Molecular Human Reproduction, Vol. 6, No. 5, 435-442,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Characterization of calcium-mobilizing, purinergic P2Y2 receptors in human ovarian cancer cells
1 Department of Obstetrics and Gynecology, University of Lubeck, Germany, D-23538 and 2 Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
Abstract
In human ovarian EFO-21 and EFO-27 carcinoma cells, extracellular ATP induced a concentration-dependent rise in intracellular calcium concentration ([Ca2+]i), suggesting the expression of a purinoreceptor. ATP and UTP were equipotent in generating [Ca2+]i signals, followed by ATP-
-S and ADP, whereas ß,-ATP, 2 methyl 1 thio-ATP, 3'-o-(4-benzoyl) benzoyl-ATP, AMP, and adenosine were ineffective. This pharmacological profile suggested the presence of the P2Y2 subtype in both cell types, and this was confirmed by reverse transcription–polymerase chain reaction (RT–PCR) analysis using P2Y2 primers. ATP-induced [Ca2+]i signals were composed of two phases: an early and extracellular calcium-independent phase, followed by a sustained plateau phase that was dependent on capacitative calcium influx. In addition to the rise in the [Ca2+]i, a time- and concentration-dependent increase in phosphatidylethanol accumulation was observed in ATP-stimulated cells, indicating an increase in phospholipase D activity. RT–PCR analysis identified the expression of a transcript for the phospholipase D-1 subtype of this enzyme. Activation of these receptors by a slowly degradable analogue, ATP--S, attenuated basal and fetal calf serum-induced cell proliferation in a time- and concentration-dependent manner. These results indicate that ATP may act as an extracellular messenger in controlling the ovarian epithelial cell cycle through P2Y2 receptors.
ATP receptors/calcium/cell proliferation/phospholipase C
Introduction
Regulation of the cell cycle is tissue specific and involves the balance of many regulatory molecules acting extracellularly and/or intracellularly. In the ovary, the periodic nature of ovulation, determined by cyclic secretion of pituitary and ovarian hormones, brings another dimension to the control of cell proliferation, differentiation, and death. Follicular growth, ovulation, and corpus luteum formation are sequential and involve a rapid switch from a highly proliferative stage in the granulosa cells to a non-proliferative, terminally differentiated phase in luteal cells (Robker and Richards, 1998
). Additionally, epithelial cells in the ovary require rapid cell division in order to repair defects in the ovarian surface induced by follicular rupture (Amsterdam and Selvaraj, 1997). Cell cycle control in ovarian epithelial and stromal cells is complex and frequently leads to mutations and generation of carcinomas. However, ovarian sex cord stromal and germ cell tumours account for only 10% of the cancers, the remaining arise from mutations in epithelial cells (Amsterdam and Selvaraj, 1997).
The growth pattern of epithelial ovarian cancers is well known (Berchuck et al., 1993). However, relatively little is known about the fundamental events controlling cell proliferation, differentiation, and apoptosis of these cells. Epidermal growth factor and transforming growth factor-alpha (TGF-
) stimulate proliferation, whereas TGF-ß inhibits the proliferation of ovarian epithelial cells. Gonadal steroid hormones also participate in controlling the epithelial cell cycle (Bast et al., 1993; Berchuck et al., 1993; Havrilesky et al., 1995; Baguley et al., 1998; Lau et al., 1999). Several G protein-coupled receptors, including gonadotrophin-releasing hormone (GnRH), FSH, and LH receptors are also expressed in human ovarian epithelial cancer cells and may participate in the control of the cell cycle (Rajaniemi et al., 1981; Heintz et al., 1985; Emons et al., 1993). Here we present evidence that ovarian epithelial cell lines EFO-21 and EFO-27 also express Ca2+-mobilizing P2Y receptors, whose activation by ATP and other agonists leads to inhibition of cell proliferation. We have characterized the subtype of these receptors and their actions on intracellular messengers in both cell lines.
Materials and methods
Chemicals
Fura-2 AM was obtained from Molecular Probes (Eugene, OR, USA). TrizolTM reagent, superscript II reverse transcriptase, oligo(dT)18 primer, and polymerase chain reaction (PCR) reagent system were from Gibco BRL (Gaithersburg, MD, USA) and pBluescript II vector from Stratagene (La Jolla, CA, USA). Sequenase version 2.0 was from Amersham (Arlington Heights, IL, USA). [9,10-3H]Oleic acid (10 Ci/mmol), and Econofluor-2 were purchased from DuPont–New England Nuclear (Boston, MA, USA). Silica gel 60 thin layer chromatography (TLC) plates were from Merck (Darmstadt, Germany). Organic reagents for TLC were high-performance liquid chromatography grade and were from J.T.Baker (Phillipsburg, NJ, USA), and liquid scintillation solution (Hydrofluor) was from National Diagnostics (Manville, NJ, USA). All other chemicals were obtained from Sigma (St Louis, MO, USA).
Ovarian cancer cell cultures
The epithelial human ovarian cancer cell lines, EFO-21 and EFO-27, used in experiments were derived from a poorly differentiated serous adenocarcinoma (EFO-21) or a mucinous papillary adenocarcinoma of intermediate differentiation (EFO-27) (Simon et al., 1983; Kunzmann and Hoelzel, 1987). Cells were cultured in minimum essential Eagle's medium (Sigma) containing 10% fetal calf serum (Gibco, Rockville, MD, USA), human insulin 40 IE/l, transferin 2.5 mg/l, and antibiotics, media was changed every 48 h. Cultures were incubated at 37°C in a fully humidified atmosphere of 5% carbon dioxide in air. Cells were subcultured, upon reaching confluence, by trypsin dissociation using 0.05% trypsin.
Measurements of calcium ion concentration in EFO-21 and EFO-27 cells
EFO-21 and EFO-27 cells (2x105 per dish) were plated on coverslips coated with poly-L-lysine. On the following day, the cells were incubated at 37°C for 60 min with 2 µmol/l fura-2 AM. After incubation, the cells were washed with Krebs–Ringer buffer. Cells were examined under a x40 oil immersion objective on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to the Attofluor Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD, USA). Cells were exposed to alternating 340 and 380 nm light beams and the intensity of light emission at 520 nm was measured. The light intensity ratio, F(340)/F(380), which reflects the intracellular Ca2+ concentrations, was monitored simultaneously in several single cells.
Reverse transcription–polymerase chain reaction (RT–PCR) analyses
Total RNA was isolated from ovarian cancer cells and rat GH3 cells using TrizolTM reagent. Total RNA (5 µg) was reverse transcribed with oligo(dT)18 primers and Superscript II RT according to the supplier's instructions. PCR amplification of 30 cycles was performed on a 5% fraction of the resulting single strand cDNA sample, in a final volume of 12.5 µl. The primer sequences used (sense: 5'-GCTTCAACGAGGACTTCAAG-3'; and antisense: 5'-CACGCTGATGCAGGTGAGGA-3') were exactly complementary to both the human (Parr et al., 1994) and rat P2Y2 receptors (Chen et al., 1996). The PCR conditions for each cycle were: denaturation at 94°C for 1 min, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by final incubation at 72°C for 10 min. PCR products were separated by 1% agarose gel electrophoresis and visualized with ethidium bromide. A PCR reaction using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers was performed on samples from the P2Y2 receptor mRNA analysis, in the same volumes (Fort et al., 1987). The sequences 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. A minimum of two independent clones, derived from separate PCR reactions, were sequenced by the dideoxy chain termination method using a Sequenase version 2.0.
To examine phospholipase D-1 expression in ovarian cancer cells, two sets of oligonucleotide primers specific to the human phospholipase D-1a subtype (Hammond et al., 1997) were designed. Nucleotide sequences for D1S1 sense and D1A1 antisense primers which correspond to the first 21 and last 20 nucleotides of human phospholipase D-1a open reading frame, respectively, and an antisense primer, D1A2, with a complementary sequence to that of 1612–1631 of phospholipase D-1a. PCR was performed with a D1S1 sense primer and either a D1A1 or a D1A2 antisense primer. The PCR temperature profiles were: denaturation at 94°C for 35 s, annealing at 48°C for 45 s, and extension at 72°C for 3 min for 30 cycles, followed by a final extension at 72 °C for 10 min.
Cell proliferation assay
Cell proliferation was examined by incorporation of [3H]thymidine into acid-precipitable material. Ovarian cells were plated at 2x105 cells/well, culture media was removed and replaced with medium lacking thymidine and fetal bovine serum. [3H]Thymidine was added at 5 µCi/ml aliquots, and cells were incubated for 4 h at 37 °C. Incorporation of radioactivity was measured and analysed according to the method described previously (Stojilkovic et al., 1994).
Phosphatidylethanol assay
Phosphatidylethanol measurements were performed in 35 mm culture dishes with 1.1 ml of Dulbecco's modified Eagle's medium (DMEM) containing 0.1% fatty acid-free bovine serum albumin (BSA), L-glutamine (292 mg/l), glucose (4.5 g/l), NaHCO3 (1.4 g/l), and 5 µCi [3H]oleic acid. After 16–24 h incubation periods, stimuli were added to the culture dishes in the presence or absence of 0.5% ethanol at indicated times. The reactions were terminated by placing the dishes on ice, removing the medium, and rinsing the dishes with 1ml ice-cold saline. Following extraction and separation, as described (Zheng et al., 1994), phosphatidic acid (PA) and phosphatidyl ethanol (PEt) were visualized either by autoradiography, for which the TLC plates were treated with EN3HANCE spray, or by iodine vapour staining. Regions corresponding to appropriate standards were scraped and placed in scintillation vials and further extracted with 1 ml of methanol–HCl (150:1). Hydroflour (9 ml) was added after the iodine stains were extinguished. Samples were kept at room temperature overnight and radioactivity was measured in a Beckman LS 9000 liquid scintillation counter.
Measurements of ATP
Degradation of ATP was measured in static cell cultures using an ATP bioluminescent assay kit (Sigma) in an AutoLumat LB 953 (Berthold, Wildbad, Germany) via injection of 100 µmol/l of assay solution into an aliquot of 100 µmol/l of sample. Calibration curves were constructed from measurements in standard solutions, which were diluted in the same medium as the corresponding solutions of unknown ATP concentration. Detection limit of the assay was 0.02 nmol/l.
Results
ATP-induced Ca2+ mobilization and entry
In single EFO-21 and EFO-27 cells, ATP (100 µmol/l) induced a rapid increase in [Ca2+]i followed by a sustained plateau-like response (Figure 1A). ATP was also able to increase [Ca2+]i in cultures bathed in Ca2+-deficient medium (Figure 1B), suggesting that these cells express a P2Y receptor, whose activation leads to Ca2+ mobilization from intracellular stores. The pattern of ATP-induced [Ca2+]i response differed in the presence and absence of extracellular Ca2+. As shown in Figure 1B, the rate of decrease in [Ca2+]i occurred faster in the absence of extracellular Ca2+, suggesting that Ca2+ entry supplements Ca2+ mobilization during the sustained agonist stimulation. Furthermore, an increase in extracellular Ca2+ concentrations from 200 nmol/l to 1.2 mmol/l was associated with an additional increase in [Ca2+]i in ATP-stimulated cells (Figure 2A). Similar Ca2+-dependence was also observed in EFO-27 cells (not shown).
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To examine the nature of these channels, cells were treated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ ATPase. Thapsigargin induced a transient rise in [Ca2+]i in cells bathed in Ca2+-deficient medium (not shown). The subsequent addition of 1.2 mmol/l Ca2+ in these cells was associated with an increase in [Ca2+]i (Figure 2B
), indicating that Ca2+ mobilization is followed by Ca2+ entry, independent of the mechanism of depletion of the intracellular Ca2+ pool. In contrast, the basal [Ca2+]i was not affected by a dihydropyridine Ca2+ channel antagonist, nifedipine, and ATP was able to induce biphasic [Ca2+]i response (Figure 2C). In cells stimulated with ATP in Ca2+-deficient and nifedipine-containing medium, extracellular Ca2+ induced a rise in [Ca2+]i comparable to that observed in controls. Finally, depolarization of EFO-21 and EFO-27 cells by a high potassium concentration was not accompanied by a rise in the [Ca2+]i (not shown).
Characterization of ATP receptor subtype
In both cell types, UTP was also an effective stimulator of Ca2+ signals. Figure 3 illustrates concentration dependence of ATP and UTP actions. The order of agonist potency for both cell types was ATP = UTP > ATP--S >> ADP. Several other agonists, including ß,-ATP, 2-MeS-ATP, BzATP, AMP, and adenosine, were ineffective in EFO-21 cells (Figure 4), as well as in EFO-27 cells (not shown). This pharmacological profile is consistent with the expression of the P2Y2 receptor subtype. The gene expression of this receptor in human ovarian carcinoma cells was examined by RT–PCR analysis. RNA from rat pituitary GH3 cells, previously reported to express the P2Y2 receptor message (Chen et al., 1996), was used as positive control. Figure 5A shows the expected size of a DNA fragment (311 bp) amplified from both ovarian carcinoma and GH3 cell cDNA. The intensities of amplified DNA from ovarian carcinoma cells were higher than those from control GH3 cells. To rule out the contamination by genomic DNA, total RNA samples without RT reaction were subjected to PCR amplification. However, no DNA fragment was detected (data not shown).
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ATP-induced activation of phospholipase D pathway
In the presence of ethanol, phospholipase D catalyses a transphosphatidylation that leads to the formation of PEt. This reaction is commonly used as a specific assay for phospholipase D activity in agonist-stimulated cells (Cesnjaj et al., 1995
). In EFO-21 cells, ATP--S induced a concentration-(Figure 6A) and time-dependent (Figure 6B) increase in PEt accumulation. In both experiments, cells were stimulated in the presence of 0.5% ethanol. These results indicate that P2Y2 receptors expressed in ovarian cancer cells also activate the phospholipase D pathway, leading to the accumulation of PEt at the expense of phosphatidic acid.
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The presence of phosphatidylcholine-hydrolysing phospholipase D1 transcripts in both types of ovarian carcinoma cell was confirmed. Figure 5B
shows two sizes of DNA fragments that correspond to an entire open reading frame (a, 3224 base pairs) and amino-terminal half (b, 1533 base pairs) of phospholipase D-1a, which were amplified by two separate PCR. Recently, a shorter form of splice variant enzyme, phospholipase D-1b, was reported (Hammond et al., 1997), that lacks a stretch of 38 amino acids. Although primers in the present study were designed for the entire open reading frame, a small difference in molecular weight between phospholipase D-1a and -1b could not be detected on 1% agarose gel, and further characterization was not performed.
Antiproliferative actions of ATP
The effects of ATP and its analogues on cell proliferation were analysed by short-term incorporation of [3H]thymidine in cell cultures. For this purpose the slow degradable ATP--S was used. As shown in Figure 7, under the cell density used in [Ca2+]i measurements degradation of ATP was observed in cultures of ovarian cells. Thus, the rapid degradation of ATP may lead to unclear conclusions about the possible effects of this compound on cell proliferation in terms of concentrations required, as well as the possibility that the degradable ATP products, such as adenosine, account for the observed effects.
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When EFO-21 and EFO-27 cells were stimulated with 100 µmol/l ATP-
-S for 3 days, a significant inhibition of cell proliferation was observed as compared to untreated cells. In further experiments, two doses of ATP--S, 50 µmol/l and 100 µmol/l, were employed and cells were cultured in medium containing 0.1% fetal calf serum at 37°C for 1, 3 or 5 days. In EFO-21 cells, [3H]thymidine incorporation was significantly lower in cultures stimulated with 100 µmol/l ATP--S for 3 and 5 days (Figure 8A, left panel). In EFO-27 cells, however, both concentrations of ATP--S caused a significant inhibition of cell growth, 100 µmol/l concentration being more effective (Figure 8A, right panel). The inhibitory effects of ATP--S on cell proliferation were also observed in cultures stimulated with increasing concentrations of fetal calf serum. As shown in Figure 8B, ATP--S (100 µmol/l) was more effective in EFO-27 cells than in EFO-21 cells.
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The differences in [3H]thymidine incorporation may indicate an inhibition of cell proliferation, but also could be explained as an increase in the rate of cell death. To examine the later possibility, a DNA fragmentation test was employed. As expected, 50 µmol/l etoposide induced fragmentation of DNA 48 h after addition in controls, indicating activation of the apoptotic cycle. In contrast ATP-
-S (2 days of 100 µmol/l or 5 mmol/l during 48 h) was unable to change the pattern of the DNA profile in EFO-27 cells. Also, the Trypan blue test of cell viability did not show any consistent difference between controls and ATP--S-treated cells (data not shown). These results indicate that it is unlikely that increase in apoptotic and/or necrotic rates accounts for a decrease in [3H]thymidine incorporation in these cells. Discussion
Human granulosa cells express purinergic receptors, whose activation leads to release of Ca2+ from intracellular pools (Kamada et al., 1994; Lee et al., 1996) calcium-mobilizing purinergic receptors are also expressed in human ovarian epithelial cancer cells, SKOV-3 (Batra and Fadeel, 1994) and OVCAR-3 (Popper and Batra, 1993). However, receptor identification and their actions on intracellular messengers have not been investigated. Here it was shown that two epithelial ovarian carcinoma cell lines, EFO-21 and EFO-27, express purinergic Ca2+-mobilizing receptors. Further analysis indicated that the Ca2+-mobilizing action of these receptors was associated with a capacitative Ca2+ entry, which sustained Ca2+ signals during prolonged agonist stimulation. Pharmacological profile and RT–PCR analysis indicated the expression of the P2Y2 subtype of these receptors in both cell types. These receptors activated both phospholipase C and the phospholipase D signalling pathways, which generates the production of several intracellular messengers. Finally, P2Y2 receptor activation is accompanied by inhibition of thymidine incorporation. Such inhibition was observed in both basal and FCS-stimulated cells.
In line with the current experiments, the growth of mice CT26 colon adenocarcinoma and human pancreatic adenocarcinoma, CAPAN-1 was inhibited by in-vivo injection of adenine nucleotides (Rapaport, 1988). Extracellular ATP also inhibited cancer growth in Ehrlich tumour cells, and this action was accompanied by a selective decrease in the glutathione content within the cancer cells in vivo (Estrela et al., 1995). In general, both the necrotic and apoptotic actions of ATP may account for slowing the growth rate in tumour and normal cells (Chow et al., 1997). ATP is also cytotoxic in microglial cells (Ferrari et al., 1997) and lymphocytes (Apasov et al., 1995). In human endometrial cancer cell lines, ATP slows the rate of cell proliferation, but this action is not associated with cytotoxicity or apoptosis (Schultze-Mosgau et al., 1998).
In contrast with the observations in this study, extracellular ATP was found to stimulate proliferation of breast cancer cells in vitro through P2Y2 receptors, whose activation leads to release of intracellular Ca2+ (Dixon et al., 1997). Extracellular ATP was also reported to stimulate cell proliferation in human ovarian cancer cell line OVCAR-3 and caused a rise in [Ca2+]i that is obligatory for stimulation of cell growth (Popper and Batra, 1993). In SKOV-3 cells, another human ovarian epithelial cell line, a bidirectional effect of ATP on cell proliferation was observed – a slight stimulatory effect in micromolar concentration range and a strong inhibitory effect in submillimolar (Batra and Fadeel, 1994). The mitogenic effects of ATP were also observed in smooth muscle cells (Erlinge, 1998) and kidney cells (Paller et al., 1988). Thus, it is likely that a Ca2+-mobilizing receptor may exhibit both proliferative and anti-proliferative actions in normal and carcinoma cells and that the direction of the agonist action is cell-specific.
Several other factors may participate in the cell-specificity of ATP action in a particular tissue. For example, the apoptotic action of ATP in lymphocytes may depend on interactions with other agonists; ATP when added alone initiated apoptosis, but antagonized the apoptotic actions of other agonists (Apasov et al., 1995). The action of ATP may also be dependent on the receptor subtype expressed in a particular tissue. In thymocytes and microglial cells expressing P2X1 and P2X7 receptor channels, respectively, ATP may induce apoptotsis (Chvatchko et al., 1996; Ferrari et al., 1997). In contrast, a transient up-regulation of P2Y2 receptors may represent a signal for differentiation of thymocytes by providing the feedback signalling from extracellular ATP (Koshiba et al., 1997). The apoptotic versus differentiation actions of ATP may also depend on its degradation to adenosine, which in turn activates another class of purinergic receptors, P1 (Abbracchio et al., 1997).
At the present time it is not clear why the activation of the same receptors in cells of the same origin, such as EFO-21/EFO-27 cells versus SKOV-3 cells, leads to such diverse ATP actions on cell growth. The antiproliferative actions of purines were also observed in SKOV-3 cells (Batra and Fadeel, 1994). In their experiments, however, ATP was used and an inhibitory effect was observed at 1 mmol/l ATP concentration. Since ectoATPase are expressed in ovarian epithelial cell lines (Figure 7), it is possible that a rapid degradation of ATP accounts for higher concentration of agonist used in their experiments. Also, the possibility that the degradable ATP products account for the stimulatory action on cell proliferation cannot be excluded. In the current experiments, a slow degradable ATP--S was used, which was effective in both cell types when applied at 100 µmol/l concentration.
In conclusion, the finding that ovarian epithelial cell lines express P2Y2 receptors, whose activation leads to a down-regulation of cell proliferation, might correspond to mechanisms that are also present in the normal ovary, such as regulation of follicular rupture. This should be addressed in additional studies with normal ovarian epithelial cells. In addition, EFO-21 and EFO-27 cells can serve as models for further investigations of the controlling mechanism of the cell cycle by purinergic receptors. Finally, the findings of this study are of potential importance for studies on chemotherapeutic and radiation control of ovarian tumour growth, including the potentiality of adjunctive treatment with slowly degradable ATP analogues. Prior to that, however, further studies should be directed towards the identification of purinergic receptor subtypes in human ovarian cancers.
Acknowledgments
We are thankful to Drs Lixin Zheng and Melanija Tomic for their help in PEt and ATP measurements and Fritz Hoelzel for providing EFO-21 and EFO-27 cells.
Notes
3 To whom correspondence should be addressed at: Section on Cellular Signaling, ERRB/NICHD, Bldg. 49, Room 6A-36, 49 Convent Drive, Bethesda, MD 20892-4510, USA 
References
Abbracchio, M.P., Ceruti, S., Brambilla, R. et al. (1997) Modulation of apoptosis by adenosine in the central nervous system: a possible role for the A3 receptors. Pathophysiological significance and therapeutic implications for neurodegenerative disorders. Ann. NY Acad. Sci., 825, 11–22.[Web of Science][Medline]
Amsterdam, A. and Selvaraj, N. (1997) Control of differentiation, transformation, and apoptosis in granulosa cells by oncogenes, oncoviruses, and tumor suppressor genes. Endocr. Rev., 18, 435–461.
Apasov, S., Koshiba, M., Redegeld, F. et al. (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.[Web of Science][Medline]
Baguley, B.C., Marshal, E.S., Holdaway, K.M. et al. (1998) Inhibition of growth of primary human tumour cell cultures by a 4-anilinoquinazoline inhibitor of the epidermal growth factor receptor family of tyrosine kinases. Eu. J. Cancer, 34, 1086–1090.
Bast, R.C., Boyer, C.M., Jacobs, I. et al. (1993) Cell growth regulation in epithelial ovarian cancer. Cancer 15, (Suppl.), 1597–1601.
Batra, S. and 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.[Web of Science][Medline]
Berchuck, A., Kohler, M.F. and Boente, N.P. et al. (1993) Growth regulation and transformation of ovarian epithelium. Cancer, 71, (Suppl.), 545–551.[Web of Science][Medline]
Cesnjaj, M., Zheng, L., Catt, K.J. et al. (1995) Dependence of stimulus-transcription coupling on phospholipase D in agonist-stimulated pituitary cells. Mol. Biol. Cell, 9, 1037–1047.
Chen, Z.-P., Krull, N., Xu, L. et al. (1996) Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor. Endocrinology, 137, 1833–1840.[Abstract]
Chow, S.C., Kass, G.E.N. and Orrenius, S. (1997) Purines and their roles in apoptosis. Neuropharmacology, 36, 1149–1156.[Web of Science][Medline]
Chvatchko, Y., Valera, S., Aubry, J.-P.R.T.B.G. et al. (1996) The involvement of an ATP-gated ion channel, P2X1, in thymocyte apoptosis. Immunity, 5, 275–283.[Web of Science][Medline]
Dixon, C.J., Bowler, W.B., Fleetwood, P. et al. (1997) Extracellular nucleotides stimulate proliferation in MCF-7 breast cancer cells via P2-purinoceptors. Br. J. Pharmacol., 75, 34–39.
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.
Erlinge, D. (1998) Extracellular ATP: a growth factor for vascular smooth muscle cells. Gen. Pharmacol., 31, 1–8.[Web of Science][Medline]
Estrela, J.M., Obrador, E., Navarro, J. et al. (1995) Elimination of Ehrlich tumours by ATP-induced growth inhibition, glutathione depletion and X-rays. Nature Med., 1, 84–88.[Web of Science][Medline]
Ferrari, D., Chiozzi, P., Falzoni, S. et al. (1997) ATP-mediated cytotoxicity in microglial cells. Neuropharmacology, 36, 1295–1301.[Web of Science][Medline]
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 Acid. Res., 15, 5657–5665.
Hammond, S.M., Jenco, J.M., Nakashima, S. et al. (1997) Characterization of two alternatively spliced forms of phospholipase D1. J. Biol. Chem., 272, 3860–3868.
Havrilesky, L.J., Hurteau, J.A., Whitaker, R.S. et al. (1995) Regulation of apoptosis in normal and malignant ovarian epithelial cells by transforming growth factor ß. Cancer Res., 55, 944–948.
Heintz, A., Hacker, N. and Lagasse, L. (1985) Epidemiology and etiology of ovarian cancer: a review. Obstet. Gynecol., 66, 127–135.[Web of Science][Medline]
Kamada, S., Blackmore, P.F., Oehninger, S. et al. (1994) Existence of P2-purinoreceptors on human and porcine gransulosa cells. J. Clin. Endocrinol. Metab., 78, 650–656.[Abstract]
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.
Kunzmann, R. and Hoelzel, F. (1987) Karyotype alterations in human ovarian carcinoma cells during long-term cultivation and nude mouse passage. Cancer Genet. Cytogenet., 28, 201–212.[Web of Science][Medline]
Lau, K.-M., Mok, S.C. and Ho, S.-M. (1999) Expression of human estrogen receptor- and -ß, progesterone receptor, and androgen receptor mRNA in normal and malignant ovarian epithelial cells. Proc. Natl. Acad. Sci. USA, 96, 5722–5727.
Lee, P.S.N., Squires, P.E., Buchan, A.M.J. et al. (1996) P2-purinoreceptor evoked changes in intracellular calcium oscillations in single isolated human granulosa-lutein cells. Endocrinology, 137, 3756–3761.[Abstract]
Paller, M.S., Schnaith, E.J. and Rosenberg, M.E. (1988) Purinergic receptors mediate cell proliferation and enhanced recovery from renal ischemia by adenosine triphosphate. J. Lab. Clin. Med., 131, 174–183.
Parr, C.E., Sullivan, D.M., Paradiso, A.M. 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.
Popper, L.D. and Batra, S. (1993) Calcium mobilization and cell proliferation activated by extracellular ATP in human ovarian tumour cells. Cell Calcium, 14, 209–218.[Web of Science][Medline]
Rajaniemi, H., Kauppila, A., Ronnberg, L., et al. (1981) LH (hCG) receptor in benign and malignant tumors of human ovary. Acta Obstet. Gynecol. Scand. 101 (Suppl.), 83–87.
Rapaport, E. (1988) Experimental cancer therapy in mice by adenine nucleotides. Eur J. Cancer Clin. Oncol., 24, 1491–1497.[Web of Science][Medline]
Robker, R.L. and Richards, J.S. (1998) Hormonal control of the cell cycle in ovarian cells: proliferation versus differentiation. Biol. Reprod., 59, 476–482.
Schultze-Mosgau, A., Koshimizu, T., Arora K.K. et al. (1998) Characterization of P2Y receptors in human cancer cells and their effector coupling. 80th Annual Endocrine Society Meeting, New Orleans, Endocrine Society, Washington, D.C., USA.
Simon, W.E., Albrecht, M., Haensel, M. et al. (1983) Cell lines derived from ovarian carcinomas: growth stimulation by gonadotropic and steroid hormones. J. Natl. Cancer Inst., 70, 839–845.
Stojilkovic, S.S., Vukicevic, S. and Luyten, F.P. (1994) Calcium signaling in endothelin- and platelet-derived growth factor-stimulated chondrocytes. J. Bone Mineral Res., 9, 705–713.[Web of Science][Medline]
Zheng, L., Stojilkovic, S.S., Hunyady, L. et al. (1994) Sequential activation of phospholipase C and phospholipase D in agonist-stimulated gonadotrophs. Endocrinology, 134, 1446–1454.
Submitted on October 15, 1999; accepted on February 28, 2000.
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