help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Katzur, A. C.
Right arrow Articles by Stojilkovic, S. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Katzur, A. C.
Right arrow Articles by Stojilkovic, S. S.
The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 11 4085-4091
Copyright © 1999 by The Endocrine Society

Original Studies

Expression and Responsiveness of P2Y2 Receptors in Human Endometrial Cancer Cell Lines

Ann C. Katzur, Taka-Aki Koshimizu, Melanija Tomic, Askan Schultze-Mosgau, Olaf Ortmann and Stanko S. Stojilkovic

Endocrinology and Reproduction Research Branch (A.C.K., T.-A.K., M.T., S.S.S.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Department of Obstetrics and Gynecology (A.S.-M., O.O.), University of Lubeck, D-23538 Lubeck, Germany

Address correspondence and requests for reprints to: Stanko Stojilkovic, Ph.D., Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In single endometrial carcinoma HEC-1A and Ishikawa cells, ATP induced a rapid and extracellular Ca2+-independent rise in cytosolic Ca2+ concentration ([Ca2+]i) in a dose-dependent manner, with an ED50 of about 10 µM. The spike phase was followed by a sustained plateau phase that was dependent on Ca2+ influx through voltage-insensitive Ca2+ channels, whose gating was controlled by a capacitative Ca2+ entry mechanism. ADP was less potent in raising the cystolic Ca2+ concentration, and AMP and adenosine were ineffective. The order of agonist potency for this receptor was ATP = UTP > ATP-{gamma}-S>>ADP. Several other agonists, including ß,{gamma}-methylene-ATP, 2-MeS-ATP, and BzATP were ineffective. This ligand-selective profile indicates the expression of the P2Y2R subtype in endometrial cells. Accordingly, reverse transcription-PCR using P2Y2 primers amplified the expected transcript from both cell lines. The coupling of these receptors to phospholipase C was confirmed by the ability of ATP to increase inositol 1,4,5-trisphosphate and diacylglycerol productions. These receptors are also coupled to the phospholipase D-1 pathway, leading to accumulation of phosphatidic acid. Activation of P2Y2 receptors by a slowly degradable ATP analog, ATP-{gamma}-S, was associated with a significant suppression of cell proliferation without affecting the cellular apoptosis. These results indicate that P2Y2 receptors may participate in control of the cell cycle of endometrial carcinoma cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ADDITION to its many intracellular functions, ATP and its degradable products act as extracellular messengers for three classes of purinergic (P) receptors: P2Y, P2X, and P1. Activation of P2Y receptors leads to phospholipase C-derived production of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG), followed by InsP3-induced Ca2+ mobilization and activation of protein kinase C (1). The members of the P2X family are plasma membrane channels permeable to Ca2+, and their activation by ATP also leads to an increase in cytosolic Ca2+ concentration ([Ca2+]i) (2). P2 receptors and receptor-channels are expressed in many cell types, including cells that cosecrete ATP along with hormones and neurotransmitters by exocytosis (2, 3, 4). The extracellular actions of ATP through P2Y and P2X receptors are controlled by ectonucleotidases, a family of enzymes that includes ecto-ATPase, ecto-ADPase, and 5'-ectonucleotidase (5). Activation of these enzymes leads to degradation of extracellular ATP to adenosine, which subsequently may act as an agonist controlling the activity of P1 receptors (6). Adenosine receptors are a complex family of proteins coupled to adenylyl cyclase and phospholipase C pathways (7).

In normal tissues expressing P receptors, ATP is locally secreted, and its actions are terminated by rapid degradation. For example, ATP is secreted by some neurons and acts as a neurotransmitter in synapses, where it is degraded by ectoATPases (8). ATP can also be released in a nonphysiological manner, such as at sites of tissue injury or inflammation. In carcinoma tissues, ATP is released during the chemotherapeutic and radiation control of tumor growth, and ATP measurements are commonly used as indicators of the efficacy of these treatments (9). Although ATP may act as an extracellular messenger, very little is known about the expression of P receptors and ectonucleotidases in carcinoma tissue. Furthermore, the coupling of P receptors (positive or negative) to cell proliferation and apoptotic cycles, as well as the identification of intracellular messenger(s) mediating this coupling, has not been studied extensively.

In this study, we used HEC-1A and Ishikawa human endometrial cancer cell lines as cell models in investigations of the possible effects of extracellular ATP on cell growth. These cells express calcium-mobilizing GnRH receptors (10, 11). In normal tissue, GnRH receptors are coupled to phospholipase C through a Gq/11 protein, as well as to the phospholipase D pathway through protein kinase C (12). In HEC-1A and Ishikawa cells, activation of GnRH receptors by GnRH analogs leads to inhibition of cell proliferation in vitro through a still uncharacterized pathway (10, 11). The focus of our study is the identification of P2 receptor subtypes expressed in these cells, characterization of the coupling of these receptors to intracellular signaling and cell cycle, and control of receptor activities by ectonucleotidases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

Fura-2 AM was obtained from Molecular Probes (Eugene, OR), suramin and adenosine-5'-O-(1-thiotriphosphate) were from Calbiochem (San Diego, CA), and pyruvate kinase and myokinase were from Boehringer Mannheim (Indianapolis, IN). TRIZOLE reagent, Superscript II reverse transcriptase, oligo(dT)18 primer, and PCR reagent system were from Gibco BRL (Gaithersburg, MD), and pBluescript II vector was from Stratagene (La Jolla, CA). Sequenase version 2.0 was from Amersham Corp. (Arlington Heights, IL). [{gamma}-32P]ATP (3000 Ci/mmol), [9,10-3H]oleic acid (10 Ci/mmol), and [5-3H]cytidine and Econofluor-2 were purchased from DuPont-New England Nuclear (Boston, MA). myo[3H]Inositol (60 Ci/mmol) was from Amersham Corp., and sn-1,2-diacylglycerol kinase of Escherichia coli was from Lipidex Inc. (Wastfield, NJ). Bovine heart cardiolipin and 1,2-dioleoyl-sn-glycero-3-phosphoethanol were from Avanti Polar-Lipids Inc. (Alabaster, AL), and ATP and 1-o-n-octyl-ß-D-glucopyranoside were from Boehinger Mannheim. Inositol-free medium 199 with Hanks’ salt solution was prepared by the NIH Media Unit (Bethesda, MD). Silica gel 60 thin-layer chromatography (TLC) plates were from Merck (Darmstadt, Germany). Organic reagents for TLC were high-pressure liquid chromatography grade and from J. T. Baker (Phillipsburg, NJ), and liquid scintillation solution (Hydrofluor) was from National Diagnostics (Manville, NJ). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).

Endometrial cell culture

Experiments were performed on endometrial HEC-1A and Ishikawa cancer cell lines. Cells were cultured in minimum essential medium Eagle (Sigma Chemical Co.) containing 10% fetal calf serum (Gibco, Rockville, MD), 40 I.E./L human insulin, 2.5 mg/l transferin, and antibiotics. Cultures were incubated at 37 C in a fully humidified atmosphere of 5% carbon dioxide in air. Media was changed every 2 days. When the cells reached confluence, they were subcultured by trypsin dissociation, using 0.05% trypsin, and plated.

Measurements of [Ca2+]i

For [Ca2+]i measurements, cells (2 x 104/dish) were plated on coverslips coated with poly-L-lysine. The next day, the cells were incubated at 37 C for 60 min with 2 µM fura-2 AM in Krebs-Ringer buffer. Coverslips with cells were washed with this buffer and mounted on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to the Attofluor Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD). Cells were examined under a x40 oil immersion objective during exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 520 nm was measured. The ratio of light intensities, F(340)/F(380), which reflects changes in Ca2+ concentration, was followed in several single cells simultaneously.

Measurements of ATP

Degradation of ATP was measured in static cell cultures, as described previously (13), using an ATP bioluminescent assay kit (Sigma Chemical Co.) in an AutoLumat LB 953 (Berthold, Wildbad, Germany) via injection of 100 µM of assay solution into an aliquot of 100 µM of sample. Calibration curves were constructed from measurements in standard solutions that were diluted in the same medium as the corresponding solutions of unknown ATP concentration. Detection limit of the assay was 0.02 nM.

RT-PCR analyses

Total RNA was isolated from endometrial cancer cell lines and rat GH3 cells using TRIZOL reagent. Total RNA (5 µg) was reverse transcribed with oligo(dT)18 primers and Superscript II reverse transcriptase according to the manufacturer’s instructions. A 5% fraction of the resulting single-strand cDNA sample was then amplified for 30 cycles with a PCR reagent system in a final volume of 12.5 µL. The primer sequences used (sense, 5'-GCTTCAACGAGGACTTCAAG-3'; antisense, 5'-CACGCTGATGCAGGTGAGGA-3') were exactly complementary to both the human P2Y2 (14) and rat P2Y2 receptors (15). The PCR conditions for each cycle were denaturation at 94 C for 1 min, annealing at 55 C for 30 sec, and extension at 72 C for 1 min, followed by a final incubation at 72 C for 10 min. PCR products were separated by an agarose gel (1%) electrophoresis and visualized with ethidium bromide. The same volume of samples used for P2Y2 receptor mRNA analysis was also subjected to a PCR reaction using glyceraldehyde-3-phosphate dehydrogenase (16)-specific primers; sequence for sense and antisense primers were 5'-GGCATCCTGGGCTACACTG-3' and 5'-TGAGGTCCACCACCCTGTT-3', respectively. The amplified PCR products were inserted into a pBluescript II vector and sequenced by the dideoxy chain termination method using Sequenase version 2.0. At least two independent clones derived from separate PCR reactions with each set of primers were sequenced.

To examine the expression level of phospholipase D-1 in endometrial cancer cells, two sets of oligonucleotide primers specific to the human phospholipase D-1a subtype (17) were designed. Nucleotide sequences for D1S1 sense and D1A1 antisense primers correspond to the first 21 and the last 20 nucleotides of human phospholipase D-1a open reading flame, respectively. An antisense primer, D1A2, has identical, but complementary, sequence to that of 1612–1631 of phospholipase D-1a. PCR was performed as D1S1 a sense primer and either D1A1 or D1A2 as an antisense primer in which temperature profiles are denatured at 94 C for 35 sec, annealing at 48 C for 45 sec, and extension at 72 C for 3 min for 30 cycles, followed by a final extension at 72 C for 10 min.

Cell proliferation and apoptotic assay

Cell proliferation was assayed by incorporation of [3H]thymidine into acid-precipitable material. Cells were plated at 2 x 105 cells/well and cultured 24 h in normal serum-containing conditions. After that, medium was removed from the cells and replaced with medium without thymidine and containing different concentrations of fetal bovine serum. [3H]thymidine was added at 5 µCi/mL, and cells were incubated for 4 h at 37 C. Incorporation of radioactivity was determined according to the method described previously (18).

For detection of DNA fragmentation, cells were plated at a density of 1.5–2 x 106 in 100-mm dishes in minimum essential medium containing 1% fetal calf serum and allowed to grow for 24 h. The ability of ATP and its analog ATP-{gamma}-S to induce apoptosis and resulting DNA fragmentation was compared with that of etoposide and tunicamycin. After the indicated period of incubation with reagents, cells were washed twice with calcium-free phosphate-buffered saline and collected in 200 µL of the same buffer. Genomic DNA was prepared by using QIA1amp blood kit (Qiagen Inc., Valencia, CA), according to manufacturer’s protocol. DNA samples (3 µg) were then separated by electrophoresis on an agarose gel (1.5%), and DNA fragments were visualized by ethidium bromide staining.

[3H]Inositol labeling and stimulation

On the 3rd day of cell culture in 4-well plates, the medium was changed to inositol-free medium 199 with Hanks’ salt solution containing 5 µCi myo[3H]inositol, NaHCO3 (1.4 g/L) and 0.1% BSA. After a 24-h incubation, the cells were washed three times with inositol-free medium 199 containing 25 mM HEPES (pH 7.4) and 0.1% BSA and were treated with 100 ATP-{gamma}-S. The radioactivity incorporated into the individual or total inositol phosphates was determined as described previously (19).

CDP-DAG assay

Cells were cultured in 4-well dishes for 2 to 3 days, as described above, and [3H]CDP-DAG formation was measured by a modification of the method of Watson and Godfrey (20). Cells were incubated in 0.45 mL DMEM containing 0.1% BSA at 37 C for 60 min with [3H]cytidine (5 µCi/mL), followed by the addition of stimuli for up to 90 min. The reactions were stopped by removal of medium and the addition of 0.5 mL dry ice-cold methanol. Cells were scraped from the plates, and lipids were extracted by vigorous vortexing with chloroform/methanol/water. After mixing with chloroform and water, the samples were centrifuged and the lower phases were transferred into new tubes and washed with methanol. Aliquots of the lipid phases containing [3H]CDP-DAG were dried under nitrogen and analyzed by liquid scintillation spectrometry after dissolving in Econofluor-2.

Phosphatidylethanol (PEt) assay

For PEt measurements, the culture medium in 35-mm culture dishes was changed to 1.1 mL DMEM containing 0.1% fatty acid-free BSA, L-glutamine, glucose (4.5 g/L), NaHCO3 (1.4 g/L), and 5 mCi [3H]oleic acid. After a 16–24 h incubation, stimuli were added to the culture dishes in the presence or absence of 0.5% ethanol for the indicated times. Treatments were terminated by placing the dishes on ice, followed by removal of the medium and rinsing the dishes with 1 mL ice-cold saline. After extraction and separation as described (19), phosphatidic acid, and PEt were visualized either by autoradiography, for which the TLC plates were treated with EN3HANCE spray, or by iodine vapor staining. The regions corresponding to the appropriate standards were scraped into scintillation vials and extracted with 1 mL methanol-HCl (150:1); hydroflour (9 mL) was added after the iodine stains were extinguished. Samples were kept at room temperature overnight, and their radioactivity was measured in a Beckman LS 9000 Liquid Scintillation Counter (Columbia, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ATP-induced Ca2+ signaling

In single HEC-1A and Ishikawa cells, ATP (100 µM) induced a rapid and sustained increase in [Ca2+]i (Fig. 1AGo). ATP was also able to increase [Ca2+]i in cultures bathed in Ca2+-deficient medium (Fig. 1BGo), suggesting that endometrial cells express a P receptor, the activation of which leads to Ca2+ mobilization from intracellular stores. The addition of thapsigargin (TG), an inhibitor of the endoplasmic reticulum (Ca2+)ATPase, increased [Ca2+]i in cells bathed in Ca2+-deficient medium (data not shown). In these cells, the subsequent addition of ATP was almost ineffective, indicating that activation of these receptors leads to a release of Ca2+ from the TG-sensitive intracellular pool [Ca2+]i ({Delta}F(340)/F(380): 100 µM ATP-treated cells = 1.89 ± 0.12 vs. ATP after 1 µM TG treatment for 30 min = 0.27 ± 0.06, P < 0.01].



View larger version (27K):
[in this window]
[in a new window]
  		Figure 1. Effects of ATP on [Ca2+]i in single endometrial carcinoma cells. A, Typical patterns of [Ca2+]i responses to 100 µM ATP in HEC-1A (left) and Ishikawa cells (right) bathed in Ca2+-containing (1.2 mM) Krebs-Ringer medium. B, Extracellular Ca2+ independence of ATP action. Extracellular Ca2+ concentration was reduced by EGTA to about 200 nM. In this and all figures, tracings shown are representative of at least 20 individual cells stimulated with ATP.

 
The pattern of ATP-induced [Ca2+]i response differs in the presence and absence of extracellular Ca2+. As shown in Fig. 1BGo, the rate of [Ca2+]i decrease occurs much faster in the absence of extracellular Ca2+, suggesting that Ca2+ entry supplements Ca2+ mobilization during the sustained agonist stimulation. Furthermore, in cells stimulated with ATP in Ca2+-deficient medium, the addition of Ca2+ during the sustained agonist stimulation was associated with a secondary increase in [Ca2+]i (Fig. 2AGo). Similarly, in TG-treated cells bathed in Ca2+-deficient medium, 1.2 mM Ca2+ induced an increase in [Ca2+]i (Fig. 2BGo). The basal [Ca2+]i (data not shown) and the ATP- and TG-induced secondary rises in [Ca2+]i were unaffected by nifedipine, a dihydropiridine calcium channel blocker (Fig. 2Go). Moreover, depolarization of HEC-1A and Ishikawa cells by a high potassium concentration was not accompanied by a rise in the [Ca2+]i (data not shown). These results indicate that endometrial carcinoma cells do not express voltage-gated calcium channels. The ability of TG to facilitate Ca2+ influx further indicates that Ca2+ mobilization is followed by Ca2+ entry that is independent of the mechanism of depletion of the intracellular calcium pool. This is consistent with the idea that a capacitative calcium influx pathway is coupled to Ca2+ release, leading to the sustained [Ca2+]i response in ATP-stimulated cells (21).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Characterization of ATP-induced Ca2+ entry in Ishikawa cells. A, Effects of addition of Ca2+ on the pattern of ATP-induced [Ca2+]i profiles. Recordings were done in Ca2+-deficient medium without nifedipine (-NIF, upper tracing) or with 1 µM nifedipine (+NIF, bottom tracing). B, Effects of TG, an inhibitor of endoplasmic reticulum (Ca2+)ATPase, on Ca2+ influx. Cells were stimulated with 1 µM +TG or with solvent dimethyl sulfoxide (-TG) for 20 minutes before recording in Ca2+-deficient medium, and 1.2 mM Ca2+ was added as indicated by the arrows. The initial extracellular Ca2+ concentration was reduced by EGTA to about 200 nM.

 
Characterization of P receptor subtypes

Before pharmacological characterization of P receptor subtypes, we examined the stability of ATP in endometrial cell cultures. In other cell cultures, ATP is degraded by ecto-ATPases to ADP, AMP, and adenosine (5). For some P receptors, ADP is more potent than ATP (22), whereas in others adenosine acts as an extracellular messenger (P1 receptors) (7, 23). Thus, the rapid degradation of ATP may lead to misleading conclusions about the receptor subtype expressed in a particular culture. Under the cell density used in [Ca2+]i measurements, degradation of ATP was also observed in cultures of endometrial cells. As shown in Fig. 3Go, degradation was more pronounced in Ishikawa cells than in HEC-1A cells, but in none of the cultures did a complete degradation occur during 3 h of stimulation at 37 C. Thus, it is unlikely that the ATP degradation products significantly participate in the Ca2+ signaling during the acute (2–3 min) stimulation with ATP and its analogs.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. The time dependence of ATP degradation in cultures of HEC-1A and Ishikawa cells. Cultures (0.5 106 cells/well) or dishes without cells were incubated at 37 C in the presence or absence of 50 µM ATP for indicated times. In cultures without added ATP, no measurable ATP was detected, and in dishes without cells no ATP degradation was detected. The values are mean ± SEM from sextuplicate determinations.

 
In further experiments, we compared the potency of ATP and several other P receptor agonists in their ability to raise [Ca2+]i during acute stimulation. In both cell types, uridine 5'-triphosphate (UTP) was also an effective stimulator of Ca2+ signals. Fig. 4Go illustrates the dose-dependence of ATP and UTP actions in HEC-1A and Ishikawa cells for both agonists. The order of agonist potency for this receptor was ATP = UTP > ATP-{gamma}-S>>ADP. In contrast, ß,{gamma}-methylene-ATP, 2-MeS-ATP, BzATP, AMP, and adenosine were ineffective (data not shown). This pharmacological profile is consistent with the expression of the P2Y2 P receptor subtype in both carcinoma cell types.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 4. Dose dependence of ATP and UTP actions in endometrial carcinoma cells. Arrows indicate the moment of ATP/UTP additions. The estimated ED50 in both cases was about 10 µM.

 
The actual expression of the P2Y2 receptor in human endometrial carcinoma cells was examined by the reverse transcription (RT) PCR analysis. For positive control, cDNA from the rat pituitary GH3 cells, originally reported to have the rat P2Y2 receptor message (15), was used. Fig. 5Go shows the expected size of a single DNA fragment (311 bp) amplified from both endometrial carcinoma and GH3 cells. The amounts of amplified DNA from endometrial carcinoma cells were more abundant than those observed in control GH3 cells. To identify the PCR product amplified from that caused by genomic contamination in the RNA preparation, total RNA samples without reverse transcriptase reaction were subjected to a PCR reaction. However, a DNA fragments were not detected (data not shown).



View larger version (48K):
[in this window]
[in a new window]
  		Figure 5. RT-PCR analysis using the P2Y2 primer in endometrial carcinoma cells and GH3 pituitary cells. The PCR products were separated in 1% agarose gel and visualized with ethidium bromide. GAPDH, glyceraldehyde phosphate dehydrogenase.

 
Coupling of P2Y2 receptors to intracellular signaling pathways

To exclude the possible impact of ATP degradation in further investigations, a slowly degradable analog, ATP-{gamma}-S, was used. In cells stimulated with this agonist for 15 min at 37 C, dose-dependent increases in InsP3 and DAG productions were observed (Fig. 6Go, A and B). The estimated ED50s were comparable with that recorded in [Ca2+]i measurements. These findings are consistent with the Ca2+-mobilizing ability of ATP and its analogs (Fig. 1BGo), i.e., with the role of InsP3 in release of Ca2+ from intracellular pools.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 6. Coupling of P2Y2 receptors to phospholipases C and D pathways. A and B, Dose-dependent effects of ATP-{gamma}-S on InsP3 and CDP-DAG accumulations. Cells were stimulated for 15 minutes at 37 C and InsP3 and CDP-DAG were measured as described in Materials and Methods. C, Dose-dependent effects of ATP-{gamma}-S on PEt accumulation. Cells were stimulated for 60 minutes at 37 C. The values are mean ± SEM from sextuplicate determinations.

 
In addition to the phospholipase C pathway, P2Y2 receptors expressed in endometrial carcinoma cells are coupled to the phospholipase D pathway. This is indicated by the ability of ATP-{gamma}-S to stimulate PEt accumulation in cultures bathed in 0.5% ethanol (Fig. 6CGo). The RT-PCR analysis indicated the presence of phosphatydilcholine-hydrolyzing phospholipase D-1 transcripts in both endometrial cancer cell types. Fig. 7Go shows two DNA fragments that correspond to the entire open reading frame (a, 3224 bp) and amino-terminal half (b, 1533 bp) of phospholipase D-1a. These fragments were amplified by two separate PCRs. Recently, a shorter form of splice variant enzyme phospholipase D-1b was reported (17). This variant form lacks a stretch of 38 amino acids from PLD1a. Although our primer set against entire the open reading frame covers a deleted part of this variant, small differences in molecular weight between PLD-1a and -1b could not be detected on 1% agarose gel, and further characterization was not performed. All together, these results indicate that P2Y2 receptors expressed in HEC-1A and Ishikawa cells do not differ from P2Y2 receptors expressed in other cell types in their coupling to intracellular messengers.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 7. Expression of phospholipase D1 transcripts in endometrial carcinoma cells. RT-PCR analysis was performed with primers against entire open reading frame (a) and amino-terminal half (b) of human phospholipase D1. Negative controls were performed in the absence of enzyme in reverse transcriptase reaction and a PCR primer set against amino-terminal half of human phospholipase D1 was used.

 
Antiproliferative actions of P2Y2 receptors

We further examined the effects of ATP-{gamma}-S on proliferation of HEC-1 and Ishikawa cells. Experiments were done in fetal calf serum-free medium, as well as in the presence of 2%, 5%, and 10% fetal calf serum. Cells were cultured for 72 h at 37 C, and ATP-{gamma}-S was added at three time points at 0, 24, and 48 h. In Ishikawa cells, [3H]thymidine incorporation was significantly (P < 0.01) lowered by ATP-{gamma}-S in all cultures (Fig. 8AGo). Comparable differences were also observed in HEC-1A cells (Fig. 8BGo). In a time-course experiment using 0.1% FCS-containing medium, a significant inhibition of [3H]thymidine incorporation was observed 24 h after the treatment with 100 µM ATP-{gamma}-S, and this inhibition was preserved for 5 days (data not shown).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 8. Antiproliferative actions of ATP-{gamma}-S in endometrial carcinoma cells. A and B, Effects of ATP-{gamma}-S on cell proliferation in Ishikawa (A) and HEC-1A cells (B) cultured in fetal calf serum (FCS)-deficient and -containing medium for 3 days. C, The lack of effects of ATP-{gamma}-S on DNA fragmentation in Ishikawa cells. 1–3, Ishikawa cells: 1, control; 2, 100 µM ATP-{gamma}-S; 3, 1 mM ATP-{gamma}-S; 4–6, GH3 cells: 4, 50 µM etoposide; 5, 100 µM adenosine; 6, 100 µM ATP; 7, markers.

 
In general, the differences in [3H]thymidine incorporation may indicate an inhibition of cell proliferation and/or an increase in receptor-controlled cell death. To examine the later possibility, we used a DNA fragmentation test. As expected, 50 µM etoposide induced fragmentation of DNA 48 h after addition in controls, indicating activation of the apoptotic cycle (Fig. 8CGo, line 4). In contrast, ATP-{gamma}-S (2 days of 100 µM or 5 mM during 48 h) was unable to change the pattern of the DNA profile in Ishikawa cells (lines 2 and 3) and in GH3 cells (line 6), which also express P2Y2 receptors (15). Also, the trypan blue test of cell viability did not show any consistent difference between controls and ATP-{gamma}-S-treated cells. Thus, it is unlikely that an increase in apoptotic and/or necrotic rates accounts for a decrease in [3H]thymidine incorporation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although ATP is released from cells under physiological conditions and during the chemotherapeutic and radiation control of tumor growth, the effects of such release on cell cycle in carcinoma tissue were not studied in detail. In general, ATP and its degradable products act as extracellular messengers for three classes of P receptors: P2Y, P2X, and P1. Each of these classes of receptors is composed of several subtypes. The whole spectrum of pathways is activated by these receptors and receptor-channels, including phospholipases C, D, A2, and adenylyl cyclase pathways, further contributing to the complexity of the actions of purines in normal and carcinoma tissues (5, 22, 24, 25). Carcinoma cells frequently express these receptors, activation of which may facilitate or inhibit cell proliferation and/or apoptosis. These actions probably depend on the receptor subtype in a particular tissue and the intracellular signaling pathways activated by purines.

In vivo injection of adenine nucleotides inhibits the growth of mice CT26 colon adenocarcinoma and human pancreatic adenocarcinoma CAPAN-1 (26). The role of extracellular ATP in inhibition of cancer growth in Ehrilich tumor cells was also reported (27). Both the necrotic and apoptotic actions of ATP were reported to be responsible for slowing the growth rate in tumor and normal cells (28). Also, ATP per se can induce apoptosis, but it can also antagonize the apoptotic actions of other agents (29). In tissue-expressing P2X1 and P2X7 receptor-channels, ATP may act as an apoptotic signal (30, 31). In contrast, a transient up-regulation of P2Y2 receptors may represent a signal for differentiation of thymocytes by providing the feedback signaling from extracellular ATP (32). The apoptotic vs. differentiation actions of ATP may also depend on its degradation to adenosine, which in turn activates P1 receptors. For example, the A3 subtype of these receptors may participate in the control of apoptosis by adenosine in the central nervous system (33).

In line with these observations, we show that the endometrial carcinoma cells HEC-1 and Ishikawa express G protein-coupled P2Y2 receptors, the activation of which leads to attenuation of cell growth. However, the antiproliferative action of ATP in these cells was not due to stimulation of the apoptotic or necrotic cycles and is probably not mediated by P1 receptors because our mitogenic assay was done with ATP-{gamma}-S, a slowly degradable ATP analog. Also, no evidence for expression of apoptotic P2X channels was observed. In further support of this, ATP was not able to activate the apoptotic cycle in GH3 cells, used as control cells in our study. These cells do not express the apoptotic P2X channels, but bear a single class of P2Y2 receptors (15). These findings are consistent with the differentiation actions of these receptors in cultural conditions used in our study.

In contrast, extracellular ATP was found to stimulate proliferation of breast cancer cells in vitro through P2Y2 receptors (34). Extracellular ATP also activates both calcium mobilization and cell proliferation in human ovarian cancer cell line OVCAR-3 (35). It has been suggested that ATP-induced rise in [Ca2+]i is obligatory for stimulation of cell growth in OVCAR-3 cells (35), but no evidence was presented to confirm that this ion is, indeed, the intracellular messenger mediating the stimulus-proliferation coupling. In SKOV-3 cells, another human ovarian carcinoma cells, a bidirectional effect of ATP on cell proliferation was observed—a low stimulatory effect in micromolar concentration range and a strong inhibitory effect in submillimolar (36). The mitogenic effects of ATP were observed in kidney and smooth muscle cells (25, 37).

Activation of G protein-coupled Ca2+-mobilizing receptors is frequently accompanied by stimulation of the mitogen-activated protein (MAP) kinase pathway and early response gene expression, leading to facilitation of cell growth and proliferation. This signaling pathway can also be activated by P2Y2 receptors in normal tissue (38, 39), as well as by endothelin-1 in ovarian carcinoma cells (40), which provides a rationale for the proliferative actions of ATP in some cells. However, although activation of the MAP kinase pathway is essential, it is not sufficient to trigger cell proliferation (41). Furthermore, the coexpression of P2Y receptors with P2X receptor-channels and P1 receptors may account for divergent actions of ATP on the cell cycle. For example, the proliferative action of ATP can be mediated by adenosine acting on the A1 receptor in human colonic adenocarcinomas (42, 43).

The antiproliferative action of P2Y2 receptors, independently of their cross-coupling to MAP kinase pathway, is not unique for calcium-mobilizing receptors. GnRH receptors expressed in endometrial cell have been used to control endometriosis and hormone-dependent cancers (44). The antiproliferative actions of GnRH were also observed in ovarian carcinoma cells (10, 11). Molecular analyses revealed the expression of GnRH receptors in endocrine tumors and cell lines derived from them (45). Furthermore, it has been shown recently that activation of high-affinity GnRH receptors transiently expressed in EcRG293 cells was accompanied by inhibition of growth and proliferation (46). In pituitary gonadotrophs, activation of these receptors leads to stimulation of phospholipases C and D pathways and MAP kinase/early gene response pathways (12). Here, we show that P2Y2 receptors are also coupled to phospholipase C and phospholipase D pathways. Thus, it is likely that activation of typical Ca2+-mobilizing receptors in endometrial cancer tissue slows the cell cycle.

In conclusion, the finding that endometrial cell lines express P2Y2 receptors, the activation of which leads to a down-regulation of cell proliferation, is of potential importance for basic investigations and may have clinical relevance. These cells can serve as good cell models for further investigations of the controlling mechanism of the cell cycle by Ca2+-mobilizing receptors. This includes investigations on up- and down-regulation of P2Y2 mRNA as an immediate early gene response in damaged cells. Our findings may also be important in chemotherapeutic and radiation control of endometrial tumor growth, including the potentiality of local injections of slow degradable ATP analogs. Therefore, further experiments should be directed toward the identification of P2Y2 receptors, as well as the other classes of P receptors, in human endometrial cancers.

Received May 19, 1999.

Accepted July 26, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chen Z-P, Levy A, Lightman SL. 1995 Nucleotides as extracellular signaling molecules. J Neuroendocrinol. 7:83–96.[CrossRef][Medline]
  2. North RA. 1996 P2X purinoceptor plethora. Semin Neurosci. 8:187–194.
  3. Illes P, Norenberg W. 1993 Neuronal ATP receptors and their mechanism of action. Trends Pharmacol Sci. 14:50–54.[CrossRef][Medline]
  4. Buell G, Collo G, Rassendren F. 1996 P2X receptors: an emerging channel family. Eur J Neurosci. 8:2221–2228.[CrossRef][Medline]
  5. Pearson JD. 1985 Ectonucleotidases: measurement of activities and use of inhibitors. Methods Pharmacol. 6:83–107.
  6. Barnard EA, Burnstock G, Webb TE. 1994 G protein-coupled receptors for ATP and other nucleotides: a new receptor family. Trends Pharmacol Sci. 15:67–70.[CrossRef][Medline]
  7. Stiles GL. 1992 Adenosine receptors. J Biol Chem. 267:6451–6454.[Abstract/Free Full Text]
  8. Evans RJ, Derkach V, Surprenant A. 1992 ATP mediates fast synaptic transmission in mammalian neurons. Nature. 357:503–505.[CrossRef][Medline]
  9. Kurbacher CM, Cree IA, Bruckner HW, et al. 1998 Use of an ex vivo ATP luminescence assay to direct chemotherapy for recurrent ovarian cancer. Anticancer Drugs. 9:51–57.[Medline]
  10. Emons G, Schroder B, Ortmann O, Westphalen S, Schulz K-D, Schally AV. 1993 High affinity binding and direct antiproliferative effects of LHRH analogues in human endometrial cancer cell lines. J Clin Endocrinol Metab. 77:1458–1464.[Abstract]
  11. Emons G, Ortmann O, Becker M, et al. 1993 High affinity binding and direct antiproliferative effects of LHRH analogues in human ovarian cancer cell lines. Cancer Res. 53:5439–5446.[Abstract/Free Full Text]
  12. Stojilkovic SS, Reinhart J, Catt KJ. 1994 GnRH receptors: structure and signal transduction pathways. Endocr Rev. 15:462–499.[Abstract/Free Full Text]
  13. Tomic M, Jobin RM, Vergara LA, Stojilkovic SS. 1996 Expression of purinergic receptor channels in their role in calcium signaling and hormone release in pituitary gonadotrophs. J Biol Chem. 271:21200–21208.[Abstract/Free Full Text]
  14. Parr CE, Sullivan DM, Paradiso AM, et al. 1994 Cloning and expression of a human P2U nucleotide receptor, a target for cystic fibrosis pharmacotherapy. Proc Natl Acad Sci USA. 91:13067–13072.[Free Full Text]
  15. Chen Z-P, Krull N, Xu L, Levy A, Lightman SL. 1996 Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor. Endocrinology. 137:1833–1840.[Abstract]
  16. Fort P, Rech J, Vie A, et al. 1987 Regulation of c-fos gene expression in hamster fibroblasts: initiation and elongation of transcription and mRNA degradation. Nucleic Acids Res. 15:5657–5665.[Abstract/Free Full Text]
  17. Hammond SM, Jenco JM, Nakashima S, et al. 1997 Characterization of two alternatively spliced forms of phospholipase D1. J Biol Chem. 272:3860–3868.[Abstract/Free Full Text]
  18. Stojilkovic SS, Vukicevic S, Luyten FP. 1994 Calcium signaling in endothelin- and platelet-derived growth factor-stimulated chondrocytes. J Bone Mineral Res. 9:705–713.[Medline]
  19. Zheng L, Stojilkovic SS, Hunyady L, Krsmanovic LZ, Catt KJ. 1994 Sequential activation of phospholipase C and phospholipase D in agonist-stimulated gonadotrophs. Endocrinology. 134:1446–1454.[Abstract/Free Full Text]
  20. Watson SP, Godfrey PP. 1988 The role of receptor-stimulated inositol phospholipid hydrolysis in the autonomic nervous system. Pharmacol Ther. 38:387–417.[CrossRef][Medline]
  21. Putney JW Jr, Bird GSJ. 1993 The inositol phosphate-calcium signaling system in nonexcitable cells. Endocr Rev. 14:610–631.[Abstract/Free Full Text]
  22. North RA, Barnard EA. 1997 Nucleotide receptors. Curr. Opin Neurobiol. 7:346–357.
  23. Jacobson KA, van Galen PJM, Williams M. 1992 Adenosine receptors: pharmacology, structure-activity relationships, and therapeutic potential. Med Chem. 35:407–422.
  24. Harden TK, Boyer JL, Nicholas RA. 1995 P2-purinergic receptors: subtype-associated signaling responses and structure. Annu Rev Pharmacol Toxicol. 35:541–579.[CrossRef][Medline]
  25. Erling D. 1998 Extracellular ATP: a growth factor for vascular smooth muscle cells. Gen Pharmacol. 31:1–8.[Medline]
  26. Rapaport E. 1988 Experimental cancer therapy in mice by adenine nucleotides. Eur J Cancer Clin Oncol. 24:1491–1497.[CrossRef][Medline]
  27. Estrela JM, Obrador E, Navarro J, De La Vega MCL, Pellicer JA. 1995 Elimination of Ehrlich tumors by ATP-induced growth inhibition, glutathione depletion and X-rays. Nat Med. 1:84–88.[CrossRef][Medline]
  28. Chow SC, Kass GEN, Orrenius S. 1997 Purines and their roles in apoptosis. Neuropharmacology. 36:1149–1156.[CrossRef][Medline]
  29. Apasov S, Koshiba M, Redegeld F, Sitkovsky MV. 1995 Role of extracellular ATP and P1 and P2 classes of purinergic receptors in T-cell development and cytotoxic T lymphocyte effector functions. Immunol Rev. 146:5–19.[CrossRef][Medline]
  30. Chvatchko Y, Valera S, Aubry J-P, Renno T, Buell G, Bonnefoy J-Y. 1996 The involvement of an ATP-gated ion channel, P2X1, in thymocyte apoptosis. Immunity. 5:275–283.[CrossRef][Medline]
  31. Ferrari D, Chiozzi P, Falzoni S, et al. 1997 ATP-mediated cytotoxicity in microglial cells. Neuropharmacology. 36:1295–1301.[CrossRef][Medline]
  32. Koshiba M, Apasov S, Sverdlov V, et al. 1997 Transient up-regulation of P2Y2 nucleotide receptor mRNA expression is an immediate early gene response in activated thymocytes. Proc Natl Acad Sci USA. 94:831–836.[Abstract/Free Full Text]
  33. Abbracchi OMP, Ceruti S, Brambilla R, et al. 1997 Modulation of apoptosis by adenosine in the central nervous system: a possible role for the A3 receptors. Ann NY Acad Sci. 825:11–22.[Medline]
  34. Dixon CJ, Bowler WB, Fleetwood P, Ginty AF, Gallagher JA, Carron JA. 1997 Extracellular nucleotides stimulate proliferation in MCF-7 breast cancer cells via P2-purinoceptors. Br J Pharmacol. 75:34–39.
  35. Popper LD, Batra S. 1993 Calcium mobilization and cell proliferation activated by extracellular ATP in human ovarian tumor cells. Cell Calcium. 14:209–218.[CrossRef][Medline]
  36. Batra S, Fadeel I. 1994 Release of intracellular calcium and stimulation of cell growth by ATP and histamine in human ovarian cancer cells (SKOV-3). Cancer Lett. 77:57–63.[CrossRef][Medline]
  37. Paller MS, Schnaith EJ, Rosenberg ME. 1988 Purinergic receptors mediate cell proliferation and enhanced recovery from renal ischemia by adenosine triphosphate. J Lab Clin Med. 131:174–183.
  38. Soltoff SP, Avraham H, Avraham S, Cantley LC. 1998 Activation of P2Y2 receptors bu UTP and ATP stimulates mitogen-activated kinase activity through a pathway that involves related adhesion focal tyrosine kinase and protein kinase C. J Biol Chem. 273:2653–2660.[Abstract/Free Full Text]
  39. Soltoff SP. 1998 Related adhesion focal tyrosine kinase and the epidermal growth factor receptor mediate the stimulation of mitogen-activated protein kinase by the G-protein-coupled P2Y2 receptor. J Biol Chem. 273:23110–23117.[Abstract/Free Full Text]
  40. Bagnato A, Tecce R, Di Castro V, Catt KJ. 1997 Activation of mitogenic signals by endothelin 1 in ovarian carcinoma cells. Cancer Res. 57:1306–1311.[Abstract/Free Full Text]
  41. Wilkie N, Morton C, Ng LL, Boarder MR. 1996 Stimulated mitogenic-activated protein kinase is necessary but not sufficient for the mitogenic response to angiotensin II: a role for phospholipase D. J Biol Chem. 271:32447–32453.[Abstract/Free Full Text]
  42. Lelievre V, Muller J-M, Falcon J. 1998 Adenosine modulates cell proliferation in human colonic adenocarcinoma. I. Possible involvement of adenosine A1 receptor subtypes in HT29 cells. Eur J Pharmacol. 341:289–297.[CrossRef][Medline]
  43. Lelievre V, Muller J-M, Falcon J. 1998 Adenosine modulates cell proliferation in human colonic carcinoma. II. Differential behavior of HT29, DLD-1, Caco-2 and SW403 cell lines. Eur J Pharmacol. 341:299–308.[CrossRef][Medline]
  44. Emons G, Schally A. 1994 The use of luteinizing hormone releasing hormone agonists and antagonists in gynecological cancers. Hum Reprod (Oxf). 9:1354–1379.
  45. Kakar SS, Grizzle WF, Neill JD. 1994 The nucleotide sequence of human GnRH receptors in breast and ovarian tumors are identical with that found in pituitary. Mol Cell Endocrinol. 106:145–149.[CrossRef][Medline]
  46. Kakar SS. 1998 Inhibition of growth and proliferation of EcRG293 cell line expressing high-affinity gonadotropin-releasing hormone (GnRH) receptor under the control of an inducible promoter by GnRH agonist (D-Lys6)GnRH and antagonist (antide). Cancer Res. 58:4558–4560.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. Schafer, F. Sedehizade, T. Welte, and G. Reiser
ATP- and UTP-activated P2Y receptors differently regulate proliferation of human lung epithelial tumor cells
Am J Physiol Lung Cell Mol Physiol, August 1, 2003; 285(2): L376 - L385.
[Abstract] [Full Text] [PDF]

Home page
 J Biomol ScreenHome page
M. U. Kassack, B. Hofgen, J. Lehmann, N. Eckstein, J. M. Quillan, and W. Sadee
Functional Screening of G Protein--Coupled Receptors by Measuring Intracellular Calcium with a Fluorescence Microplate Reader
J Biomol Screen, June 1, 2002; 7(3): 233 - 246.
[Abstract] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
R. Liu, P. D. Bell, J. Peti-Peterdi, G. Kovacs, A. Johansson, and A. E. G. Persson
Purinergic Receptor Signaling at the Basolateral Membrane of Macula Densa Cells
J. Am. Soc. Nephrol., May 1, 2002; 13(5): 1145 - 1151.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Katzur, A. C.
Right arrow Articles by Stojilkovic, S. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Katzur, A. C.
Right arrow Articles by Stojilkovic, S. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals