Abstract

Extracellular ATP (eATP) and its metabolites have emerged as key modulators of different diseases and comprise a complex pathway called purinergic signaling. An increased number of tools have been developed to study the role of nucleotides and nucleosides in cell proliferation and migration, influence on the immune system and tumor progression. These tools include receptor agonists/antagonists, engineered ectonucleotidases, interference RNAs and ectonucleotidase inhibitors that allow the control and quantification of nucleotide levels. NTPDase1 (also called apyrase, ecto-ATPase and CD39) is one of the main enzymes responsible for the hydrolysis of eATP, and purified enzymes, such as apyrase purified from potato, or engineered as soluble CD39 (SolCD39), have been widely used in in vitro and in vivo experiments. However, the commercial apyrase had its effects recently questioned and SolCD39 exhibits limitations, such as short half-life and need of high doses to reach the expected enzymatic activity. Therefore, this study investigated a non-viral method to improve the overexpression of SolCD39 and evaluated its impact on other enzymes of the purinergic system. Our data demonstrated that PiggyBac transposon system proved to be a fast and efficient method to generate cells stably expressing SolCD39, producing high amounts of the enzyme from a limited number of cells and with high hydrolytic activity. In addition, the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other enzymes from the purinergic system. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.

Introduction

Extracellular nucleotides are involved in the control of numerous pathophysiological mechanisms and influence a wide range of processes through purinergic receptors which include immune responses, inflammation, platelet aggregation, vasodilatation, cell proliferation, migration, cell death and many others [1,2]. Extracellular ATP (eATP) and their metabolites together with other nucleotides have emerged as key modulators of tumor biology and inflammatory diseases since their concentrations in the microenvironment of the tissue of these diseases are much higher than in healthy tissues [3].

Following its release into the extracellular space, eATP is mainly hydrolyzed by ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and subsequently to adenosine (ADO) by the ecto-5′-nucleotidase (also known as CD73) [4]. ADO has received special attention, mainly due to its strong influence in generating an immunosuppressive environment. Nonetheless, eATP also has important roles depending on the receptor it activates [5].

Different studies have revealed the participation of eATP as an inflammatory mediator in the pathophysiology of several infectious [6], autoimmune [7], neurodegenerative diseases [8], as well as cancer [5]. Consequently, an increased number of tools have been developed to better understand these mechanisms, as well as methods that allow in vivo [9] and in vitro [10,11] quantification of nucleotides and tools to control the levels of nucleotide in order to prevent associated pathological effects [12]. Commercial apyrase has been widely used to modulate eATP levels. This enzyme is purified from potato or recombinant organisms and is used to deplete endogenous nucleotides in biological preparations [13]. However, Madry et al. [14] recently demonstrated that processes associated with eATP, after the use of apyrase, could be reflected by cellular depolarization caused by K+ contamination and not by ATP depletion, generating effects independent of the intended enzymatic activity.

Another strategy commonly used is the recombinant soluble NTPDase1/CD39 (SolCD39). NTPDase1, also known as ecto-ATPase, CD39 and apyrase, is a membrane glycoprotein that hydrolyzes both ATP and ADP [4]. To produce the soluble form, the transmembrane domains that anchor NTPDase1/CD39 to the cell surface were removed [15]. The SolCD39 is commonly used as a potent platelet aggregation inhibitor [1517], due to its capacity to hydrolyze ADP, the main platelet-activating and -recruiting agent. Nowadays, its therapeutic benefit has also been documented in disease models such as cardiac ischemia [18,19], transplantation [20,21], renal ischemia–reperfusion injury [22,23], inhibition of metastasis [24] and stroke [25], indicating effects associated with antithrombotic, anti-inflammatory and cardioprotection activity. However, the half-life of SolCD39 is ∼48 h and repeated applications of the enzyme are required for attaining efficient and long lasting enzymatic activity [15,26].

Furthermore, although these studies suggest that the presence of SolCD39 does not cause changes in signaling pathways implicated in homeostasis [27], little is known about the behavior of the other enzymes of the purinergic signaling in the presence of this soluble enzyme. In addition to its enzymatic functions, NTPDase1 may also perform functions by interacting with others proteins [28].

Therefore, the objective of this study was to evaluate the production of SolCD39 by cancer cells through a non-viral method that is faster and cheaper viral methods. This system can facilitate the application of this tool to explore the role of extracellular nucleotides in different in vitro and in vivo conditions. For that, we chose a PiggyBac-based transposon system, which is a non-viral genome modification system that can generate stable cell lines more easily when compared with other gene delivery systems generally used [29,30]. In addition, the cells were analyzed in order to investigate whether the gene insertion produced changes in cellular parameters such as proliferation, migration and adhesion, and its influence in the purinergic signaling and metabolism of extracellular nucleotides.

Materials and methods

Cell culture

C6 rat glioma cells were obtained from the American Tissue Culture Collection (ATCC) and were cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco, Massachusetts, EUA) supplemented with 10% fetal bovine serum (FBS; Gibco, Massachusetts, EUA), 1% penicillin–streptomycin and 0.1% amphotericin B (Sigma Chemicals, MO, U.S.A.). Cells were kept at 37°C, humidity of 95% and 5% CO2 in air.

Plasmids

We used a recombinant soluble form of Rattus norvegicus Entpd1 (SolCD39), as described previously [15]. Briefly, the Entpd1 sequence was constructed comprising the rat coding sequence from Thr38 to Thr476, which removed the nucleotides coding for the hydrophobic regions at the N- and C-terminals, and fused to a sequence coding for the rat IL-2-derived leader sequence that allows the enzyme to be secreted, as previously described [31]. Similarly, a mutated Entpd1 [SolCD39 (E174A)] was synthesized as a control based on a study which demonstrated that site-directed mutagenesis of glutamate 174 to alanine (E174A) resulted in complete loss of enzymatic activity [32]. All plasmids pPB[SolCD39], pPB[SolCD39 (E174A)], pPB[Empty vector] and helper plasmid pRP[PBase], which contains a PiggyBac (PB) transposase gene (PBase), were custom made from VectorBuilder (CA, EUA).

Cell transfection

C6 glioma cells stably expressing the SolCD39, SolCD39 (E174A) or Empty vector were generated by non-viral transfection with transposon PiggyBac-based method. Briefly, 3 × 104 C6 glioma cells were seeded into each well of a 12-well-plate 3 days prior to transfection and cultured as described above. For each well, 1 µg of the plasmid of interest and 0.4 µg of the PBase were co-transfected using Lipofectamine® LTX Reagent according to standard protocols (ThermoFisher Scientific Inc., Rockford, U.S.A.). Three days after transfection, the drug selection with G-418 antibiotic (2 mg/ml — Gibco, Massachusetts, EUA) was performed for 2 weeks.

To verify cell purity, transfected cells were submitted to fluorescence-activated cell sorting (FACS; BD Biosciences, San Jose, U.S.A.) for phytochrome-based near-infrared fluorescent protein 713 (iRFP-713) with 645 lasers for excitation and 713 emission filter. Immunofluorescence and analyzes using InCell Analyzer 6000 (GE Healthcare Life Sciences, Pittsburgh, EUA) were also performed to confirm the efficiency of cell transfection. Non-transfected C6 glioma cells were used as control.

Ectonucleotidases assay

NTPDase activity

To determine the ATP/ADPase activities of the soluble enzymes, all cells [C6 cell, SolCD39, SolCD39 (E174A) and Empty vector] were cultured in 24-well microplates until confluence, washed three times with incubation medium (IM) containing (final concentration) 2 mM CaCl2, 120 mM NaCl, 5 mM KCl, 10 mM glucose, 20 mM HEPES (Sigma–Aldrich, St. Louis, U.S.A.), pH 7.4 and incubated 300 µl of IM for 6 h at 37°C, 5% CO2 in a humidified incubator. Next, the supernatant of each cell was centrifuged at 14 000 g for 5 min and 180 μl of the supernatant was transferred to a new microcentrifuge tube. The reaction was started by the addition of 20 μl of the nucleotides (ATP or ADP) (Sigma–Aldrich, St. Louis, U.S.A.) to a final concentration of 2 mM at 37°C during 30 min [33]. To stop the reaction, 200 μl of trichloroacetic acid (TCA) was added to a final concentration of 5% (w/v). The production of inorganic phosphate (Pi) was measured using the Malachite green method [34] and controls to correct for non-enzymatic hydrolysis were performed by adding the sample after stopping the reaction. ATP and ADP hydrolysis are expressed as nmol Pi/min/ml.

To determine the ATP/ADP/AMPase activities of the membrane enzymes, the cells were cultivated as described above, washed three times with IM and the reaction was started by the addition of 200 μl of the IM containing 2 mM of ATP, ADP or AMP. For AMP hydrolysis, the same IM was used with the exception that the 2 mM MgCl2 was used instead of CaCl2. At the end reaction, an aliquot was transferred to a tube containing TCA (5% w/v) previously placed on ice and the release of inorganic phosphate (Pi) was evaluated as previously described [33,34].

Nucleotide and nucleoside analysis using HPLC

The cells were cultivated as described above and the supernatant incubated with ATP, except that the final concentration was 100 μM. To stop the reaction, an aliquot of the medium was transferred to a tube on ice and centrifuged at 4°C for 15 min at 16 000 g. Aliquots of 20 μl were applied to a reverse phase HPLC system using a C18 Shimadzu column (Shimadzu, Japan) with absorbance measured at 250 nm. The mobile phase was 60 mM KH2PO4, 5 mM tetrabutylammoniumchloride, pH 5.0, in 30% methanol (Sigma–Aldrich, MO, U.S.A.) as described [33]. Retention times were assessed using standard samples of nucleotide and purines, and concentrations are expressed as μMol of nucleotide.

To evaluate the role of membrane ectonucleotidases together with soluble enzymes, we also incubate the nucleotide in direct contact with the cells after concentrating the soluble enzymes in cell supernatant. The cells were incubated as described above, except that the ATP (final concentration of 100 μM) was added directly on the 24-well plates. At the end of each incubation time, the supernatant was collected, centrifuged and frozen for further analysis in HPLC.

Comparison of the enzymatic activity of the SolCD39 enzyme and apyrase from potato

To evaluate the specific activity between SolCD39 and apyrase from potato, SolCD39 cells were cultured in 100 mm plates until reaching confluence (∼9 × 106 cells). Next, the cells were washed with IM and incubated with 5 ml of this IM for 6 h. The supernatant was collected, centrifuged at 14 000 g for 5 min to remove non-adhered cells and concentrated with Centricon (Microcon-30kDa, Merk, Darmstadt, Germany; 4× concentrate). Protein content obtained and of the apyrase was measured by BCA (Bicinchoninic Acid) protein assay (ThermoFisher Scientific Inc., IL, U.S.A.). For the enzymatic assay, the protein concentration of both enzymes was adjusted to 0.03 mg/ml (concentration obtained in the SolCD39 enzyme). The reaction was started by the addition of 10 μl of the enzymes in 190 μl IM contain ATP (final concentration of 2 mM) at 37°C during 10 min. At the end, the reaction was added 200 μl TCA (5% w/v) previously placed on ice and the release of inorganic phosphate (Pi) was evaluated as previously described [33,34]. ATP hydrolysis is expressed as nmol Pi/min/mg protein.

RNA extraction, cDNA synthesis and RT-PCR analysis

Total RNA from cells was isolated with Trizol reagent (Life Technologies, Carlsbad, CA, U.S.A.) in accordance with the manufacturer's instructions. Complementary DNA was synthesized with M-MLV reverse transcriptase (Promega, Madison, WI, U.S.A.) from 1 μg of total RNA according to the manufacturer's instructions.

To real-time PCR, the samples were prepared in 12.5 μl (final volume) which was composed by 6.25 μl SYBR Green Master Mix (Applied Biosystems, CA, U.S.A.), 0.2 μl primer pair solution (0.2 μM final concentration of each primer; Supplementary Table S1), 5.05 μl of water and 1 μl of diluted cDNA. Real-time PCRs were carried out in the Applied Biosystem Step One Plus Real-Time PCR cycler and performed in duplicates. The relative gene expression values were determined using the formula as described previously [35] and GAPDH was used as housekeeping gene for normalization.

Migration, adhesion and proliferation analysis

To evaluate in vitro cell characteristics after cell transfection, all cells were characterized for migration, adhesion and proliferation potential. In brief, for migration, the cells were analyzed by the scratch wound healing method. The cells were plated in 24-well culture plates and grown incomplete medium with 1% FBS until reaching 90% of confluence. Then, the monolayer was scratched using a 200 μl sterile plastic pipette tip and washed with PBS. Cells were kept at 37°C, humidity of 95% and 5% CO2 in air and the scratch wound closure was monitored by phase microscopy at 0, 6, 12 and 24 h using a 40× magnification. To evaluate the role of ATP and its metabolites, cells were allowed to produce soluble enzymes overnight and after scratched the cell monolayer, 100 µM or 1 mM ATP was added at time zero (without exchanging the culture medium to ensure the presence of the secreted enzymes) and the scratch wound closure analyzed as described.

To the adhesion assay, cells were seed at a density of 3 × 104 cells/well in 96-well plates and incubated for 1 h at 37°C with a 5% CO2-enriched atmosphere. The non-adherent cells were removed by washing with PBS. Adherent cells were fixed with 4% paraformaldehyde (PFA) for 10 min and stained for 10 min with 100μl 0.5% crystal violet diluted in 20% methanol. The cells were washed again and the stain was eluted in 100 μl 10% acetic acid (v/v). Cell adhesion was analyzed by measuring optical density (OD) at 570 nm in a microplate reader.

To measure proliferation rates in a long-term culture, cells were evaluated by the cumulative population doublings (cPD) method. Cells were plated in 12-well plates at a concentration of 1 × 104 cells/well and when reached cellular confluence were passaged, and PDs were determined according to the formula PD = [log2N(t) − log2N]. N(t) is the number of cells per well at the time of passage and N(t0) is the number of cells seeded at the previous passage. The sum of PDs was then plot against the time of culture. Cells were followed until day 20. Sulforhodamine B (SRB) assay was used to determine the cell proliferation and an ATP dose curve was used (1, 10, 100 and 1000 μM ATP). Briefly, cells were seemed and permitted to adhere with complete culture medium. Next, cells were starved by reducing FBS to 0.5% overnight to concentrate the soluble enzymes and added ATP at time zero and evaluated in 6, 12, 24 and 48 h. The control cells were submitted to only DMEM 0.5% FBS or DMEM 10% FBS. The cells were fixated with 50% (wt/vol) TCA at 4°C for 30 min, washed, air-dry at room temperature and stained with 0.4% (wt/vol) SRB solution by 30 min. The SRB was washed with 1% (vol/vol) acetic acid and 100 μl of 10 mM Tris base solution (pH 10.5) was added to each well and shacked on an orbital shaker for 10 min to solubilize the protein-bound dye. The SRB was measured in the absorbance at 510 nm in a microplate reader.

Flow cytometry analysis

Immunophenotypic characterization of cells for purinergic system enzymes was performed by flow cytometry using the following antibodies: rabbit or Guinea Pig anti-rat NTPDase1 (rN1-6LI5), NTPDase2 (rN2-6L), NTPDase3 (rN3-1LI5), CD73 (rNu-9LI5) and NPP1 (mNPP1-2cL5) (http://ectonucleotidases-ab.com). For staining, at least 1 × 106 cells were washed once with PBS and incubated with primary antibody (1:200) followed by 30 min incubation with anti-rabbit or Guinea Pig secondary antibody Alexa Fluor 488, with a minimum of two washes after each incubation. At least 10 000 events were acquired on a FACS Calibur flow cytometer (Becton Dikinson, CA, EUA).

Western blot

Protein was concentrated from the supernatant of Control, SolCD39, SolCD39 (E174A) and Empty vector cells. Briefly, cells were cultured in 100 mm plates until reaching confluence (∼9 × 106 cells). Next, cells were washed with IM (see section Ectonucleotidases assay) and incubated with 10 ml of this IM for 6 h. The supernatant was collected, centrifuged at 14 000 g for 5 min to remove non-adhered cells and then concentrated with Amicon Ultra-15 (10 kDa cutoff) centrifugal filter unit (Merck Millipore). To cell lysate, cells were homogenized in RIPA buffer containing 50 mM (pH 7,4) Tris–HCl, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.25% deoxycholic acid and 1% NP-40 (Merck Millipore). Lysates were centrifuged at 16 000 g for 20 min at 4°C, and supernatants were used for the assays. Protein content was measured by BCA (Bicinchoninic Acid) protein assay (ThermoFisher Scientific Inc., IL, U.S.A.).

Proteins were separated in 10% SDS-polyacrylamide gel, in which 50 µl of each protein from the supernatant and 20 µg from cell lysate were used, followed by electrotransfer to a PVDF membrane. The standard molecular mass marker (Novex Sharp Pre-stained Protein Standard, ThermoFisher Scientific Inc., IL, U.S.A.) was used. Membranes were stained with Coomassie R-250, imaged and a representative band was used as a loading control. Afterwards, the membrane was blocked by incubation with Tris-buffered saline containing 0.1% Tween 20 and 1% BSA for 2 h at room temperature and then incubated overnight at + 4°C with rabbit anti-rat CD39 antibody (dilution 1:500) (19229-1-AP, Proteintech, Rosemont, U.S.A.). Primary antibody was detected by secondary antibody (1:10 000, for 2 h) using ECL western blotting substrate (ThermoFisher Scientific Inc., U.S.A.) and ChemiDoc XRS+ system with Image Lab software (Bio-Rad Laboratories, CA, U.S.A.).

Statistical analysis

Student's t-test was performed to compare two groups while one-way or two-way ANOVA with post hoc Bonferroni was used to compare three or more groups. Results were considered significant when P < 0.05 (*), P < 0.001 (**) and P < 0.0001 (***).

Results

Efficient PiggyBac-mediated transposition of the soluble enzymes

Initially, we performed cell transfection with transposon PiggyBac-based vectors (Figure 1A) in C6 cells, which are key cells to investigate the mechanisms involved in the progression of in vitro and in vivo glioblastoma, brain tumors that are strongly influenced by the purinergic signaling [3639].

PiggyBac transposon system and expression on transfected cells.

Figure 1.
PiggyBac transposon system and expression on transfected cells.

(A) Schematic representation of transposon constructs. Donor constructs were driven by EF1A promoter, followed by genes of Entpd1 (SolCD39) or Mutated Entpd1 (SolCD39 (E174A)) and iRFP-713. The Empty vector construct contained only iRFP sequence and all vectors had neomycin resistance cassette. (B) Representative flow cytometry histograms showing the presence of iRFP-713 fluorochrome in all cells after cell sorting. Non-transfected cells were used as control. (C) Gene expression analysis by real-time PCR demonstrated the increase in Entpd1 mRNA in SolCD39 and SolCD39 (E174A) cells, in comparison with control and Empty vector cells. (D) Western blot of NTPDase1/CD39 in Control, SolCD39, SolCD39 (E174A) and Empty Vector cells. Albumin was also used as a negative control to antibody. The loading control (LC) shows representative bands of the Coomassie-stained membrane. Results of gene expression were expressed as mean ± SD of three independent experiments carried out in duplicate. One-way analysis of variance with a post hoc test of Bonferroni's was used (*) P < 0.05.

Figure 1.
PiggyBac transposon system and expression on transfected cells.

(A) Schematic representation of transposon constructs. Donor constructs were driven by EF1A promoter, followed by genes of Entpd1 (SolCD39) or Mutated Entpd1 (SolCD39 (E174A)) and iRFP-713. The Empty vector construct contained only iRFP sequence and all vectors had neomycin resistance cassette. (B) Representative flow cytometry histograms showing the presence of iRFP-713 fluorochrome in all cells after cell sorting. Non-transfected cells were used as control. (C) Gene expression analysis by real-time PCR demonstrated the increase in Entpd1 mRNA in SolCD39 and SolCD39 (E174A) cells, in comparison with control and Empty vector cells. (D) Western blot of NTPDase1/CD39 in Control, SolCD39, SolCD39 (E174A) and Empty Vector cells. Albumin was also used as a negative control to antibody. The loading control (LC) shows representative bands of the Coomassie-stained membrane. Results of gene expression were expressed as mean ± SD of three independent experiments carried out in duplicate. One-way analysis of variance with a post hoc test of Bonferroni's was used (*) P < 0.05.

After transfection, cells were selected by cell sorting for the iRFP-713 fluorochrome, in order to ensure that only cells producing the soluble enzymes were subsequently analyzed. Cell purity was determined by flow cytometry, which confirmed that more than 90% of cells expressed the iRFP-713 fluorescent protein (SolCD39 = 93.3%, SolCD39 (E174A) = 97.2% and Empty Vector = 90.1%; Figure 1B). In addition, the cells transfected with the SolCD39 and SolCD39 (E174A) vectors showed a significant increase in Entpd1 mRNA when compared with control and Empty vector cells (Figure 1C). Finally, to verify that a soluble form of NTPDase1 was secreted, we concentrated the proteins from cell supernatant and evaluated by western blot, which demonstrated the presence of soluble NTPDase1/CD39 enzyme in both SolCD39 and SolCD39 (E174A) cells (Figure 1D). Most importantly, we detected a high protein concentration from the supernatant concentrate, with ∼0.02 mg/ml from of ∼9 × 106 cells to both SolCD39 and SolCD39 (E174A) cells, while control cells (C6 cells and Empty vector) showed levels almost undetectable. NTPDase1/CD39 protein also was detected only in cell lysate from SolCD39 and SolCD39 (E174A) cells (Supplementary Figure S1).

The immunofluorescence images reveal that iRFP-713 fluorescent protein was located throughout the cellular cytoplasm (Figure 2). In addition, as the NTPDase1/CD39 has already been associated with the modulation of cell migration and proliferation in other cell types [40,41], we evaluated these parameters after insertion of the soluble enzyme gene. We observed no changes in cell migration by scratch wound healing method (Figure 3A), proliferation by population doubling assay (Figure 3B) and adhesion (Figure 3C). These results demonstrated that transfected cells in vitro maintained the same characteristics of their wild type counterparts, meaning that neither the transfection protocol nor the NTPDase1/CD39 expression affected the cell biology parameters.

iRFP-713 immunofluorescence in transfected C6 cells.

Figure 2.
iRFP-713 immunofluorescence in transfected C6 cells.

Representative images of C6 cell culture after transfection showed that iRFP-713 fluorescent protein was located throughout the cellular cytoplasm. All images presented are at ×400 magnification.

Figure 2.
iRFP-713 immunofluorescence in transfected C6 cells.

Representative images of C6 cell culture after transfection showed that iRFP-713 fluorescent protein was located throughout the cellular cytoplasm. All images presented are at ×400 magnification.

Characterization of cell biology parameters in transfected cells.

Figure 3.
Characterization of cell biology parameters in transfected cells.

Cell migration (A), proliferation (B) and adhesion (C) analysis of control and transfected cells. Results were expressed as mean ± SD of three independent experiments carried out in triplicate.

Figure 3.
Characterization of cell biology parameters in transfected cells.

Cell migration (A), proliferation (B) and adhesion (C) analysis of control and transfected cells. Results were expressed as mean ± SD of three independent experiments carried out in triplicate.

Transfected cells secreted an enzymatically active soluble NTPDase1/CD39

Next, we investigated the functionality of the recombinant enzyme secreted by the transfected cells through their capacity to hydrolyze ATP and ADP (Figure 4A). For this, 180 µl IM obtained from ∼5 × 105 cells of each group was incubated with the nucleotides for 20 min, as described in the Material and methods section. The enzymatic activity from the cell supernatant demonstrated that SolCD39 cells secreted an enzyme with ATPase activity of 4.5 ± 0.8 nmol Pi/min/ml and ADPase of 2.5 ± 0.3 nmol Pi/min/ml, producing an ATPase/ADPase ratio of ∼2:1 (Figure 4B). This enzymatic activity was ∼5 and 3-fold higher than control cells for the degradation of ATP and ADP, respectively. In addition, the enzymatic activity observed in the supernatant of the cells transfected with the SolCD39 (E174A) enzyme was similar to that of the control and empty vector transfected cells, confirming the loss of enzymatic activity as expected.

Nucleotides catabolism by the soluble CD39 enzyme from cell supernatants.

Figure 4.
Nucleotides catabolism by the soluble CD39 enzyme from cell supernatants.

(A) Schematic representation of enzymatic activity assay from the cell supernatant. The detailed protocol was described in the Material and methods section. (B) Enzymatic specific activities from the supernatant of control and transfected cells measured by release of inorganic phosphate after incubation with ATP or ADP (Malachite Green) (n = 5). Data are express as nmol Pi/min/ml. (C) Kinetics of the nucleotides and nucleosides in the absence or presence of soluble enzymes from cell supernatant. Cell supernatant was incubated with 100 µM ATP, an aliquot was withdrawn at 0, 1, 10, 15, 30, 60, 120 and 180 min and the presence of ATP, ADP, AMP, ADO, inosine and hypoxanthine components were determined after separation by HPLC. Data are represented as µMol of nucleotide and expressed as mean ± SD of three independent experiments carried out in duplicate, ***P < 0.001. (Two-way ANOVA followed by Bonferroni's multiple comparison post hoc test.)

Figure 4.
Nucleotides catabolism by the soluble CD39 enzyme from cell supernatants.

(A) Schematic representation of enzymatic activity assay from the cell supernatant. The detailed protocol was described in the Material and methods section. (B) Enzymatic specific activities from the supernatant of control and transfected cells measured by release of inorganic phosphate after incubation with ATP or ADP (Malachite Green) (n = 5). Data are express as nmol Pi/min/ml. (C) Kinetics of the nucleotides and nucleosides in the absence or presence of soluble enzymes from cell supernatant. Cell supernatant was incubated with 100 µM ATP, an aliquot was withdrawn at 0, 1, 10, 15, 30, 60, 120 and 180 min and the presence of ATP, ADP, AMP, ADO, inosine and hypoxanthine components were determined after separation by HPLC. Data are represented as µMol of nucleotide and expressed as mean ± SD of three independent experiments carried out in duplicate, ***P < 0.001. (Two-way ANOVA followed by Bonferroni's multiple comparison post hoc test.)

HPLC analyzes showed that the supernatant of the SolCD39 cells completely hydrolyzed 100 µM of ATP in 1 min, leading to the accumulation of AMP and a small increase in ADO (Figure 4C).

Apyrase from potato is a potent enzyme widely used to modulate eATP levels in vitro and in vivo studies. We compared the specific activity of the apyrase enzyme with our SolCD39 enzyme. As expected, apyrase from potato showed a high ATP nucleotide degradation rate (51 403 ± 680.4 nmol Pi/min/mg of protein), while SolCD39 exhibited a specific activity of 1014 ± 21.1 nmol Pi/min/mg of protein (Supplementary Figure S2).

Characterization of purinergic signaling after insertion of soluble CD39

NTPDase1/CD39 is known as an enzyme exclusively present and active on the cell membrane [42]. Therefore, we investigated whether the presence of a recombinant soluble form could alter other enzymes that metabolize nucleotides and nucleosides. First, we evaluated by gene expression of other enzymes responsible for the hydrolysis of ATP and ADP (Supplementary Table S1). As shown in Figure 5A, the Entpd3, 5 and 6 mRNA levels were similar between transfected and control cells. The Entpd2 and Entpd8 showed no detectable levels in the RT-qPCR (Figure 5A). The ecto-5′-nucleotidase (CD73) mRNA level was also not altered. In addition, we did not observe significant differences in the gene expression of Npp enzymes (Figure 5B).

Gene expression of Entpd and Npp enzymes in transfected and control cells.

Figure 5.
Gene expression of Entpd and Npp enzymes in transfected and control cells.

(A) Quantitative RT-PCR shows similar gene expression levels of main Entpdases responsible for ATP and ADP degradation. ND, not detectable. (B) No statistical difference was observed comparing Npp gene expression. Quantitative PCR measurements of gene expression levels were normalized against GAPDH levels and determined using the formula as described in [35]. The ANOVA test was used to determine the statistical difference. All experiments were repeated at least three times.

Figure 5.
Gene expression of Entpd and Npp enzymes in transfected and control cells.

(A) Quantitative RT-PCR shows similar gene expression levels of main Entpdases responsible for ATP and ADP degradation. ND, not detectable. (B) No statistical difference was observed comparing Npp gene expression. Quantitative PCR measurements of gene expression levels were normalized against GAPDH levels and determined using the formula as described in [35]. The ANOVA test was used to determine the statistical difference. All experiments were repeated at least three times.

To verify the expression of these proteins at the cell surface, we performed flow cytometry analyzes. The representative histograms show that the transfected cells did not express any of the main membrane-bound NTPDases (1, 2 and 3) and expressed CD73 protein. In agreement with the qRT-PCR, the cells expressed NPP1 on its surface (Figure 6).

Characterization of membrane-bound purinergic enzymes on transfected cells.

Figure 6.
Characterization of membrane-bound purinergic enzymes on transfected cells.

Representative histograms of the NTPDases 1, 2, 3, CD73 and NPP1 from control and transfected cells. Gray histograms are cells incubated only with secondary antibody. Transfected C6 cells with membrane NTPDase1 and rat astrocytes were used as a positive control to NTPDase 1 and NTPDase 2 antibodies, respectively, as demonstrated in inserts in each histogram.

Figure 6.
Characterization of membrane-bound purinergic enzymes on transfected cells.

Representative histograms of the NTPDases 1, 2, 3, CD73 and NPP1 from control and transfected cells. Gray histograms are cells incubated only with secondary antibody. Transfected C6 cells with membrane NTPDase1 and rat astrocytes were used as a positive control to NTPDase 1 and NTPDase 2 antibodies, respectively, as demonstrated in inserts in each histogram.

These results are in accordance with the specific enzymatic activity measured on intact cells (Figure 7A) which demonstrated the hydrolysis of AMP and low hydrolysis levels of ATP and ADP in all transfected cells (Figure 7B). This profile of nucleotide hydrolysis by C6 glioma cells correlated with our previously published data [37].

Analysis of nucleotides metabolism on C6 cells transfected with SolCD39.

Figure 7.
Analysis of nucleotides metabolism on C6 cells transfected with SolCD39.

(A) Membrane-bound ectonucleotidase activity assays. (B) The control and transfected cells exhibited low ATPase and ADPase activities and higher AMP hydrolysis. The result was expressed as specific activity (nmol Pi/min/mg of protein). (C) To evaluate the ATP metabolism by membrane-bound ectonucleotidases, when soluble CD39 was present, 100 µM of ATP was added in cell supernatant, after concentrating the soluble enzyme for 6 h. (D) An aliquot was withdrawn at 0, 30, 60, 120 and 180 min. The presence of nucleotides/nucleosides was determined by HPLC and presented as µMol of nucleotide. Data are expressed as mean ± SD of three independent experiments carried out in duplicate.

Figure 7.
Analysis of nucleotides metabolism on C6 cells transfected with SolCD39.

(A) Membrane-bound ectonucleotidase activity assays. (B) The control and transfected cells exhibited low ATPase and ADPase activities and higher AMP hydrolysis. The result was expressed as specific activity (nmol Pi/min/mg of protein). (C) To evaluate the ATP metabolism by membrane-bound ectonucleotidases, when soluble CD39 was present, 100 µM of ATP was added in cell supernatant, after concentrating the soluble enzyme for 6 h. (D) An aliquot was withdrawn at 0, 30, 60, 120 and 180 min. The presence of nucleotides/nucleosides was determined by HPLC and presented as µMol of nucleotide. Data are expressed as mean ± SD of three independent experiments carried out in duplicate.

To further investigate the pattern of ATP hydrolysis in these cells, after the insertion of SolCD39, we also evaluated the ATP metabolism by membrane-bound enzymes in the presence of soluble NTPDase1/CD39. For this, the soluble enzyme was firstly concentrated from the supernatant of a cell monolayer for 6 h and then the nucleotide was added, as represented in the scheme of Figure 7C. The degradation metabolites from ATP were measured by HPLC. Similarly to previous results, it demonstrated that SolCD39 cells were able to rapidly degrade 100 µM of ATP, leading to accumulation mainly of ADO and hypoxanthine at the end of 3 h (Figure 7D). On the other hand, the control cells slowly hydrolyzed ATP, with ∼50% degradation in 2 h, but similarly to SolCD39 cells, accumulating ADO and hypoxanthine at the end of 3 h.

ATP did not significantly affect proliferation or migration in the control or transfected cells, suggesting that the differences in the nucleotide and nucleoside profile do not have an impact on proliferation and migration potential (Supplementary Figure S3).

Discussion

The knowledge about the purinergic signaling has opened up new treatment opportunities to a wide variety of diseases. These advances are partly due to the development of important tools that allow the modulation of purinergic agents such as eATP, ADO and other extracellular nucleotides with engineered ectonucleotidases, interference RNAs (SiRNAs) and ectonucleotidase inhibitors [43].

The goal of this study was to produce a stable rat soluble CD39 in cells also expressing an iRFP-713 fluorescent protein and optimize the cell expression of such construct by using the PiggyBac transposon non-viral system. Cells expressing the plasmids were analyzed and the produced enzymes were enzymatically characterized. Also, we evaluated its interaction with purinergic signaling on the cells used.

The NTPDase1/CD39, together with CD73, are the main membrane-bound enzymes responsible for controlling the levels of eATP and ADO [5]. Thus, the soluble form of NTPDase1 (SolCD39) appears as an important tool to modulate the eATP in substitution for the commercial apyrase enzyme, which has recently had its effects questioned and demonstrating the need for dialysis to decrease K+ contamination [14]. The SolCD39 has already been used for some years as a potent platelet aggregation inhibitor [17,18,23]. However, limitations such as short half-life and need of high doses to reach the expected enzymatic activity, mainly on in vivo models, restricts its use [15,26,44]. In this context, another limitation is the putative immunostimulation by proteins produced by very different species that could affect the biological alterations produced. Indeed, it was shown that additional injections of human SolCD39 in mouse induced a neutralizing immune response against SolCD39 [26], demonstrating the need to use enzymes of the same species mainly in the in vivo studies.

Although viral vectors continue to be widely used for gene transfer, non-viral methods have gained space due to their numerous advantages. We chose the PiggyBac transposon system, which are discrete elements of DNA that have the distinctive ability to move from one chromosomal location to another [45]. Whereas viral methods require packaging cells, demanding time and cost, this method is based on ‘cut and paste’ mechanism. The delivery of the target sequence is done together with the transposase in a single step, reducing costs, time, side effects and immunogenicity that are inherent to viral methods [29]. Furthermore, gene transfer based on the PiggyBac system enables greater cargo capacity (up to 15 kb), it has a different genomic target sequence, possibility of seamless excision of the transposon by transposase and does not suffer from overproduction inhibition, which are especially interesting for generation of cultured cells stably expressing one or more recombinant gene sequences [30].

Indeed, we used a construct that had in addition to the target genes [SolCD39 and SolCD39 (E174A)], a selection gene and a fluorescent protein (iRFP-713), which may be useful to in vivo experiments, generating a vector of ∼11 kb. Despite its large size, more than 70% of the cells expressed the iRFP-713 fluorescent protein after antibiotic selection (data not shown), reaching almost 100% purity after cell sorting. In addition, we observed a significant increase in the NTPDase1/CD39 gene expression after stable transfection, which was corroborated by the detection of the high amount of the protein in cell supernatant. Other methods have reached the production of ∼0.001–0.003 mg SolCD39/ml in CHO cells after purification [15], while using a centricon system, we observed a concentration of 0.02 mg SolCD39/ml. More important is that the enzyme produced has a high capacity to hydrolyze ATP and ADP. When compared with apyrase from potato, although the latter has a 50× greater activity, the SolCD39 enzyme showed a high-specific activity (>1000 nmol Pi/min/mg protein). This value was comparable to the ATP hydrolyzes rate measured in the surface of rat astrocytes, which is one of the highest activity described in biological samples [33]. In addition, the enzymatic assay was corroborated by HPLC analysis, which demonstrated that 100 µM ATP was degraded in less than 1 min when in contact with the SolCD39 enzyme. Therefore, although studies show that withdrawal of the transmembrane domains affects the enzymatic activity of NTPDase1/CD39 [4648], we produced here an enzymatically active enzyme with high hydrolysis potential.

The catalytic activity is undoubtedly the main function associated with the enzymes of the purinergic system. However, non-enzymatic functions were described for some of these enzymes, such as CD73, which is involved, for example, with T-cell activation and cell–cell adhesion [49,50]. Similarly, NTPDase1/CD39 may exhibit functions beyond its enzymatic activity, by physical interactions with other molecules, such as RanBPM scaffolding protein, which is associated with regulation of some cellular processes as apoptosis, cell adhesion and migration [28,51]. In spite of these reports, we did not observe cellular alterations in C6 cells after overexpression of SolCD39 or SolCD39 (E174A), demonstrating that its structure did not compromise parameters such as cell proliferation, migration or adhesion.

Previous studies have already investigated associations between the components of purinergic signaling through FRET microscopy, revealing potential combinations among purinergics receptors and enzymes, such as the possibility of interaction between NTPDase1 and NTPDase2 [52,53]. Thus, we wondered if overexpression of a soluble form of NTPDase1/CD39 could lead to changes in the expression and function of the other enzymes of the purinergic system since protein overexpression might lead to the presence of others proteins in the membrane, which would not appear under native conditions [52].

Our results of gene expression analysis did not reveal significative differences to NTPDases and NPPs mRNA in cells after the overexpression of SolCD39. Similarly, flow cytometry experiments proved that there were no changes in the expression profile of the purinergic enzymes. Furthermore, C6 cells are known to have a slow degradation rate of the ATP and ADP nucleotides, mainly due to the low expression of NTPDase1 and NTPDase2, and high expression and hydrolytic activity of CD73 [37,54]. This hydrolysis profile of the membrane-bound enzymes was maintained after overexpression of SolCD39 and SolCD39 (E174A), demonstrating that neither the catalytic activity nor the structure of the SolCD39 promoted changes in the profile of the purinergic enzymes. On the other hand, when the secreted enzymes were maintained during the enzymatic activity assay, cells secreting SolCD39 quickly exhibited a peak of ADO, while SolCD39 (E174A) cells showed the same profile of control cells.

Several lines of evidence highlight the importance of ADO in tumor progression, mainly due to its influence in the generation of an immunosuppressive environment, recruiting tumor-infiltrating immune cells, such as MDSCs, macrophages type M2 and TRegs, and preventing an efficient response of natural killer (NK), dendritic cells and effector T cell. At the same time, studies suggest that Ado can stimulate tumor cells proliferation, migration and angiogenesis [55,56]. Despite all this evidence, in our in vitro analysis, we did not see a significant difference in proliferation and migration potential after the addition of exogenous ATP and generation of ADO by the enzymes produced by the cells. Indeed, likewise eATP, Ado may show pro- or anti-tumor effects depending on its levels, expressed receptor and tumor type [5]. Although the in vitro results only reflect the direct contribution of the cancer cells in terms of proliferation and migration, the in vivo role of Ado in the tumor microenvironment complexity has to be taken into account. Therefore, SolCD39 will be an important tool to investigate and determinate of the roles of eATP and Ado in the tumor microenvironment in different tumor types. Any potential therapeutic use of this tool has to consider the increase in the concentration of Ado, which is an immunosuppressor and can have pro-tumor effects.

In conclusion, our data demonstrated that the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other purinergic enzymes. In addition, the PiggyBac non-viral system proved to be an extremely efficient, fast and cheap method to generate cells stably producing high amounts of the SolCD39 enzyme and with a high-specific activity. The possibility of using cells constantly producing this enzyme in in vivo studies, without the need to purify the enzyme, without repeated applications and with the advantage of performing excision of the transposon, being able to remove the enzyme and its constant catalytic activity even in vivo, makes this system extremely interesting. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.

Abbreviations

     
  • ADO

    adenosine

  •  
  • ADP

    adenosine diphosphate

  •  
  • AMP

    adenosine monophosphate

  •  
  • eATP

    extracellular adenosine triphosphate

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • FBS

    fetal bovine serum

  •  
  • FACS

    fluorescence-activated cell sorting

  •  
  • IM

    incubation medium

  •  
  • cPD

    cumulative population doublings

  •  
  • TCA

    trichloroacetic acid

  •  
  • iRFP-713

    phytochrome-based near-infrared fluorescent protein 713.

  •  
  • NTPDases

    ecto-nucleoside triphosphate diphosphohydrolase

  •  
  • SRB

    sulforhodamine B

  •  
  • SolCD39

    soluble NTPDase1/CD39

Author Contribution

L.R.B. performed stable transfection cell, cell culture experiments, HPLC assays and wrote the manuscript. G.R.O. assisted the stable transfection cell assays. I.C.I. and D.M.F. performed enzymatic assay and cell migration, adhesion and doubling population experiments. A.P.S.B. performed the western blot assay. J.S. contributed to the interpretation of the results and provided the NTPDases antibodies. M.R.W. and G.L. designed and supervised the experiments, assisted in drafting and critical reading. All the authors discussed the results and contributed to the writing of the manuscript.

Funding

All students are recipients of fellowships from CAPES (Coordenação de Aperfeicoamento de Pessoal de Nível Superior) and M.R.W. and G.L. are recipients of research fellowship from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). This study was supported by CAPES, PROCAD (158819); ICGEB (405231/2015-6 MCTI/CNPq-ICGEB); and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS — Pronex16/2551-0000473-0). J.S. received support from the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN-2016-05867) and he was also the recipient of a ‘Chercheur National’ research award from the Fonds de recherche du Québec – Santé (FRQS).

Acknowledgements

The authors thank Dr José Artur Bogo Chies, Drª. Jacqueline Valverde-Villegas and Valéria de Lima Kaminski (Laboratório de Imunogenética/UFRGS) for their assistance with cell sorting analysis. The authors also gratefully acknowledge Dr. Fabricio Figueiro and Juliete Nathali Scholl (Departamento de Bioquímica/UFRGS) for assistance with NPP1 cytometry analysis.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Supplementary data