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

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 P2X7receptor 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.

Human and mouse leukocytes express the purinergic P2X 7 receptor (1)(2)(3)(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)(12)(13)(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 P2X 7 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 P2X 7 receptors could also mediate a proliferation signal. During the last 2 years, cDNAs encoding the rat, mouse, and human P2X 7 receptor have become available, thus allowing investigation of the effect of P2X 7 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 P2X 7 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 P2X 7 -transfected K562 and LG14 cells. Our data show that P2X 7 transfection enhances cell proliferation in the absence of exogenous growth factors and that this effect depends on autocrine/paracrine stimulation by released ATP.
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 P2X 7 . 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 P2X 7 -specific internal oligoprobe, P2X 7 cDNA was visualized by chemiluminescent detection after incubation with a dilution of anti-digoxigenin Fab fragments conjugated to alkaline phosphatase.
P2X 7 Transfectants-The vector used for transfection was pcDNA3 from Invitrogen (ampR) with a NotI-NotI insert for the human P2X 7 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 Ca 2ϩ Concentration Measurements-Changes in [Ca 2ϩ ] i were measured with the fluorescent indicator Fura-2/AM, as described previously (7). Briefly, cells were loaded for 15 min with 2 M Fura-2/AM and incubated in a thermostat-controlled (37°C), magnetically stirred fluorometer microcuvette (LS50, Perkin-Elmer) at a concentration of 10 6  ATP Measurement-Cells (25,000/well) were seeded in microtiter plastic dishes in a total volume of culture medium of 100 l, then rinsed and supplemented with 100 l 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 l of a luciferinluciferase solution (FireZyme) was added, and light emission was recorded.
Data Presentation-Data shown in the graphs are quadruplicate determinations Ϯ 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
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 P2X 7 receptor mRNA. We therefore used those cells as a model to investigate the effect of P2X 7 transfection on human leukemic cell responses. P2X 7 transfection rendered both K562 and LG14 cells fully sensitive to activation by ATP, as shown by changes in the cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] i ) (Fig. 1), whereas wild type cells were unresponsive. The resting [Ca 2ϩ ] i level of P2X 7 transfectants was consistently higher than that of wild type cells, but the difference was not statistically significant.
Under standard culture conditions, the growth rates of mockand P2X 7 -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 P2X 7 insert progressively declined, but the growth of P2X 7 -transfected cells (both K562 and LG14) was much less affected (Fig. 2, B and D). After 72 h, the number of K562P2X 7 and LG14P2X 7 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.
There are currently very few pharmacological blockers of the P2X 7 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 P2X 7 . We therefore tested the effect of oATP on wild type and P2X 7 -transfected cells. Fig. 3 shows that treatment with an optimal concentration of 300 M oATP inhibited proliferation of the P2X 7 -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 P2X 7 expression, as in the presence of this agent serum-starved LG14P2X 7 and K562P2X 7 exhibited a proliferation rate very similar to that of the serum-starved wild type parental cells.
These observations suggested that it was the activation of P2X 7 that enabled transfected LG14 and K562 cells to overcome serum starvation. Because the only known physiologic ligand for P2X 7 is ATP, and ATP is known to be a mitogenic agent in several cell systems (11)(12)(13)(14)(15), we measured whether ATP was released in the supernatants of LG14 and K562 cells. The luciferin-luciferase assay showed that K562wt and K562P2X 7 released 17.68 Ϯ 3.75 (average Ϯ S.D.; n ϭ 9) and 15.26 Ϯ 2.8 g of ATP/10 6 cells, whereas LG14wt and LG14P2X 7 released 3.5 Ϯ 1.5 and 12.74 Ϯ 5.98 g of ATP/10 6 cells. It is therefore reasonable to hypothesize that extracellular ATP provided a stimulus that LG14P2X 7 and K562P2X 7 , 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 LG14P2X 7 reverted to that of the wild type, whereas the growth rate of control LG14P2X 7 cells treated with inactivated apyrase was not reduced.
Over the last few years, extracellular ATP has been implicated in different lymphocyte responses such as gene expression (22), proliferation (11)(12)(13)(14)(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 P2X 1 or P2X 7 receptor stimulation (25). Ca 2ϩ 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 P2X 7 agonist, and inhibited by oATP, thus pointing to an involvement of P2X 7 in lymphocyte proliferation. Our present data support the hypothesis that the P2X 7 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 P2X 7 -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 LG14P2X 7 cells appear to release significantly more ATP than their wild type counter part. Many tumor cell lines exhibit high P2X 7 receptor levels, and early studies by Heppel and co-workers (27) suggested that transformation increases expression of this receptor. P2X 7 receptor expression may therefore be a factor that confers a selective advantage to tumor cells and improves their survival in an unfavorable environment.