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

Increased Proliferation Rate of Lymphoid Cells Transfected with the P2X7 ATP Receptor THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 47, Issue of November 19, pp. 33206 –33208, 1999 Communication © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. last 2 years, cDNAs encoding the rat, mouse, and human P2X Increased Proliferation Rate of receptor have become available, thus allowing investigation of Lymphoid Cells Transfected with the effect of P2X transfection on cell proliferation (16 –19). In our laboratory we have thoroughly characterized the P2 recep- ATP Receptor* the P2X tor of several human lymphoid cell lines, among which two, K562 and LG14, lack endogenous P2X receptors as well as (Received for publication, July 12, 1999, and in revised form, functional P2Y receptors (19). In the present study we have September 9, 1999) compared the proliferation rate and measured ATP release O. Roberto Baricordi‡§, Loredana Melchiorri‡, from wild type and P2X -transfected K562 and LG14 cells. Our Elena Adinolfi‡, Simonetta Falzoni¶, data show that P2X transfection enhances cell proliferation in Paola Chiozzi¶, Gary Buelli, 7 the absence of exogenous growth factors and that this effect and Francesco Di Virgilio§¶** depends on autocrine/paracrine stimulation by released ATP. From the Sections of ‡Medical Genetics and ¶General Pathology, Department of Experimental and Diagnostic EXPERIMENTAL PROCEDURES Medicine and the §Biotechnology Center, University of Ferrara, I-44100 Ferrara, Italy and iAres-Serono, Cells and Solutions—K562 leukemic cells and the LG14 B-lympho- 1228 Geneva, Switzerland blastoid cell line were grown in Iscove’s medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, and 10 units/ml Human leukocytes can express the P2X purinergic penicillin. [Ca ] measurements were performed in a solution contain- receptor, an ionic channel gated by extracellular ATP, ing 300 mM sucrose, 1 mM MgCl ,1mM K HPO ,5mM KHCO , 5.5 mM 2 2 4 3 for which the physiological role is only partially under- glucose, 1 mM CaCl , and 20 mM Hepes (pH adjusted to 7.4 with 21 1 cDNA into lymphoid cells stood. Transfection of P2X Tris-HCl). A low sodium solution was chosen to minimize Ca /Na competition for permeation through P2X (19). that lack this receptor sustains their proliferation in RNA Isolation and RT-PCR—Total cytoplasmic RNA was extracted serum-free medium. Increased proliferation of serum- by the acid guanidinium thiocyanate phenol chloroform method, as transfectants is abolished by the P2X re- starved P2X 7 7 described by Chomczynski and Sacchi (20). The reverse transcriptase- ceptor blocker oxidized ATP or by the ATP hydrolase polymerase chain reaction (RT) was performed following the method of -transfected lymphoid apyrase. Both wild type and P2X Rappolee et al. (21). Amplification primers and probes were chosen on cells release large amounts of ATP into the culture me- the basis of cDNA sequences of the human P2X receptor: sense dium. These data suggest the operation of an ATP-based amplimer, 59-AGATCGTGGAGAATGGAGTG-39; antisense amplimer, autocrine/paracrine loop that supports lymphoid cell 59-TCCTCGTGGTGTAGTTGTGG-39; and probe, 59-TCATGCACTACA- growth in the absence of serum-derived growth factors. CACCTTCC-39. Amplification primers for b-actin used as a reference for the amount of total mRNA loaded were: 59 primer, 59-TGACGGGGTCACCCACAC- TGTGCCCATCTA-39;39 primer, 59-AGTCATAGTCCGCCTAGAAGCA- Human and mouse leukocytes express the purinergic P2X TTTGCGGT-39. receptor (1– 4). There is evidence that this plasma membrane All oligonucleotides were synthesized by Genenco Life Science Lab- receptor/pore participates in various macrophage, microglia, oratories (Genenco Medical, Firenze, Italy). Blots and labeling of probes and dendritic cell responses such as plasma membrane perme- were carried out under standard conditions described in digoxigenin labeling and detection protocols from Roche Molecular Biochemicals. abilization, cytokine release, multinucleated giant cell forma- RT-PCR amplification (30 cycles) produced a fragment of the expected tion, and apoptosis (5–10), but its physiological function in size, 399 base pairs, for P2X . RT-PCR products were separated in 1.2% lymphocytes is unknown. Several authors have proposed extra- agarose gel and transferred to a positively charged nylon membrane cellular ATP as a novel mediator of cell proliferation, and (Roche Molecular Biochemicals) by a vacuum blotter system (Bio-Rad evidence for such a role also in lymphoid cells has been pre- Laboratories, Hercules, CA) for 2 h. After hybridization with the digoxi- sented in the past (11–14). Although the P2 receptor subtype genin-labeled P2X -specific internal oligoprobe, P2X cDNA was visu- 7 7 alized by chemiluminescent detection after incubation with a dilution of involved has never been clearly defined, it is generally believed anti-digoxigenin Fab fragments conjugated to alkaline phosphatase. that the growth stimulating effects of ATP are mediated by P2X Transfectants—The vector used for transfection was pcDNA3 P2Y receptors. In a previous study we showed that human from Invitrogen (ampR) with a NotI-NotI insert for the human P2X peripheral T lymphocytes express a purinergic receptor of the cDNA. The plasmid was transfected into K562 and LG14 cells by P2X subtype, and we made the surprising observation that its electroporation (Bio-Rad gene pulsar) at 250 V, 960 microfarads. After blockade severely decreased the proliferation stimulated by 24 h, cells were selected with 0.6 mg/ml geneticin. Proliferation—Cells were resuspended in Iscove’s medium in the anti-CD3 antibodies, phytohemagglutinin, or allogeneic cells presence or absence of 10% fetal calf serum and seeded in 96-well (15). Therefore, we put forward the suggestion that P2X re- Falcon 3072 plates (Becton Dickinson, Lincoln Park, NJ) in triplicate at ceptors could also mediate a proliferation signal. During the 37 °C. After various times, the cell number was established by counting with a phase-contrast Leitz microscope. Cytoplasmic Free Ca Concentration Measurements—Changes in * This work was supported by the Italian Ministry for Scientific Research (40 and 60%), the National Research Council of Italy (Target [Ca ] were measured with the fluorescent indicator Fura-2/AM, as Project on Biotechnology), the Italian Association for Cancer Research described previously (7). Briefly, cells were loaded for 15 min with 2 mM (AIRC), the IX AIDS Project, the II Tuberculosis Project, and Telethon Fura-2/AM and incubated in a thermostat-controlled (37 °C), magneti- of Italy. The costs of publication of this article were defrayed in part by cally stirred fluorometer microcuvette (LS50, Perkin-Elmer) at a con- the payment of page charges. This article must therefore be hereby centration of 10 /ml in the presence of 250 mM sulfinpyrazone. The marked “advertisement” in accordance with 18 U.S.C. Section 1734 intracellular Ca concentration was determined with the 340/380 ex- solely to indicate this fact. ** To whom correspondence should be addressed: Dept. of Experi- mental and Diagnostic Medicine, Section of General Pathology, Univer- The abbreviations used are: RT-PCR, reverse transcriptase-polym- sity of Ferrara, Via Borsari 46, I-44100 Ferrara, Italy. Tel.: 39-0532- erase chain reaction; [Ca ] , cytosolic free calcium concentration; 291353; Fax: 39-0532-247278; E-mail: FDV@dns.unife.it. oATP, oxidized ATP. 33206 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. P2X Receptor and Lymphocytes 33207 FIG.3. Effect of oATP on K562 and LG14 cell proliferation. Cells (10 /well) were incubated for 72 h in Iscove’s medium under different experimental conditions. Samples treated with oATP (300 mM) were kept in the presence of this inhibitor throughout the experiment. A, K562 cells. B, LG14 cells. Closed bars, wild type cells; open bars, P2X -transfected cells. FIG.1. Effect of ATP on [Ca ] changes in wild type and P2X - i 7 transfected K562 and LG14 cells. 10 Cells/ml were incubated in the fluorometer cuvette and stimulated with 1 mM ATP. Ionomycin (iono) was 1 mM. A, LG14wt (lower trace) and LG14P2X (upper trace). B, K562wt (lower trace) and K562P2X (upper trace). The inset shows P2X 7 7 mRNA expression in wild type and P2X -transfected cells; human macrophages, MF, are also shown as control. FIG.4. Effect of apyrase on growth rate of LG14 cells. Cells (10 /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 heat- ing for 10 min at 100 °C. Closed bars, LG14wt cells; open bars, LG14P2X . Diego, CA) to stabilize extracellular ATP, and placed directly in the test chamber of a luminometer (FireZyme). Then, 100 ml of a luciferin- luciferase solution (FireZyme) was added, and light emission was recorded. Data Presentation—Data shown in the graphs are quadruplicate FIG.2. Proliferation rates of P2X and mock-transfected K562 determinations 6 S.D. from a single experiment representative of three and LG14 cells. Cells (10 /well) were incubated for the indicated times similar experiments. In some graphs, error bars are not shown because in complete Iscove’s medium (A and C) or in Iscove’s medium without their width exceeded the dimensions of the symbol. 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 RESULTS P2X insert (mock-transfected); open circles, cells transfected with P2X 7 7 cDNA. K562 and LG14 cells have been shown previously to be insensitive to stimulation with extracellular ATP (19). RT-PCR citation ratio at an emission wavelength of 505 nm. analysis (Fig. 1, inset) showed that these cells do not express ATP Measurement—Cells (25,000/well) were seeded in microtiter the P2X receptor mRNA. We therefore used those cells as a plastic dishes in a total volume of culture medium of 100 ml, then rinsed 7 and supplemented with 100 ml of diluent buffer (FireZyme Ltd., San model to investigate the effect of P2X transfection on human 7 33208 P2X Receptor and Lymphocytes leukemic cell responses. P2X transfection rendered both K562 thymocytes do not express functional P2Y receptors (15, 26). A and LG14 cells fully sensitive to activation by ATP, as shown few reports on the mitogenic effect of ATP in mouse lympho- 21 21 by changes in the cytoplasmic Ca concentration ([Ca ] ) cytes have appeared in the past (12, 13), and a study on human (Fig. 1), whereas wild type cells were unresponsive. The resting T lymphocytes was published by our laboratory in 1996 (15). In [Ca ] level of P2X transfectants was consistently higher this study we showed that ATP potentiated the mitogenic effect i 7 than that of wild type cells, but the difference was not statis- of phytohemagglutinin and anti-CD3 antibodies, although by tically significant. itself ATP had no effect on lymphocyte proliferation. We also Under standard culture conditions, the growth rates of mock- observed that this activity was mimicked by benzoyl-ATP, a and P2X -transfected cells did not significantly differ (Fig. 2, A selective P2X agonist, and inhibited by oATP, thus pointing to and C). However, in the absence of serum, the proliferation of an involvement of P2X in lymphocyte proliferation. Our pres- LG14 and K562 cells transfected with the plasmid vector with- ent data support the hypothesis that the P2X receptor in out the P2X insert progressively declined, but the growth of 7 lymphoid cells mediates a growth-promoting signal, but only P2X -transfected cells (both K562 and LG14) was much less 7 under those conditions in which other, maybe more powerful, affected (Fig. 2, B and D). After 72 h, the number of K562P2X growth factors are absent. It appears that under serum star- and LG14P2X cells was 2- and 4-fold higher, respectively, 7 vation P2X -transfected cells are able to take advantage of an than that of control cells. The growth rates of mock-transfected autocrine/paracrine loop based on the leak of ATP into the and wild type cells were the same whether in the presence or pericellular milieu and the consequent purinergic receptor absence of serum. stimulation. In this respect, an incidental observation, being There are currently very few pharmacological blockers of the followed up in our laboratory, is that LG14P2X cells appear to P2X receptor. The most potent, KN-62, an isoquinoline deriv- release significantly more ATP than their wild type counter ative, is however also a calmodulin inhibitor and is thus un- part. Many tumor cell lines exhibit high P2X receptor levels, suited for long term studies on cell proliferation. In this re- and early studies by Heppel and co-workers (27) suggested that spect, a better, albeit less potent, agent is oxidized ATP (oATP), transformation increases expression of this receptor. P2X re- an ATP analog that covalently inhibits and thus irreversibly ceptor expression may therefore be a factor that confers a blocks P2X . We therefore tested the effect of oATP on wild type selective advantage to tumor cells and improves their survival and P2X -transfected cells. in an unfavorable environment. Fig. 3 shows that treatment with an optimal concentration of 300 mM oATP inhibited proliferation of the P2X -transfected REFERENCES clones, whereas a similar treatment had no effect on the wild 1. Wiley, J. S., and Dubyak, G. R. (1989) Blood 73, 1316 –1323 type cells. It is intriguing that oATP appeared to block exactly 2. Di Virgilio, F., Bronte, V., Collavo, D., and Zanovello, P. (1989) J. Immunol. 143, 1955–1960 the extra proliferation resulting from P2X expression, as in 3. Di Virgilio, F. (1995) Immunol. Today 16, 524 –528 the presence of this agent serum-starved LG14P2X and 7 4. Chused, T. M., Apasov, S., and Sitkovsky, M. V. (1996) J. Immunol. 157, 1371–1380 K562P2X exhibited a proliferation rate very similar to that of 5. Steinberg, T. H., and Silverstein, S. C. (1987) J. Biol. Chem. 262, 8884 – 8888 the serum-starved wild type parental cells. 6. Ferrari, D., Chiozzi, P., Falzoni, S., Dal Susino, M., Melchiorri, L., Baricordi, These observations suggested that it was the activation of O. R., and Di Virgilio, F. (1997) J. Immunol. 159, 1451–1458 7. Falzoni, S., Munerati, M., Ferrari, D., Spisani, S., Moretti, S., and Di Virgilio, P2X that enabled transfected LG14 and K562 cells to over- F. (1995) J. Clin. Invest. 95, 1207–1216 come serum starvation. Because the only known physiologic 8. Zanovello, P., Bronte, V., Rosato, A., Pizzo, P., and Di Virgilio, F. (1990) ligand for P2X is ATP, and ATP is known to be a mitogenic J. Immunol. 145, 1545–1550 9. Ferrari, D., Villalba. M., Chiozzi, P., Falzoni, S., Ricciardi-Castagnoli, P., and agent in several cell systems (11–15), we measured whether Di Virgilio, F. (1996) J. Immunol. 156, 1531–1539 ATP was released in the supernatants of LG14 and K562 cells. 10. Coutinho-Silva, R., Persechini, P. M., Bisaggio, R. D., Perfettini, J. L., Neto, A. C., Kanellopulos, J. M., Motta-Ly, I., Dautry-Varsat, A., and Ojcius, The luciferin-luciferase assay showed that K562wt and D. M. (1999) Am. J. Physiol. 276, C1139 –C1147 K562P2X released 17.68 6 3.75 (average 6 S.D.; n 5 9) and 11. Huang, N., Wang, D., Heppel, L. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 15.26 6 2.8 mg of ATP/10 cells, whereas LG14wt and 7904 –7908 12. Ikehara, S., Pahwa, R. P., Lunzer, D. G., Good, R. A., Modak, M. J. (1981) LG14P2X released 3.5 6 1.5 and 12.74 6 5.98 mg of ATP/10 J. Immunol. 127, 1834 –1842 cells. It is therefore reasonable to hypothesize that extracellu- 13. Gregory, S. H., and Kern, M. (1978) Biochem. Biophys. Res. Commun. 83, lar ATP provided a stimulus that LG14P2X and K562P2X , 1111–1115 7 7 14. Neary, J. T., Kang, Y., Bu, Y., Ye, E., Akong, K., and Peters, C. M. (1999) but not LG14wt and K562wt, were able to exploit to sustain J. Neurosci. 19, 4211– 4220 proliferation in the absence of serum-derived growth factors. 15. Baricordi, O. R., Ferrari, D., Melchiorri, L., Chiozzi, P., Hanau, S., Chiari, E., An implication of this interpretation is that agents such as Rubini, M., and Di Virgilio, F. (1996) Blood 87, 682– 690 16. Surprenant, A., Rassendren, F., Kawashima, E., North, R. A., and Buell, G. apyrase that hydrolyze ATP should mimic the effect of oATP. (1996) Science 272, 735–738 The experiments shown in Fig. 4 confirm this prediction be- 17. Rassendren, F., Buell, G. N., Virginio, C., Collo, G., North, R. A., and Surprenant, A. (1997) J. Biol. Chem. 272, 5482–5486 cause in the presence of this ATP-hydrolyzing enzyme, the 18. Michel, A. D., Chessel, I. P., Hibell, A. D., Simon, J., and Humphrey, P. P. A. growth rate of serum-starved LG14P2X reverted to that of the (1998) Br. J. Pharmacol. 125, 1194 –1201 wild type, whereas the growth rate of control LG14P2X cells 19. Ferrari, D., Munerati, M., Melchiorri, L., Hanau, S., Di Virgilio, F., and Baricordi, O. R. (1994) Am. J. Physiol. 267, C886 –C892 treated with inactivated apyrase was not reduced. 20. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156 –159 Over the last few years, extracellular ATP has been impli- 21. Rappolee, D. A., Wang, A., Mark, D., and Werb, Z. (1989) J. Cell. Biochem. 39, cated in different lymphocyte responses such as gene expres- 1–11 22. Padeh, S., Cohen, A., and Roifman, C. M. (1991) J. Immunol. 146, 1626 –1632 sion (22), proliferation (11–15), thymic selection (23, 24), and 23. Chvatchko, Y., Valera, S., Aubry, J. P., Renno, T., Buell, G., and Bonnefoy, cytotoxicity (3, 8). However, plasma membrane receptors re- J. Y. (1996) Immunity 5, 275–283. 24. Freedman, B. D., Liu, Q. H., Gaulton, G., Kotlikoff, M. I., Hescheler, J., and sponsible for these various and often contrasting effects are Fleischmann, B. K. (1999) Eur. J. Immunol. 29, 1635–1646 only now being identified. It is generally thought that the 25. Di Virgilio, F., Chiozzi, P., Falzoni, S., Ferrari, D., Sanz, J. M., Vishwanath, V., growth-promoting activity of ATP depends on P2Y receptor and Baricordi, O. R. (1998) Cell Death Differ. 5, 191–199 26. Chused, T. M., Apasov, S., and Sitkovsky, M. (1996) J. Immunol. 157, activation, whereas cytotoxic or cytostatic effects are caused by 1371–1380 P2X or P2X receptor stimulation (25). Ca response studies 1 7 27. Rozengurt, E., Heppel, L. A., and Friedberg, I. (1977) J. Biol. Chem. 252, have shown that human peripheral T lymphocytes and mouse 4584 – 4590 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

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

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 47, Issue of November 19, pp. 33206 –33208, 1999 Communication © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. last 2 years, cDNAs encoding the rat, mouse, and human P2X Increased Proliferation Rate of receptor have become available, thus allowing investigation of Lymphoid Cells Transfected with the effect of P2X transfection on cell proliferation (16 –19). In our laboratory we have thoroughly characterized the P2 recep- ATP Receptor* the P2X tor of several human lymphoid cell lines, among which two, K562 and LG14, lack endogenous P2X receptors as well as (Received for publication, July 12, 1999, and in revised form, functional P2Y receptors (19). In the present study we have September 9, 1999) compared the proliferation rate and measured ATP release O. Roberto Baricordi‡§, Loredana Melchiorri‡, from wild type and P2X -transfected K562 and LG14 cells. Our Elena Adinolfi‡, Simonetta Falzoni¶, data show that P2X transfection enhances cell proliferation in Paola Chiozzi¶, Gary Buelli, 7 the absence of exogenous growth factors and that this effect and Francesco Di Virgilio§¶** depends on autocrine/paracrine stimulation by released ATP. From the Sections of ‡Medical Genetics and ¶General Pathology, Department of Experimental and Diagnostic EXPERIMENTAL PROCEDURES Medicine and the §Biotechnology Center, University of Ferrara, I-44100 Ferrara, Italy and iAres-Serono, Cells and Solutions—K562 leukemic cells and the LG14 B-lympho- 1228 Geneva, Switzerland blastoid cell line were grown in Iscove’s medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, and 10 units/ml Human leukocytes can express the P2X purinergic penicillin. [Ca ] measurements were performed in a solution contain- receptor, an ionic channel gated by extracellular ATP, ing 300 mM sucrose, 1 mM MgCl ,1mM K HPO ,5mM KHCO , 5.5 mM 2 2 4 3 for which the physiological role is only partially under- glucose, 1 mM CaCl , and 20 mM Hepes (pH adjusted to 7.4 with 21 1 cDNA into lymphoid cells stood. Transfection of P2X Tris-HCl). A low sodium solution was chosen to minimize Ca /Na competition for permeation through P2X (19). that lack this receptor sustains their proliferation in RNA Isolation and RT-PCR—Total cytoplasmic RNA was extracted serum-free medium. Increased proliferation of serum- by the acid guanidinium thiocyanate phenol chloroform method, as transfectants is abolished by the P2X re- starved P2X 7 7 described by Chomczynski and Sacchi (20). The reverse transcriptase- ceptor blocker oxidized ATP or by the ATP hydrolase polymerase chain reaction (RT) was performed following the method of -transfected lymphoid apyrase. Both wild type and P2X Rappolee et al. (21). Amplification primers and probes were chosen on cells release large amounts of ATP into the culture me- the basis of cDNA sequences of the human P2X receptor: sense dium. These data suggest the operation of an ATP-based amplimer, 59-AGATCGTGGAGAATGGAGTG-39; antisense amplimer, autocrine/paracrine loop that supports lymphoid cell 59-TCCTCGTGGTGTAGTTGTGG-39; and probe, 59-TCATGCACTACA- growth in the absence of serum-derived growth factors. CACCTTCC-39. Amplification primers for b-actin used as a reference for the amount of total mRNA loaded were: 59 primer, 59-TGACGGGGTCACCCACAC- TGTGCCCATCTA-39;39 primer, 59-AGTCATAGTCCGCCTAGAAGCA- Human and mouse leukocytes express the purinergic P2X TTTGCGGT-39. receptor (1– 4). There is evidence that this plasma membrane All oligonucleotides were synthesized by Genenco Life Science Lab- receptor/pore participates in various macrophage, microglia, oratories (Genenco Medical, Firenze, Italy). Blots and labeling of probes and dendritic cell responses such as plasma membrane perme- were carried out under standard conditions described in digoxigenin labeling and detection protocols from Roche Molecular Biochemicals. abilization, cytokine release, multinucleated giant cell forma- RT-PCR amplification (30 cycles) produced a fragment of the expected tion, and apoptosis (5–10), but its physiological function in size, 399 base pairs, for P2X . RT-PCR products were separated in 1.2% lymphocytes is unknown. Several authors have proposed extra- agarose gel and transferred to a positively charged nylon membrane cellular ATP as a novel mediator of cell proliferation, and (Roche Molecular Biochemicals) by a vacuum blotter system (Bio-Rad evidence for such a role also in lymphoid cells has been pre- Laboratories, Hercules, CA) for 2 h. After hybridization with the digoxi- sented in the past (11–14). Although the P2 receptor subtype genin-labeled P2X -specific internal oligoprobe, P2X cDNA was visu- 7 7 alized by chemiluminescent detection after incubation with a dilution of involved has never been clearly defined, it is generally believed anti-digoxigenin Fab fragments conjugated to alkaline phosphatase. that the growth stimulating effects of ATP are mediated by P2X Transfectants—The vector used for transfection was pcDNA3 P2Y receptors. In a previous study we showed that human from Invitrogen (ampR) with a NotI-NotI insert for the human P2X peripheral T lymphocytes express a purinergic receptor of the cDNA. The plasmid was transfected into K562 and LG14 cells by P2X subtype, and we made the surprising observation that its electroporation (Bio-Rad gene pulsar) at 250 V, 960 microfarads. After blockade severely decreased the proliferation stimulated by 24 h, cells were selected with 0.6 mg/ml geneticin. Proliferation—Cells were resuspended in Iscove’s medium in the anti-CD3 antibodies, phytohemagglutinin, or allogeneic cells presence or absence of 10% fetal calf serum and seeded in 96-well (15). Therefore, we put forward the suggestion that P2X re- Falcon 3072 plates (Becton Dickinson, Lincoln Park, NJ) in triplicate at ceptors could also mediate a proliferation signal. During the 37 °C. After various times, the cell number was established by counting with a phase-contrast Leitz microscope. Cytoplasmic Free Ca Concentration Measurements—Changes in * This work was supported by the Italian Ministry for Scientific Research (40 and 60%), the National Research Council of Italy (Target [Ca ] were measured with the fluorescent indicator Fura-2/AM, as Project on Biotechnology), the Italian Association for Cancer Research described previously (7). Briefly, cells were loaded for 15 min with 2 mM (AIRC), the IX AIDS Project, the II Tuberculosis Project, and Telethon Fura-2/AM and incubated in a thermostat-controlled (37 °C), magneti- of Italy. The costs of publication of this article were defrayed in part by cally stirred fluorometer microcuvette (LS50, Perkin-Elmer) at a con- the payment of page charges. This article must therefore be hereby centration of 10 /ml in the presence of 250 mM sulfinpyrazone. The marked “advertisement” in accordance with 18 U.S.C. Section 1734 intracellular Ca concentration was determined with the 340/380 ex- solely to indicate this fact. ** To whom correspondence should be addressed: Dept. of Experi- mental and Diagnostic Medicine, Section of General Pathology, Univer- The abbreviations used are: RT-PCR, reverse transcriptase-polym- sity of Ferrara, Via Borsari 46, I-44100 Ferrara, Italy. Tel.: 39-0532- erase chain reaction; [Ca ] , cytosolic free calcium concentration; 291353; Fax: 39-0532-247278; E-mail: FDV@dns.unife.it. oATP, oxidized ATP. 33206 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. P2X Receptor and Lymphocytes 33207 FIG.3. Effect of oATP on K562 and LG14 cell proliferation. Cells (10 /well) were incubated for 72 h in Iscove’s medium under different experimental conditions. Samples treated with oATP (300 mM) were kept in the presence of this inhibitor throughout the experiment. A, K562 cells. B, LG14 cells. Closed bars, wild type cells; open bars, P2X -transfected cells. FIG.1. Effect of ATP on [Ca ] changes in wild type and P2X - i 7 transfected K562 and LG14 cells. 10 Cells/ml were incubated in the fluorometer cuvette and stimulated with 1 mM ATP. Ionomycin (iono) was 1 mM. A, LG14wt (lower trace) and LG14P2X (upper trace). B, K562wt (lower trace) and K562P2X (upper trace). The inset shows P2X 7 7 mRNA expression in wild type and P2X -transfected cells; human macrophages, MF, are also shown as control. FIG.4. Effect of apyrase on growth rate of LG14 cells. Cells (10 /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 heat- ing for 10 min at 100 °C. Closed bars, LG14wt cells; open bars, LG14P2X . Diego, CA) to stabilize extracellular ATP, and placed directly in the test chamber of a luminometer (FireZyme). Then, 100 ml of a luciferin- luciferase solution (FireZyme) was added, and light emission was recorded. Data Presentation—Data shown in the graphs are quadruplicate FIG.2. Proliferation rates of P2X and mock-transfected K562 determinations 6 S.D. from a single experiment representative of three and LG14 cells. Cells (10 /well) were incubated for the indicated times similar experiments. In some graphs, error bars are not shown because in complete Iscove’s medium (A and C) or in Iscove’s medium without their width exceeded the dimensions of the symbol. 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 RESULTS P2X insert (mock-transfected); open circles, cells transfected with P2X 7 7 cDNA. K562 and LG14 cells have been shown previously to be insensitive to stimulation with extracellular ATP (19). RT-PCR citation ratio at an emission wavelength of 505 nm. analysis (Fig. 1, inset) showed that these cells do not express ATP Measurement—Cells (25,000/well) were seeded in microtiter the P2X receptor mRNA. We therefore used those cells as a plastic dishes in a total volume of culture medium of 100 ml, then rinsed 7 and supplemented with 100 ml of diluent buffer (FireZyme Ltd., San model to investigate the effect of P2X transfection on human 7 33208 P2X Receptor and Lymphocytes leukemic cell responses. P2X transfection rendered both K562 thymocytes do not express functional P2Y receptors (15, 26). A and LG14 cells fully sensitive to activation by ATP, as shown few reports on the mitogenic effect of ATP in mouse lympho- 21 21 by changes in the cytoplasmic Ca concentration ([Ca ] ) cytes have appeared in the past (12, 13), and a study on human (Fig. 1), whereas wild type cells were unresponsive. The resting T lymphocytes was published by our laboratory in 1996 (15). In [Ca ] level of P2X transfectants was consistently higher this study we showed that ATP potentiated the mitogenic effect i 7 than that of wild type cells, but the difference was not statis- of phytohemagglutinin and anti-CD3 antibodies, although by tically significant. itself ATP had no effect on lymphocyte proliferation. We also Under standard culture conditions, the growth rates of mock- observed that this activity was mimicked by benzoyl-ATP, a and P2X -transfected cells did not significantly differ (Fig. 2, A selective P2X agonist, and inhibited by oATP, thus pointing to and C). However, in the absence of serum, the proliferation of an involvement of P2X in lymphocyte proliferation. Our pres- LG14 and K562 cells transfected with the plasmid vector with- ent data support the hypothesis that the P2X receptor in out the P2X insert progressively declined, but the growth of 7 lymphoid cells mediates a growth-promoting signal, but only P2X -transfected cells (both K562 and LG14) was much less 7 under those conditions in which other, maybe more powerful, affected (Fig. 2, B and D). After 72 h, the number of K562P2X growth factors are absent. It appears that under serum star- and LG14P2X cells was 2- and 4-fold higher, respectively, 7 vation P2X -transfected cells are able to take advantage of an than that of control cells. The growth rates of mock-transfected autocrine/paracrine loop based on the leak of ATP into the and wild type cells were the same whether in the presence or pericellular milieu and the consequent purinergic receptor absence of serum. stimulation. In this respect, an incidental observation, being There are currently very few pharmacological blockers of the followed up in our laboratory, is that LG14P2X cells appear to P2X receptor. The most potent, KN-62, an isoquinoline deriv- release significantly more ATP than their wild type counter ative, is however also a calmodulin inhibitor and is thus un- part. Many tumor cell lines exhibit high P2X receptor levels, suited for long term studies on cell proliferation. In this re- and early studies by Heppel and co-workers (27) suggested that spect, a better, albeit less potent, agent is oxidized ATP (oATP), transformation increases expression of this receptor. P2X re- an ATP analog that covalently inhibits and thus irreversibly ceptor expression may therefore be a factor that confers a blocks P2X . We therefore tested the effect of oATP on wild type selective advantage to tumor cells and improves their survival and P2X -transfected cells. in an unfavorable environment. Fig. 3 shows that treatment with an optimal concentration of 300 mM oATP inhibited proliferation of the P2X -transfected REFERENCES clones, whereas a similar treatment had no effect on the wild 1. Wiley, J. S., and Dubyak, G. R. (1989) Blood 73, 1316 –1323 type cells. It is intriguing that oATP appeared to block exactly 2. Di Virgilio, F., Bronte, V., Collavo, D., and Zanovello, P. (1989) J. Immunol. 143, 1955–1960 the extra proliferation resulting from P2X expression, as in 3. Di Virgilio, F. (1995) Immunol. Today 16, 524 –528 the presence of this agent serum-starved LG14P2X and 7 4. Chused, T. M., Apasov, S., and Sitkovsky, M. V. (1996) J. Immunol. 157, 1371–1380 K562P2X exhibited a proliferation rate very similar to that of 5. Steinberg, T. H., and Silverstein, S. C. (1987) J. Biol. Chem. 262, 8884 – 8888 the serum-starved wild type parental cells. 6. Ferrari, D., Chiozzi, P., Falzoni, S., Dal Susino, M., Melchiorri, L., Baricordi, These observations suggested that it was the activation of O. R., and Di Virgilio, F. (1997) J. Immunol. 159, 1451–1458 7. Falzoni, S., Munerati, M., Ferrari, D., Spisani, S., Moretti, S., and Di Virgilio, P2X that enabled transfected LG14 and K562 cells to over- F. (1995) J. Clin. Invest. 95, 1207–1216 come serum starvation. Because the only known physiologic 8. Zanovello, P., Bronte, V., Rosato, A., Pizzo, P., and Di Virgilio, F. (1990) ligand for P2X is ATP, and ATP is known to be a mitogenic J. Immunol. 145, 1545–1550 9. Ferrari, D., Villalba. M., Chiozzi, P., Falzoni, S., Ricciardi-Castagnoli, P., and agent in several cell systems (11–15), we measured whether Di Virgilio, F. (1996) J. Immunol. 156, 1531–1539 ATP was released in the supernatants of LG14 and K562 cells. 10. Coutinho-Silva, R., Persechini, P. M., Bisaggio, R. D., Perfettini, J. L., Neto, A. C., Kanellopulos, J. M., Motta-Ly, I., Dautry-Varsat, A., and Ojcius, The luciferin-luciferase assay showed that K562wt and D. M. (1999) Am. J. Physiol. 276, C1139 –C1147 K562P2X released 17.68 6 3.75 (average 6 S.D.; n 5 9) and 11. Huang, N., Wang, D., Heppel, L. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 15.26 6 2.8 mg of ATP/10 cells, whereas LG14wt and 7904 –7908 12. Ikehara, S., Pahwa, R. P., Lunzer, D. G., Good, R. A., Modak, M. J. (1981) LG14P2X released 3.5 6 1.5 and 12.74 6 5.98 mg of ATP/10 J. Immunol. 127, 1834 –1842 cells. It is therefore reasonable to hypothesize that extracellu- 13. Gregory, S. H., and Kern, M. (1978) Biochem. Biophys. Res. Commun. 83, lar ATP provided a stimulus that LG14P2X and K562P2X , 1111–1115 7 7 14. Neary, J. T., Kang, Y., Bu, Y., Ye, E., Akong, K., and Peters, C. M. (1999) but not LG14wt and K562wt, were able to exploit to sustain J. Neurosci. 19, 4211– 4220 proliferation in the absence of serum-derived growth factors. 15. Baricordi, O. 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Published: Nov 1, 1999

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