3.7. mRNA expression of MRP4 and MRP5 is similar in HEL and K-562 cells The mRNA expression of MRP4 and MRP5 in HEL and K-562 cells was analyzed by quantitative real-time PCR (qPCR). The DCq of MRP4 (ABCC4) mRNA expression in HEL cells was lower than in K-562 cells but this difference was not significant (p > 0.05) (Table 1). DCq values of MRP5 (ABCC5) expression were similar in HEL and K-562 cells (p > 0.05) (Table 1). 3.8. Inhibition of MRPs increases apoptosis induced by cNMP-AMs HEL and K-562 cells were pre-treated with the MRP inhibitor probenecid [21]. Thereby, the apoptosis-inducing effect of cAMPAM and cUMP-AM were significantly enhanced in HEL cells after

24 h (p < 0.0001) (Fig. 8A). For K-562 cells, the pre-treatment resulted in a significant induction of apoptosis by all tested cNMP-AMs (p < 0.0001), whereas apoptosis was not induced without pre-treatment after 48 h (p > 0.05) (Fig. 8B). 4. Discussion In a previous study, our group examined the effects of cNMPAMs in S49 wild-type and kinase-deficient mouse lymphoma cell lines [6]. Therein, we showed for the first time that cCMP-AM induces apoptosis via the intrinsic, mitochondrial pathway and via the endoplasmatic reticulum (ER) stress pathway, whereas the extrinsic apoptotic pathway is not involved [6]. Many proteins that are linked to cAMP-induced apoptosis in S49 cells could be excluded as cCMP targets [6]. Therefore, we were interested if the effects of cCMP-AM on these mouse lymphoma cells can be observed in human cell lines as well. cNMPs lacking the AM-group had no effect on HEL cells (Fig. 4C) as previously observed in S49 cells [6], although it is well known that extracellularly added cNMPs can exert biological effects [13,14]. Since only the membrane-permeant analogs (cNMP-AMs) exhibited biological effects in HEL cells (Fig. 4A), extracellular targets can be excluded and we conclude that membrane penetration of cNMPs is necessary to address as yet unknown intracellular targets (Fig. 9A).

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F. Dittmar et al. / Biochemical Pharmacology 112 (2016) 13–23

A

C

BFA

250

x-fold of untreated cells

3.5 3.0 2.5 2.0 1.5 1.0 0.5

150 100 75 50 25

E

B

5 FA µM

cU

1 5 MP 0 -A µM M

50 Pµ M AM

cG 1 M

cC

1 5 MP 0 -A µM M

50 PµM AM

cA 1 M

50 4-A µ M M3

B

5 FA µM

cU

1 5 MP 0 -A µM M

cC

cG 1 M

50 Pµ M AM

15 MP 0 -A µM M

cA 1 M

PO

50 PµM AM

0 50 4-A µ M M3

0.0

200

PO

x-fold of untreated cells

PO 4-AM3

cleaved Caspase 3

4.0

HEL cells 5× 10

4

4× 10 4

***

** E(400/505)

cAMP-AM

D

Caspase 3

cUMP-AM

α tubulin

cGMP-AM

α tubulin

cCMP-AM

cleaved caspase 3

untreated

caspase 3

BFA

PO4 -AM3

cUMP-AM

cGMP-AM

cCMP-AM

untreated

cAMP-AM

B

****

*

3× 10 4

2× 10 4

1× 10 4

µM

B FA 5

50 PµM AM

50 Pµ M AM

cU 1 M

cG 1 M

PA µM M 0

cC M

15

50 PµM AM

cA 1 M

50 4-A µM M3

PO

un

tr ea te d

0

Fig. 5. Analysis of caspase 3 activation in HEL cells. (A, B) 50 lg protein of HEL cell lysates obtained from cells incubated with 150 lM cNMP-AMs, 50 lM PO4-AM3 and 5 lM BFA for 18 h were used for western blot analysis. (C, D) Images were analyzed with Image Studio Lite software, values were referred to a tubulin expression and normalized to untreated cells. (C) Data shown are mean ± SD of three independent experiments and were assessed by a one-way ANOVA with Dunnett’s post-test. (D) Data shown are mean ± SD of five independent experiments and were assessed by a one-way ANOVA with Dunnett’s post-test. (E) HEL cells were incubated with 150 lM cNMP-AMs, 50 lM PO4-AM3 and 5 lM BFA for 20 h. Data shown are mean ± SD of five independent experiments and were assessed by a one-way ANOVA with Dunnett’s post-test (⁄, p < 0.05; ⁄⁄ , p < 0.01; ⁄⁄⁄, p < 0.001; ⁄⁄⁄⁄, p < 0.0001).

The pro-apoptotic effects of cNMP-AMs in HEL cells are specific for the following reasons: (i) Different cNMP-AMs showed differential effects in various assays with HEL cells. (ii) The control compound PO4-AM3 which is used to test for potential side effects of the cleaved acetoxymethylester group, had no significant effects. (iii) In contrast to HEL cells, K-562 cells were resistant to the apoptosis-inducing effects of cNMP-AMs (Figs. 2D–F, 3D– F, 4B and 9, Table 2). To investigate if the resistance of K-562 cells toward the apoptosis-inducing effect by cNMP-AM is due to their rapid export via MRPs, we determined the mRNA expression levels of MRP4 and MRP5 in HEL and K-562 cells. Since we could not notice a significant difference in the mRNA expression levels of both cell lines (Table 1), we additionally examined the effect of probenecid, a MRP inhibitor [21]. After pre-treatment of K-562

cells with probenecid, apoptosis was also induced by all examined cNMP-AMs (Fig. 8B). Thus, cNMP-AMs cannot induce apoptosis in K-562 cells because they are rapidly exported via MRPs, is confirmed (Fig. 9B). Furthermore, we show that cCMP-AM reduces the number of viable cells (Fig. 1A) and induces apoptosis (Figs. 2A–C, 3A– C and 4A) in HEL cells. For determination of the number of viable cells, we used 100 lM cNMP-AMs and obtained significant decreases in HEL cells (Fig. 1A). However, regarding the detection of apoptosis via flow cytometry using annexinV/PI-staining, only weak and fluctuating effects could be determined in HEL cells when using 100 lM cNMP-AMs (Fig. 3A–C). Therefore, we increased the concentration of cNMP-AMs and recorded a concentration-response curve (Fig. 3), which revealed that the

20

F. Dittmar et al. / Biochemical Pharmacology 112 (2016) 13–23

A

B

HEL cells

HEL cells

60

60

45

*

40 35

***

30

*

*

25 20 15 10

55

cells with depolarized Δψ [%]

****

50

50 45 40 35

15 10

5 FA µM

untreated

cU M

B

15 P-A 0 µM M

cG M

15 P0 AM µM

cC M

15 P0 AM µM

cA

M

15 P0 AM µM

0

PO

**

20

5 4 50 - A M µM 3

**

25

0

un

*

30

5

tre at ed

cells with depolarized Δψ [%]

55

Z-VAD-FMK 20 µM

-

+

PO4 -AM3 50 µM +

cCMP-AM 150 µM +

-

BFA 5 µM -

+

Fig. 6. Analysis of mitochondrial membrane potential of HEL cells. (A) HEL cells were incubated with 150 lM cNMP-AMs, 50 lM PO4-AM3 and 5 lM BFA for 24 h. Data shown are mean ± SD of three independent experiments and were assessed by a one-way ANOVA with Dunnett’s post-test (⁄, p < 0.05; ⁄⁄⁄, p < 0.001; ⁄⁄⁄⁄, p < 0.0001). Bonferroni’s post-test was additionally used to compare cAMP-AM and cCMP-AM. (B) HEL cells were treated with 150 lM cCMP-AM, 50 lM PO4-AM3 and 5 lM BFA for 24 h and preincubated with 20 lM Z-VAD-FMK for 1 h. Data shown are mean ± SD of three independent experiments and were assessed by a one-way ANOVA with Dunnett’s post-test (⁄, p < 0.05; ⁄⁄, p < 0.01). Bonferroni’s post-test was additionally used to compare both approaches.

B

subG1 phase

G0/G1 phase

70

70

****

60

60 50

cells [%]

****

40

**

***

40 30 20

0 tr ea te d PO 4 5 0 -A µ M cA M 3 M 15 P 0 -A cC µM M M 15 P 0 -A cG µM M M 15 P0 A cU µM M M 15 P 0 -A µM M

B

µM

un

un

C

D

S phase

G2/M phase

70

70

60

60 50

cells [%]

50

*

40 30

40

10

0

0

**

** ****

un

µM

B 5 FA

tr ea te d PO 4 5 0 -A µ M cA M 3 M 15 P0 A cC µM M M 15 P 0 -A cG µM M M 15 P0 A cU µM M M 15 P 0 -A µM M

20

10

un

*

30

20

tre at ed PO 4 5 0 -A µ M cA M 3 M 15 P0 A cC µM M M 15 P 0 -A cG µM M M 15 P0 A cU µM M M 15 P 0 -A µM M

cells [%]

B

10

0 tre at ed PO 4 50 - A µ M cA M 3 M 15 P 0 -A cC µ M M M 15 P 0 -A cG µ M M M 15 P0 A cU µ M M M 15 P 0 -A M

10

5 FA µM

20

**

B

30

5 FA µM

cells [%]

50

5 FA µM

A

Fig. 7. Cell cycle analysis of HEL cells. HEL cells were incubated with 150 lM cNMP-AMs, 50 lM PO4-AM3 and 5 lM BFA for 24 h. Cell cycle analysis was performed using flow cytometry where results are displayed as a histogram. (A–D) Intervals for the different phases were used to calculate statistics: subG1, G0/G1, S and G2/M. Data shown are mean ± SD of at least three independent experiments and were assessed by a one-way ANOVA with Dunnett’s post-test (⁄, p < 0.05; ⁄⁄, p < 0.01; ⁄⁄⁄, p < 0.001; ⁄⁄⁄⁄, p < 0.0001). Bonferroni’s post-test was additionally used to compare cAMP-AM and cCMP-AM.

Table 1 mRNA expression of MRP4 and MRP5. DCq values were calculated according to the following equation: DCq = Cq (target gene) – Cq (reference gene). GUSB was used as reference gene. There is no significant difference between the DCq values of MRP4 (ABCC4) and MRP5 (ABCC5) expression in HEL and K-562 cells (p > 0.05). Data shown are mean ± SD of three independent experiments and were assessed by unpaired t-test with Welch’s correction. ABCC, ATP-binding cassette sub-family C; Cq, quantification cycle; GUSB, bglucuronidase; MRP, multidrug resistance-associated protein. Cells

GUSB Cq

MRP4 Cq

MRP4 DCq

MRP5 Cq

MRP5 DCq

HEL K-562

27.49 ± 0.43 26.05 ± 0.25

25.74 ± 0.20 28.18 ± 0.64

1.74 ± 0.27 2.13 ± 0.39

29.67 ± 0.25 27.97 ± 0.52

2.19 ± 0.21 1.92 ± 0.27

F. Dittmar et al. / Biochemical Pharmacology 112 (2016) 13–23

A

HEL cells

apoptotic cells [%]

$$$$

$$$$

100 90

early apoptotic

80

late apoptotic / necrotic

70

n.s.

####

####

**** ****

60

n.s.

####

####

****

50

****

40 30

n.s.

20 10 0 PROB 500 µM

untreated

-

+

PO4 -AM3

cAMP-AM

cCMP-AM

cGMP-AM

50 µM

150 µM

150 µM

150 µM

-

-

B

+

-

+

-

cUMP-AM 150 µM

+

-

+

K-562 cells

apoptotic cells [%]

100 90

early apoptotic

80

late apoptotic / necrotic

70

$$$$

$$$$

$$$$ ####

$$$$

####

#### ####

60 50 40 30

n.s.

20 10 0 PROB 500 µM

untreated

-

+

PO4 -AM3

cAMP-AM

cCMP-AM

cGMP-AM

50 µM

150 µM

150 µM

150 µM

-

-

+

-

+

-

cUMP-AM 150 µM

+

-

+

Fig. 8. Analysis of apoptosis of HEL and K-562 cells. Cells were treated with 150 lM cNMP-AMs and 50 lM PO4-AM3 for indicated points of time and pre-incubated with 500 lM probenecid (PROB) for 5 min. Data shown are mean ± SD of four independent annexinV/PI-staining experiments and were assessed by a one-way ANOVA with Dunnett’s post-test (n.s., not significant, p > 0.05; ⁄⁄⁄⁄/####, p < 0.0001). Statistical analysis of the approach without PROB is compared to untreated cells and marked with ⁄, whereas statistical analysis of the approach with PROB is compared to untreated cells + PROB and marked with #. Bonferroni’s posttest was additionally used to compare both approaches (marked with $; $$$$, p < 0.0001). (A) HEL cells treated for 24 h. (B) K-562 cells treated for 48 h.

effects with 150 lM cNMP-AMs were larger while the effect of 50 lM PO4-AM3, which is used as control compound, was moderate. In contrast, with 66 lM PO4-AM3, which is corresponding to 200 lM cNMP-AMs, the effect was too pronounced. Therefore, we decided to perform all further experiments with 150 lM cNMP-AMs and 50 lM PO4-AM3. The effects of PO4-AM3 on the number of viable cells may be due to the release of toxic formaldehyde from the compound [16]. Apoptosis induced by cNMP-AMs in HEL cells is caspasedependent (Fig. 4A) and is induced via the intrinsic and mitochondria-dependent pathway (Figs. 6 and 9A). This supports the observations in S49 cells. In these cells, the lack of caspase 8 activation pointed to an intrinsic pathway, and cytochrome c release as well as caspase 9 activation are indicative for activation of the mitochondria-dependent pathway [6]. In our present study, we additionally examined the mitochondrial membrane potential in HEL cells and found that it becomes most effectively depolarized by cCMP-AM compared to the other cNMP-AMs (Fig. 6A). Thus, the regulation of the mitochondrial membrane potential is a novel function of cCMP. We also examined the effects of cNMP-AMs on the cell cycle of HEL cells and observed an increase in subG1 population, which represents apoptotic cells, by cCMP and cGMP; whereas G2/M population was simultaneously decreased. This is in accordance with the concept that disruption of Dw is associated with the appearance of the

21

subG1 cell population representing apoptotic cells and that the decrease of Dw coincides with a decrease of the G2 population [28]. A decrease in the G2/M population could be explained by a decrease in the S cell entrance in G2 phase due to apoptosis since accumulation of topoisomerase-mediated DNA strand breaks likely causes the failure of DNA repair as well as subsequent inactivation of the G2-checkpoint mechanism and concomitantly switches on the cell death signal [28]. Furthermore, we performed western blots for caspase 3 and cleaved caspase 3 since the results in S49 cells for cleaved caspase 3 were promising [6]. Unfortunately, according to the statistical analysis, the differences in HEL cells were neither significant for caspase 3 (Fig. 5C) nor for cleaved caspase 3 (Fig. 5D). Thus, we alternatively performed a caspase 3 activation assay and obtained more conclusive results, which show that caspase 3 is activated after treatment with cCMP-AM, cAMP-AM and cGMP-AM (Fig. 5E). This discrepancy might be related to a higher sensitivity of the activation assay. Induction of caspase-dependent apoptosis using 150 lM cNMP-AMs is dependent on the cell line: S49 cells are only sensitive to cCMP-AM with higher sensitivity of wild-type than kin- cells, whereas K-562 cells are insensitive and HEL cells are sensitive to all tested cNMP-AMs (Table 2). Hence, we revealed unexpected structure-activity relationships for cNMP-AMs: (i) cCMP-AM had the largest biological effect regarding mitochondrial membrane potential (Fig. 6A). (ii) cCMP-AM and cGMP-AM effectively modulated cell cycle, but cAMP-AM and cUMP-AM were inactive (Fig. 7). (iii) Determination of the number of viable cells showed that cCMP-AM and cAMP-AM were most effective (Fig. 1A), whereas the effects of all cNMP-AMs in the apoptosis assay were comparable (Fig. 4A). These findings suggest a different regulation of individual steps of cell growth and apoptosis for each cNMP. These data point to the involvement of various unknown target proteins in these processes that are differentially activated by the individual cNMPs. In this context, it is noteworthy that cNMPs differentially activate dynamic mass distribution [15], protein kinases A and G [12,29,30] and hyperpolarizationactivated, cNMP-gated ion channels [31–33]. Thus, the concept is emerging that any given cNMP plays a unique second messenger role [13,14]. This concept is further supported by the fact that cNMPs are differentially transported by MRPs [11] and cleaved by PDEs [10,34]. Cell membrane-permeable cAMP analogs induce apoptosis in peripheral blood mononuclear cells (PBMCs) from patients with chronic lymphocytic leukemia (CLL) [35]. Since cAMP analogs cannot be used clinically, targeting of proteins that are involved in cAMP generation, degradation, metabolism and signaling, like PDEs, G-protein coupled receptors (GPCRs) and adenylyl cyclases (ACs), came into focus as therapeutic strategies for leukemia therapy [35]. Our pro-apoptotic data on cCMP indicate that this cNMP could also play a role in future tumor therapy. Future experiments should focus on the identification of the hitherto unknown cCMP targets. Future experiments should also extend the proliferation studies by colony formation assay [36] and metabolic labeling methods (BrdU and 3H-thymidine incorporation) [37,38]. Additionally, more in-depth mechanistic questions should be addressed by examining the effects of PKA and PKG inhibitors [15], the adenylyl cyclase activator forskolin [39] and dibutyrylated analogs of cNMPs (DB-cNMPs) [29] in future studies. In conclusion, cCMP effectively induces apoptosis in HEL cells, cCMP export by MRPs prevents apoptosis of K-562 cells and cNMP-AMs differentially regulate various aspects of apoptosis, cell growth and mitochondrial function. Our data support the concept of distinct second messenger functions of cAMP, cGMP, cCMP and cUMP.

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F. Dittmar et al. / Biochemical Pharmacology 112 (2016) 13–23

A

B

HEL cells cNMP-AMs

cAMP, cUMP

cNMP-AMs

cNMPs

MRPs 4, 5

MRPs 4, 5

NMPs

AM PDEs

? JC-1

cNMPs

Bax

Δψ

? ER

Bax

? Bak

stress

Bcl-xL

?

mitochondrium

?

Caspase 12

cytochrome c

?

Bim cross talk ?

mitochondrium

Bcl-xL

Z-VAD-FMK

Δψ

?

cross talk ?

Caspase 9

JC-1

cNMPs Bak

Bim

Caspase 12

?

?

? stress

esterases

probenecid

AM PDEs

NMPs

esterases

probenecid

ER

K-562 cells

cytochrome c

Caspase 9

APAF-1

APAF-1

Caspase 9

Caspase 9 Caspase 3

Caspase 3 Caspase 6

Caspase 7

Caspase 6

?

?

?

Caspase 7

?

PARP

PARP

apoptosis

apoptosis

Fig. 9. cNMP-signaling pathways in HEL and K-562 cells based on the results of our studies. The involvement of all components indicated with a question mark within the cNMP signaling pathway in HEL and K-562 cells remains unclear, e.g. hydrolysis by PDEs as an additional mechanism to eliminate cNMPs. (A) In HEL cells, cNMP-AMs penetrate the cell membrane, the AM-group is cleaved by esterases inside the cell and cNMPs then induce apoptosis via the intrinsic and mitochondria-dependent pathway. The mitochondrial membrane potential Dw is depolarized and caspase 3 is activated resulting in apoptosis. This effect can be decreased using the caspase-inhibitor Z-VADFMK and increased by using the MRP-inhibitor probenecid. (B) In K-562 cells, cNMP-AMs penetrate the cell membrane and the AM-group is cleaved by esterases inside the cell but cNMPs are then rapidly exported via MRPs (e.g. MRP 4 and 5). Blocking this pathway using the MRP-inhibitor probenecid also results in induction of apoptosis in K-562. Since K-562 cells are resistant to the extrinsic apoptotic pathway [40,41], this mechanism is not shown. APAF-1, apoptotic protease-activating factor 1; PARP, poly (ADP-ribose) polymerase.

Table 2 Significance of early apoptotic cells induced by cNMP-AMs compared to untreated cells. Cells were treated with 150 lM cNMP-AMs and 50 lM PO4-AM3 for indicated periods of time. Data shown are derived from the statistical analysis of three independent annexinV/PI-staining experiments where cells were analyzed by flow cytometry. Shown are the significances of the percentage of early apoptotic cells from treated cells compared to the percentage of early apoptotic cells from untreated cells. Original data of S49 cells were obtained from Wolter et al. (2015) [6] and reevaluated with a newer version of GraphPad Prism software, data for HEL and K-562 cells were obtained from Fig. 4A and B and p-values were recalculated for early apoptotic cells only by a one-way ANOVA with Dunnett’s post-test (⁄, p < 0.05; ⁄⁄⁄, p < 0.001; ⁄⁄⁄⁄ , p < 0.0001). Treatment

HEL 24 h

K-562 48 h

cAMP-AM 150 lM cCMP-AM 150 lM cGMP-AM 150 lM cUMP-AM 150 lM PO4-AM3 50 lM

⁄⁄⁄⁄

n.s. n.s. n.s. n.s. n.s.

⁄⁄⁄⁄ ⁄⁄⁄⁄ ⁄⁄⁄⁄ ⁄

S49 wt 24 h/48 h

S49 kin24 h/48 h

n.s.

n.s.

⁄⁄⁄⁄

⁄⁄⁄

n.s. n.s. n.s.

n.s. n.s. n.s.

Conflict of interest The authors declare no conflict of interest.

[4]

[5]

[6]

[7]

[8]

[9] [10] [11]

[12]

[13] [14]

Acknowledgments [15]

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