J Biol Chem, Vol. 274, Issue 47, 33206-33208, November 19, 1999

COMMUNICATION
Increased Proliferation Rate of Lymphoid Cells Transfected with the P2X₇ ATP Receptor^*

O. Roberto Baricordi^§, Loredana Melchiorri, Elena Adinolfi, Simonetta Falzoni^¶, Paola Chiozzi^¶, Gary Buell, and Francesco Di Virgilio^§¶**

From the Sections of  Medical Genetics and ^¶ General Pathology, Department of Experimental and Diagnostic Medicine and the ^§ Biotechnology Center, University of Ferrara, I-44100 Ferrara, Italy and  Ares-Serono, 1228 Geneva, Switzerland

	ABSTRACT

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Human leukocytes can express the P2X₇ purinergic receptor, an ionic channel gated by extracellular ATP, for which the physiological role is only partially understood. Transfection of P2X₇ cDNA into lymphoid cells that lack this receptor sustains their proliferation in serum-free medium. Increased proliferation of serum-starved P2X₇ transfectants is abolished by the P2X₇ receptor blocker oxidized ATP or by the ATP hydrolase apyrase. Both wild type and P2X₇-transfected lymphoid cells release large amounts of ATP into the culture medium. These data suggest the operation of an ATP-based autocrine/paracrine loop that supports lymphoid cell growth in the absence of serum-derived growth factors.

	INTRODUCTION

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Human and mouse leukocytes express the purinergic P2X₇ receptor (1-4). There is evidence that this plasma membrane receptor/pore participates in various macrophage, microglia, and dendritic cell responses such as plasma membrane permeabilization, cytokine release, multinucleated giant cell formation, and apoptosis (5-10), but its physiological function in lymphocytes is unknown. Several authors have proposed extracellular ATP as a novel mediator of cell proliferation, and evidence for such a role also in lymphoid cells has been presented in the past (11-14). Although the P2 receptor subtype involved has never been clearly defined, it is generally believed that the growth stimulating effects of ATP are mediated by P2Y receptors. In a previous study we showed that human peripheral T lymphocytes express a purinergic receptor of the P2X₇ subtype, and we made the surprising observation that its blockade severely decreased the proliferation stimulated by anti-CD3 antibodies, phytohemagglutinin, or allogeneic cells (15). Therefore, we put forward the suggestion that P2X₇ receptors could also mediate a proliferation signal. During the last 2 years, cDNAs encoding the rat, mouse, and human P2X₇ receptor have become available, thus allowing investigation of the effect of P2X₇ transfection on cell proliferation (16-19). In our laboratory we have thoroughly characterized the P2 receptor of several human lymphoid cell lines, among which two, K562 and LG14, lack endogenous P2X₇ receptors as well as functional P2Y receptors (19). In the present study we have compared the proliferation rate and measured ATP release from wild type and P2X₇-transfected K562 and LG14 cells. Our data show that P2X₇ transfection enhances cell proliferation in the absence of exogenous growth factors and that this effect depends on autocrine/paracrine stimulation by released ATP.

	EXPERIMENTAL PROCEDURES

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Cells and Solutions-- K562 leukemic cells and the LG14 B-lymphoblastoid cell line were grown in Iscove's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, and 10 units/ml penicillin. [Ca²⁺]_i measurements were performed in a solution containing 300 mM sucrose, 1 mM MgCl₂, 1 mM K₂HPO₄, 5 mM KHCO₃, 5.5 mM glucose, 1 mM CaCl₂, and 20 mM Hepes (pH adjusted to 7.4 with Tris-HCl). A low sodium solution was chosen to minimize Ca²⁺/Na⁺ competition for permeation through P2X₇ (19).

RNA Isolation and RT-PCR-- Total cytoplasmic RNA was extracted by the acid guanidinium thiocyanate phenol chloroform method, as described by Chomczynski and Sacchi (20). The reverse transcriptase-polymerase chain reaction (RT)¹ was performed following the method of Rappolee et al. (21). Amplification primers and probes were chosen on the basis of cDNA sequences of the human P2X₇ receptor: sense amplimer, 5'-AGATCGTGGAGAATGGAGTG-3'; antisense amplimer, 5'-TCCTCGTGGTGTAGTTGTGG-3'; and probe, 5'-TCATGCACTACACACCTTCC-3'.

Amplification primers for -actin used as a reference for the amount of total mRNA loaded were: 5' primer, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3'; 3' primer, 5'-AGTCATAGTCCGCCTAGAAGCATTTGCGGT-3'.

All oligonucleotides were synthesized by Genenco Life Science Laboratories (Genenco Medical, Firenze, Italy). Blots and labeling of probes were carried out under standard conditions described in digoxigenin labeling and detection protocols from Roche Molecular Biochemicals. RT-PCR amplification (30 cycles) produced a fragment of the expected size, 399 base pairs, for P2X₇. RT-PCR products were separated in 1.2% agarose gel and transferred to a positively charged nylon membrane (Roche Molecular Biochemicals) by a vacuum blotter system (Bio-Rad Laboratories, Hercules, CA) for 2 h. After hybridization with the digoxigenin-labeled P2X₇-specific internal oligoprobe, P2X₇ cDNA was visualized by chemiluminescent detection after incubation with a dilution of anti-digoxigenin Fab fragments conjugated to alkaline phosphatase.

P2X₇ Transfectants-- The vector used for transfection was pcDNA3 from Invitrogen (ampR) with a NotI-NotI insert for the human P2X₇ cDNA. The plasmid was transfected into K562 and LG14 cells by electroporation (Bio-Rad gene pulsar) at 250 V, 960 microfarads. After 24 h, cells were selected with 0.6 mg/ml geneticin.

Proliferation-- Cells were resuspended in Iscove's medium in the presence or absence of 10% fetal calf serum and seeded in 96-well Falcon 3072 plates (Becton Dickinson, Lincoln Park, NJ) in triplicate at 37 °C. After various times, the cell number was established by counting with a phase-contrast Leitz microscope.

Cytoplasmic Free Ca²⁺ Concentration Measurements-- Changes in [Ca²⁺]_i were measured with the fluorescent indicator Fura-2/AM, as described previously (7). Briefly, cells were loaded for 15 min with 2 µM Fura-2/AM and incubated in a thermostat-controlled (37 °C), magnetically stirred fluorometer microcuvette (LS50, Perkin-Elmer) at a concentration of 10⁶/ml in the presence of 250 µM sulfinpyrazone. The intracellular Ca²⁺ concentration was determined with the 340/380 excitation ratio at an emission wavelength of 505 nm.

ATP Measurement-- Cells (25,000/well) were seeded in microtiter plastic dishes in a total volume of culture medium of 100 µl, then rinsed and supplemented with 100 µl of diluent buffer (FireZyme Ltd., San Diego, CA) to stabilize extracellular ATP, and placed directly in the test chamber of a luminometer (FireZyme). Then, 100 µl of a luciferin-luciferase solution (FireZyme) was added, and light emission was recorded.

Data Presentation-- Data shown in the graphs are quadruplicate determinations ± S.D. from a single experiment representative of three similar experiments. In some graphs, error bars are not shown because their width exceeded the dimensions of the symbol.

	RESULTS

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K562 and LG14 cells have been shown previously to be insensitive to stimulation with extracellular ATP (19). RT-PCR analysis (Fig. 1, inset) showed that these cells do not express the P2X₇ receptor mRNA. We therefore used those cells as a model to investigate the effect of P2X₇ transfection on human leukemic cell responses. P2X₇ transfection rendered both K562 and LG14 cells fully sensitive to activation by ATP, as shown by changes in the cytoplasmic Ca²⁺ concentration ([Ca ²⁺]_i) (Fig. 1), whereas wild type cells were unresponsive. The resting [Ca²⁺]_i level of P2X₇ transfectants was consistently higher than that of wild type cells, but the difference was not statistically significant.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effect of ATP on [Ca2+]i changes in wild type and P2X7-transfected K562 and LG14 cells. 106 Cells/ml were incubated in the fluorometer cuvette and stimulated with 1 mM ATP. Ionomycin (iono) was 1 µM. A, LG14wt (lower trace) and LG14P2X7 (upper trace). B, K562wt (lower trace) and K562P2X7 (upper trace). The inset shows P2X7 mRNA expression in wild type and P2X7-transfected cells; human macrophages, M, are also shown as control.

Under standard culture conditions, the growth rates of mock- and P2X₇-transfected cells did not significantly differ (Fig. 2, A and C). However, in the absence of serum, the proliferation of LG14 and K562 cells transfected with the plasmid vector without the P2X₇ insert progressively declined, but the growth of P2X₇-transfected cells (both K562 and LG14) was much less affected (Fig. 2, B and D). After 72 h, the number of K562P2X₇ and LG14P2X₇ cells was 2- and 4-fold higher, respectively, than that of control cells. The growth rates of mock-transfected and wild type cells were the same whether in the presence or absence of serum.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Proliferation rates of P2X7 and mock-transfected K562 and LG14 cells. Cells (105/well) were incubated for the indicated times in complete Iscove's medium (A and C) or in Iscove's medium without serum (panels B and D). A and B, K562 cells. C and D, LG14 cells. Closed circles, cells transfected with the PcDNA3 plasmid without the P2X7 insert (mock-transfected); open circles, cells transfected with P2X7 cDNA.

There are currently very few pharmacological blockers of the P2X₇ receptor. The most potent, KN-62, an isoquinoline derivative, is however also a calmodulin inhibitor and is thus unsuited for long term studies on cell proliferation. In this respect, a better, albeit less potent, agent is oxidized ATP (oATP), an ATP analog that covalently inhibits and thus irreversibly blocks P2X₇. We therefore tested the effect of oATP on wild type and P2X₇-transfected cells.

Fig. 3 shows that treatment with an optimal concentration of 300 µM oATP inhibited proliferation of the P2X₇-transfected clones, whereas a similar treatment had no effect on the wild type cells. It is intriguing that oATP appeared to block exactly the extra proliferation resulting from P2X₇ expression, as in the presence of this agent serum-starved LG14P2X₇ and K562P2X₇ exhibited a proliferation rate very similar to that of the serum-starved wild type parental cells.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of oATP on K562 and LG14 cell proliferation. Cells (105/well) were incubated for 72 h in Iscove's medium under different experimental conditions. Samples treated with oATP (300 µM) were kept in the presence of this inhibitor throughout the experiment. A, K562 cells. B, LG14 cells. Closed bars, wild type cells; open bars, P2X7-transfected cells.

These observations suggested that it was the activation of P2X₇ that enabled transfected LG14 and K562 cells to overcome serum starvation. Because the only known physiologic ligand for P2X₇ is ATP, and ATP is known to be a mitogenic agent in several cell systems (11-15), we measured whether ATP was released in the supernatants of LG14 and K562 cells. The luciferin-luciferase assay showed that K562wt and K562P2X₇ released 17.68 ± 3.75 (average ± S.D.; n = 9) and 15.26 ± 2.8 µg of ATP/10⁶ cells, whereas LG14wt and LG14P2X₇ released 3.5 ± 1.5 and 12.74 ± 5.98 µg of ATP/10⁶ cells. It is therefore reasonable to hypothesize that extracellular ATP provided a stimulus that LG14P2X₇ and K562P2X₇, but not LG14wt and K562wt, were able to exploit to sustain proliferation in the absence of serum-derived growth factors. An implication of this interpretation is that agents such as apyrase that hydrolyze ATP should mimic the effect of oATP. The experiments shown in Fig. 4 confirm this prediction because in the presence of this ATP-hydrolyzing enzyme, the growth rate of serum-starved LG14P2X₇ reverted to that of the wild type, whereas the growth rate of control LG14P2X₇ cells treated with inactivated apyrase was not reduced.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effect of apyrase on growth rate of LG14 cells. Cells (105/ml) were incubated for 72 h in serum-free Iscove's medium. Apyrase was added at the beginning of the incubation and left in samples throughout the experiment. Apyrase was inactivated by heating for 10 min at 100 °C. Closed bars, LG14wt cells; open bars, LG14P2X7.

Over the last few years, extracellular ATP has been implicated in different lymphocyte responses such as gene expression (22), proliferation (11-15), thymic selection (23, 24), and cytotoxicity (3, 8). However, plasma membrane receptors responsible for these various and often contrasting effects are only now being identified. It is generally thought that the growth-promoting activity of ATP depends on P2Y receptor activation, whereas cytotoxic or cytostatic effects are caused by P2X₁ or P2X₇ receptor stimulation (25). Ca²⁺ response studies have shown that human peripheral T lymphocytes and mouse thymocytes do not express functional P2Y receptors (15, 26). A few reports on the mitogenic effect of ATP in mouse lymphocytes have appeared in the past (12, 13), and a study on human T lymphocytes was published by our laboratory in 1996 (15). In this study we showed that ATP potentiated the mitogenic effect of phytohemagglutinin and anti-CD3 antibodies, although by itself ATP had no effect on lymphocyte proliferation. We also observed that this activity was mimicked by benzoyl-ATP, a selective P2X₇ agonist, and inhibited by oATP, thus pointing to an involvement of P2X₇ in lymphocyte proliferation. Our present data support the hypothesis that the P2X₇ receptor in lymphoid cells mediates a growth-promoting signal, but only under those conditions in which other, maybe more powerful, growth factors are absent. It appears that under serum starvation P2X₇-transfected cells are able to take advantage of an autocrine/paracrine loop based on the leak of ATP into the pericellular milieu and the consequent purinergic receptor stimulation. In this respect, an incidental observation, being followed up in our laboratory, is that LG14P2X₇ cells appear to release significantly more ATP than their wild type counter part. Many tumor cell lines exhibit high P2X₇ receptor levels, and early studies by Heppel and co-workers (27) suggested that transformation increases expression of this receptor. P2X₇ receptor expression may therefore be a factor that confers a selective advantage to tumor cells and improves their survival in an unfavorable environment.

	FOOTNOTES

^* This work was supported by the Italian Ministry for Scientific Research (40 and 60%), the National Research Council of Italy (Target Project on Biotechnology), the Italian Association for Cancer Research (AIRC), the IX AIDS Project, the II Tuberculosis Project, and Telethon of Italy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, Via Borsari 46, I-44100 Ferrara, Italy. Tel.: 39-0532-291353; Fax: 39-0532-247278; E-mail: FDV@dns.unife.it.

	ABBREVIATIONS

The abbreviations used are: RT-PCR, reverse transcriptase-polymerase chain reaction; [Ca²⁺]_i, cytosolic free calcium concentration; oATP, oxidized ATP.

	REFERENCES

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