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Purine nucleosides and nucleotides stimulate proliferation of a wide range of cell types

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Summary

Presumptive astrocytes isolated from 10-day white Leghorn chick embryos, Factor VIII-positive human brain capillary endothelial cells, meningeal fibroblasts from 10-day chick embryos, Swiss mouse 3T3 cells, and human astrocytoma cell lines, SKMG-1 and U373, were rendered quiescent when placed in culture medium that contained 0 or 0.2% serum for 48 h; their proliferation was markedly reduced and they incorporated [3H]thymidine at a low rate. [3H]Thymidine incorporation and cell proliferation were induced in all types of cells by addition of guanosine, GMP, GDP, GTP, and to a lesser extent, adenosine, AMP, ADP or ATP to the culture medium. The stimulation of proliferation by adenosine and guanosine was abolished by 1,3-dipropyl-7-methylxanthine (DPMX), an adenosine A2 receptor antagonist, but not by 1,3,-dipropyl-8-(2-amino-4-chorophenyl)xanthine (PACPX), an A1 antagonist. Stimulation of proliferation by the nucleotides was not abolished by either DPMX or PACPX. The P2 receptor agonists,α,β-methyleneATP and 2-methylthioATP, also stimulated [3H]thymidine incorporation into the cells with peak activity at approximately 3.5 and 0.03 nM, respectively. These data imply that adenosine and guanosine stimulate proliferation of these cell types through activation of an adenosine A2 receptor, and the stimulation of cell proliferation by the nucleotides may be due to the activation of purinergic P2y receptors. As the primary cultures grew older their growth rate slowed. The capacity of the purine nucleosides and nucleotides to stimulate their growth diminished concomitantly. The 3T3 cells showed neither decreased growth with increased passages nor reduced response to the purines. In contrast, although the doubling time of the immortalized human astrocytoma cell lines SKMG-1 and U373 remained constant, the responsiveness to purinergic stimulation of the U373 cells decreased but that of the SKMG-1 did not. These data are compatible with a decrease in the number, or the ligand-binding affinity of the purinergic receptors, or a decreased coupling of purinergic receptors to intracellular mediators in primary cells aged in tissue culture.

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References

  1. Butcher, S. P.; Roberts, P. J.; Collins, J. F. Purine nucleotides inhibit the binding of DL-[3H] 2-amino-4-phosphonobutyrate (DL-[3H]APB) tol-glutamate-sensitive sites on rat brain membranes. Biochem. Pharmacol. 35:991–994; 1986.

    Article  PubMed  CAS  Google Scholar 

  2. Carlone, R. L.; Kim, J.-K.; Rathbone, M. P. Purification of a chick brain-derived growth factor by reversed phase high performance liquid chromatography. J. Neurosci. 7:2163–2167; 1987.

    PubMed  CAS  Google Scholar 

  3. Carlone, R. L.; Waters, B. W.; Leonard, S. M., et al. A low-molecular weight chick brain-derived growth factor is mitogenic for cultured astroglia from the chick embryo. Dev. Brain Res. 39:97–104; 1988.

    Article  Google Scholar 

  4. Centelles, J. J.; Franco, R.; Bozal, J. Purification and partial characterization of brain ADA: inhibition by purine compounds and by drugs. J. Neurosci. Res. 19:258–267; 1988.

    Article  PubMed  CAS  Google Scholar 

  5. Choo, A. F.; Logan, D. M.; Rathbone, M. P. Neurotrophic regulation of protein synthesis in regeneration blastemata in vitro: the partial purification and characterization of trophic factors embryonic brain. Exp. Neurol. 73:558–570; 1981.

    Article  PubMed  CAS  Google Scholar 

  6. Costello, P. C.; DelMaestro, R. F. Human cerebral endothelium: isolation and characterization of cells derived from microvessels of nonneoplastic and malignant glial tissue. J. Neuro-Oncol. 8:231–243; 1990.

    Article  CAS  Google Scholar 

  7. Cusack, N. J.; Hourani, S. M. O. Sub-types of P2-purinoceptors. Ann. NY Acad. Sci. 603:172–181; 1990.

    Article  PubMed  CAS  Google Scholar 

  8. Debault, L. E.; Cancilla, P. A. Some properties of isolated endothelial cells in culture. In: Eisenberg, H. M.; Suddith, R. L., eds. The cerebral microvasculature: investigations of the blood-brain barrier. Advances in experimental medicine and biology series, vol. 131. New York: Plenum Press; 1980:257–269.

    Google Scholar 

  9. Engstrom, W.; Zetterberg, A. The relationship between purines, pyrimidines, nucleosides and glutamine for fibroblast cell proliferation. J. Cell Physiol. 120:233–240; 1984.

    Article  PubMed  CAS  Google Scholar 

  10. Folkman, J.; Haudenschild, C. C.; Zetter, B. R. Long-term culture of capillary endothelial cells. Proc. Natl. Acad. Sci. USA 76:5217–5221; 1979.

    Article  PubMed  CAS  Google Scholar 

  11. Gallo, R. C.; Whang-Peng, J.; Perry, S. Isopentenyl adenosine stimulates and inhibits mitosis of human lymphocytes treated with phytohemagglutinin. Science 165:400–402; 1969.

    Article  PubMed  CAS  Google Scholar 

  12. Geiger, J. D.; Nagy, J. I. Adenosine deaminease [3H] and nitrobenzylthioinosine as markers of adenosine metabolism and transport in central purinergic systems. In: Williams, M., ed. Adenosine and adenosine receptors. Clifton, NJ: The Humana Press Inc.; 1990:225–288.

    Google Scholar 

  13. Giulian, D.; Baker, J. S.; Shih, L. N., et al. Interleukin I of the central nervous system is produced by ameboid microglia. J. Exp. Med. 164:594–604; 1986.

    Article  PubMed  CAS  Google Scholar 

  14. Gospodarowicz, D.; Bialecki, H.; Greenburg, G. Purification of the fibroblast growth factor activity from bovine brain. J. Biol. Chem. 253:3736–3743; 1978.

    PubMed  CAS  Google Scholar 

  15. Gysbers, J. W.; Rathbone, M. P. The role of purine nucleotides in the response of brain to injury: II. Stimulation of neurite outgrowth. Abstracts of the 10th Annual Marcus Singer Symposium for Regeneration and Limb Development. 10:9; 1991. Publisher: M. W. Egar, Indiana University School of Med. Indianapolis, IN.

    Google Scholar 

  16. Hagberg, H.; Anderson, P.; Lacadewicz, J., et al. Extracellular adenosine, inosine, hypoxanthine and xanthine in relation to tissue nucleotides and purines in rat striatum during transient ischemia. J. Neurochem.,49:227–2331; 1987.

    Article  PubMed  CAS  Google Scholar 

  17. Hanson, G. R.; Iverson, P. L.; Partlow, L. M. Preparation and partial characterization of highly purified primary cultures of neuronal and non-neuronal (glial) cells from embryonic chick cerebral hemispheres and several other regions of the nervous system. Dev. Brain Res. 3:529–545; 1982.

    Article  Google Scholar 

  18. Heppel, L. A.; Wang, D.; Huang, N., et al. Extracellular ATP is a mitogen for 3T3, 3T6, A431, DDT1-MF2, BALB/MK, NIE-115 and HFF cells. Ann. NY Acad. Sci. 603:432–434; 1990.

    Article  Google Scholar 

  19. Kawamoto, T.; Nishi, M.; Takahashi, K., et al. Stimulation by transforming growth factor-β of epidermal growth factor-dependent growth of aged human fibroblasts: recovery of high affinity EGF receptors and growth stimulation by EGF. In Vitro Cell. Dev. Biol. 25:965–970; 1989.

    Article  PubMed  CAS  Google Scholar 

  20. Kim, J.-K.; Rathbone, M. P.; Middlemiss, P. J., et al. Purinergic stimulation of astroblast proliferation: guanosine and its nucleotides stimulate cell division in chick astroblasts. J. Neurosci. Res. 28:442–455; 1991.

    Article  PubMed  CAS  Google Scholar 

  21. Laloue, M.; Terrine, C.; Guern, J. Cytokinins: metabolism and activity of N6-(Δ2-isopententyl)adenosine and N6-(Δ2-isopentenyl)adenine in tobacco cells and callus. Plant Physiol. 59:478–483; 1977.

    Article  PubMed  CAS  Google Scholar 

  22. Laurenssen, C. M.; Middlemiss, P. J.; Rathbone, M. P. The role of purine nucleosides in the response of brain and other tissues to injury: I. Mechanisms of action of guanosine, adenosine metabolites and deoxynucleosides and nucleotides. Abstracts of the 10th Annual Marcus Singer Symposium for Regeneration and Limb Development. 10:8; 1991. Publisher: M. W. Egar, Indian University School of Med. Indianapolis, IN.

    Google Scholar 

  23. Lukacsko, P.; Krell, R. D. The effects of nucleotides on the response of the isolated guinea-pig urinary bladder to nonadrenergic noncholinergic nerve stimulation. Can. J. Physiol. Pharmacol. 59:1199–1201; 1982.

    Google Scholar 

  24. Meininger, C. J.; Schelling, M. E.; Granger, H. J. Adenosine and hypoxia stimulate proliferation and migration of endothelial cells. Am. J. Physiol. 255:H554-H562; 1988.

    PubMed  CAS  Google Scholar 

  25. Meininger, C.; Granger, J. H. Mechanisms leading to adenosine-stimulated proliferation of microvascular endothelial cells. Am. J. Physiol. 258:H198-H206; 1990.

    PubMed  CAS  Google Scholar 

  26. Middlemiss, P. J.; Rathbone, M. P.; Hooftman, E. Purinergic stimulation of astrocyte proliferation: evidence for involvement of P1-A2 and P2y receptors. Can. Coll. Neuro. Pharm. Abstracts 13:7; 1990.

    Google Scholar 

  27. Norenberg, M. D.; Neary, J. T.; Baker, L., et al. Actions of extracellular ATP on astrocytes in primary culture: protential role in reactive gliosis. Abstract #279. Soc. Neurosci. Abst. 16(1):667; 1990.

    Google Scholar 

  28. Okajima, F.; Tokumitsu, Y.; Kondo, Y., et al. P2-purinergic receptors are coupled to two signal transduction systems leading to inhibition of cAMP generation and to production of inositol triphosphate in rat hepatocytes. J. Biol. Chem. 262:13483–13490; 1987.

    PubMed  CAS  Google Scholar 

  29. Rathbone, M. P.; Middlemiss, P. J.; Kim, J.-K., et al. Stimulation of astrocyte proliferation by purine nucleosides and nucleotides through adenosine A2 and purine P2y receptors. Abstract #413.21. Soc. Neurosci. Abst. 16(2):1000; 1990.

    Google Scholar 

  30. Rathbone, M. P.; Middlemiss, P. J.; Kim, J.-K., et al. Adenosine and its nucleotides stimulate proliferation of chick astroblasts and human astrocytoma cells. Neurosci. Res. 13:1–17; 1992.

    Article  PubMed  CAS  Google Scholar 

  31. Rathbone, M. P.; DeForge, S.; DeLuca, B., et al. Purinergic stimulation of cell division and differentiation: mechanisms and pharmacological implications. Med. Hypotheses. 37:213–219; 1992.

    Article  PubMed  CAS  Google Scholar 

  32. Rozengurt, E. Adenosine receptor activation in quiescent Swiss 3T3 cells. Exp. Cell Res. 139:71–78; 1982.

    Article  PubMed  CAS  Google Scholar 

  33. Schor, S.; Rozengurt, E. Enhancement by purine nucleosides and nucleotides of serum-induced DNA synthesis in quiescent 3T3 cells. J. Cell Physiol. 81:339–346; 1973.

    Article  PubMed  CAS  Google Scholar 

  34. Stone, T. W.; Newby, A. C.; Lloyd, H. G. E. Adenosine release. In: Williams, M., ed. Adenosine and adenosine receptors. Clifton, NJ: The Humana Press, Inc.; 1990:173–223.

    Google Scholar 

  35. Todero, G. J.; Green, H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell. Biol. 17:299–313; 1963.

    Article  Google Scholar 

  36. Vinters, H. V.; Reave, S.; Costello, P. C., et al. Isolation and culture of cells derived from human cerebral microvessels. Cell Tissue Res. 249:657–667; 1987.

    Article  PubMed  CAS  Google Scholar 

  37. Williams, M. Purine receptors in mammalian tissues: pharmacology and functional significance. Ann. Rev. Pharmacol. Toxicol. 27:315–345; 1987.

    Article  CAS  Google Scholar 

  38. Xiong, Y.; Xu, S.; Slakey, L. Modulation of response to adenosine in vascular smooth muscle cells cultured in defined medium. In Vitro Cell. Dev. Biol. 27A:355–362; 1991.

    PubMed  CAS  Google Scholar 

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Rathbone, M.P., Middlemiss, P.J., Gysbers, J.W. et al. Purine nucleosides and nucleotides stimulate proliferation of a wide range of cell types. In Vitro Cell Dev Biol - Animal 28, 529–536 (1992). https://doi.org/10.1007/BF02634137

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