Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 Aug;12(8):3600–3608. doi: 10.1128/mcb.12.8.3600

Evidence that glucocorticoid- and cyclic AMP-induced apoptotic pathways in lymphocytes share distal events.

D R Dowd 1, R L Miesfeld 1
PMCID: PMC364626  PMID: 1378529

Abstract

WEHI7.2 murine lymphocytes undergo apoptotic death when exposed to glucocorticoids or elevated levels of intracellular cyclic AMP (cAMP), and these pathways are initiated by the glucocorticoid receptor (GR) and protein kinase A, respectively. We report the isolation and characterization of a novel WEHI7.2 variant cell line, WR256, which was selected in a single step for growth in the presence of dexamethasone and arose at a frequency of approximately 10(-10). The defect was not GR-related, as WR256 expressed functional GR and underwent GR-dependent events associated with apoptosis, such as hormone-dependent gene transcription and inhibition of cell proliferation. Moreover, the glucocorticoid-resistant phenotype was stable in culture and did not revert after treatment with 5-azacytidine or upon stable expression of GR cDNA. In addition, WR256 did not exhibit the diminished mitochondrial activity commonly associated with apoptosis. Interestingly, WR256 was also found to be resistant to 8-bromo-cAMP and forskolin despite having normal levels of protein kinase A activity and the ability to induce cAMP-dependent transcription. We examined the steady-state transcript levels of bcl-2, a gene whose protein product acts dominantly to inhibit thymocyte apoptosis, to determine whether elevated bcl-2 expression could account for the resistant phenotype. Our data showed that bcl-2 RNA levels were similar in the two cell lines and not altered by either dexamethasone or 8-bromo-cAMP treatment. These results suggest that WR256 exhibits a "deathless" phenotype and has a unique defect in a step of the apoptotic cascade that may be common to the glucocorticoid- and cAMP-mediated cell death pathways.

Full text

PDF

Images in this article

3603Image
on p.3603
3604Image
on p.3604
3605Image
on p.3605
3606Image
on p.3606

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baughman G., Harrigan M. T., Campbell N. F., Nurrish S. J., Bourgeois S. Genes newly identified as regulated by glucocorticoids in murine thymocytes. Mol Endocrinol. 1991 May;5(5):637–644. doi: 10.1210/mend-5-5-637. [DOI] [PubMed] [Google Scholar]
  2. Bettuzzi S., Troiano L., Davalli P., Tropea F., Ingletti M. C., Grassilli E., Monti D., Corti A., Franceschi C. In vivo accumulation of sulfated glycoprotein 2 mRNA in rat thymocytes upon dexamethasone-induced cell death. Biochem Biophys Res Commun. 1991 Mar 29;175(3):810–815. doi: 10.1016/0006-291x(91)91637-r. [DOI] [PubMed] [Google Scholar]
  3. Bourgeois S., Newby R. F. Diploid and haploid states of the glucocorticoid receptor gene of mouse lymphoid cell lines. Cell. 1977 Jun;11(2):423–430. doi: 10.1016/0092-8674(77)90060-5. [DOI] [PubMed] [Google Scholar]
  4. Briehl M. M., Miesfeld R. L. Isolation and characterization of transcripts induced by androgen withdrawal and apoptotic cell death in the rat ventral prostate. Mol Endocrinol. 1991 Oct;5(10):1381–1388. doi: 10.1210/mend-5-10-1381. [DOI] [PubMed] [Google Scholar]
  5. Chi C. W., Ip M. M. Combined therapy with 5-azacytidine and hydrocortisone in glucocorticoid-sensitive and -resistant mouse P1798 lymphosarcoma. J Natl Cancer Inst. 1988 Aug 17;80(12):912–918. doi: 10.1093/jnci/80.12.912. [DOI] [PubMed] [Google Scholar]
  6. Cleary M. L., Smith S. D., Sklar J. Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell. 1986 Oct 10;47(1):19–28. doi: 10.1016/0092-8674(86)90362-4. [DOI] [PubMed] [Google Scholar]
  7. Daniel V., Litwack G., Tomkins G. M. Induction of cytolysis of cultured lymphoma cells by adenosine 3':5'-cyclic monophosphate and the isolation of resistant variants. Proc Natl Acad Sci U S A. 1973 Jan;70(1):76–79. doi: 10.1073/pnas.70.1.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Danielsen M., Peterson D. O., Stallcup M. R. Immunological selection of variant mouse lymphoid cells with altered glucocorticoid responsiveness. Mol Cell Biol. 1983 Jul;3(7):1310–1316. doi: 10.1128/mcb.3.7.1310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Danielsen M., Stallcup M. R. Down-regulation of glucocorticoid receptors in mouse lymphoma cell variants. Mol Cell Biol. 1984 Mar;4(3):449–453. doi: 10.1128/mcb.4.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Vita V. T., Jr, Hubbard S. M., Longo D. L. The chemotherapy of lymphomas: looking back, moving forward--the Richard and Hinda Rosenthal Foundation award lecture. Cancer Res. 1987 Nov 15;47(22):5810–5824. [PubMed] [Google Scholar]
  11. Delegeane A. M., Ferland L. H., Mellon P. L. Tissue-specific enhancer of the human glycoprotein hormone alpha-subunit gene: dependence on cyclic AMP-inducible elements. Mol Cell Biol. 1987 Nov;7(11):3994–4002. doi: 10.1128/mcb.7.11.3994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dieken E. S., Miesfeld R. L. Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis. Mol Cell Biol. 1992 Feb;12(2):589–597. doi: 10.1128/mcb.12.2.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dowd D. R., MacDonald P. N., Komm B. S., Haussler M. R., Miesfeld R. Evidence for early induction of calmodulin gene expression in lymphocytes undergoing glucocorticoid-mediated apoptosis. J Biol Chem. 1991 Oct 5;266(28):18423–18426. [PubMed] [Google Scholar]
  14. Edelman A. M., Blumenthal D. K., Krebs E. G. Protein serine/threonine kinases. Annu Rev Biochem. 1987;56:567–613. doi: 10.1146/annurev.bi.56.070187.003031. [DOI] [PubMed] [Google Scholar]
  15. Friedrich U., Coffino P. Mutagenesis in S49 mouse lymphoma cells: induction of resistance to ouabain, 6-thioguanine, and dibutyryl cyclic AMP. Proc Natl Acad Sci U S A. 1977 Feb;74(2):679–683. doi: 10.1073/pnas.74.2.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Galili U., Peleg A., Milner Y., Galili N. Be13, a human T-leukemia cell line highly sensitive to dexamethasone-induced cytolysis. Cancer Res. 1984 Oct;44(10):4594–4601. [PubMed] [Google Scholar]
  17. Gasson J. C., Bourgeois S. A new determinant of glucocorticoid sensitivity in lymphoid cell lines. J Cell Biol. 1983 Feb;96(2):409–415. doi: 10.1083/jcb.96.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gehring U., Coffino P. Independent mechanisms of cyclic AMP and glucocorticoid action. Nature. 1977 Jul 14;268(5616):167–169. doi: 10.1038/268167a0. [DOI] [PubMed] [Google Scholar]
  19. Gruol D. J., Campbell N. F., Bourgeois S. Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function. J Biol Chem. 1986 Apr 15;261(11):4909–4914. [PubMed] [Google Scholar]
  20. Gruol D. J., Dalton D. K. Phenothiazines cause a shift in the cAMP dose-response: selection of resistant variants in a murine thymoma line. J Cell Physiol. 1984 Apr;119(1):107–118. doi: 10.1002/jcp.1041190118. [DOI] [PubMed] [Google Scholar]
  21. Harbour D. V., Chambon P., Thompson E. B. Steroid mediated lysis of lymphoblasts requires the DNA binding region of the steroid hormone receptor. J Steroid Biochem. 1990 Jan;35(1):1–9. doi: 10.1016/0022-4731(90)90137-h. [DOI] [PubMed] [Google Scholar]
  22. Harmon J. M., Norman M. R., Fowlkes B. J., Thompson E. B. Dexamethasone induces irreversible G1 arrest and death of a human lymphoid cell line. J Cell Physiol. 1979 Feb;98(2):267–278. doi: 10.1002/jcp.1040980203. [DOI] [PubMed] [Google Scholar]
  23. Harris A. W., Bankhurst A. D., Mason S., Warner N. L. Differentiated functions expressed by cultured mouse lymphoma cells. II. Theta antigen, surface immunoglobulin and a receptor for antibody on cells of a thymoma cell line. J Immunol. 1973 Feb;110(2):431–438. [PubMed] [Google Scholar]
  24. Hockenbery D., Nuñez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990 Nov 22;348(6299):334–336. doi: 10.1038/348334a0. [DOI] [PubMed] [Google Scholar]
  25. Horibata K., Harris A. W. Mouse myelomas and lymphomas in culture. Exp Cell Res. 1970 Apr;60(1):61–77. doi: 10.1016/0014-4827(70)90489-1. [DOI] [PubMed] [Google Scholar]
  26. Huet-Minkowski M., Gasson J. C., Bourgeois S. Induction of glucocorticoid-resistant variants in a murine thymoma line by antitumor drugs. Cancer Res. 1981 Nov;41(11 Pt 1):4540–4546. [PubMed] [Google Scholar]
  27. Insel P. A., Bourne H. R., Coffino P., Tomkins G. M. Cyclic AMP-dependent protein kinase: pivotal role in regulation of enzyme induction and growth. Science. 1975 Nov 28;190(4217):896–898. doi: 10.1126/science.171770. [DOI] [PubMed] [Google Scholar]
  28. Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol. 1975 Jul;66(1):188–193. doi: 10.1083/jcb.66.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lanotte M., Riviere J. B., Hermouet S., Houge G., Vintermyr O. K., Gjertsen B. T., Døskeland S. O. Programmed cell death (apoptosis) is induced rapidly and with positive cooperativity by activation of cyclic adenosine monophosphate-kinase I in a myeloid leukemia cell line. J Cell Physiol. 1991 Jan;146(1):73–80. doi: 10.1002/jcp.1041460110. [DOI] [PubMed] [Google Scholar]
  30. Lemaire I., Coffino P. Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP. J Cell Physiol. 1977 Sep;92(3):437–445. doi: 10.1002/jcp.1040920311. [DOI] [PubMed] [Google Scholar]
  31. Lemaire I., Coffino P. Cyclic AMP-induced cytolysis in S49 cells: selection of an unresponsive "deathless" mutant. Cell. 1977 May;11(1):149–155. doi: 10.1016/0092-8674(77)90325-7. [DOI] [PubMed] [Google Scholar]
  32. Mayhew E. Ion transport of ouabain resistant and sensitive Ehrlich ascites carcinoma cells. J Cell Physiol. 1972 Jun;79(3):441–452. doi: 10.1002/jcp.1040790314. [DOI] [PubMed] [Google Scholar]
  33. McConkey D. J., Nicotera P., Hartzell P., Bellomo G., Wyllie A. H., Orrenius S. Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca2+ concentration. Arch Biochem Biophys. 1989 Feb 15;269(1):365–370. doi: 10.1016/0003-9861(89)90119-7. [DOI] [PubMed] [Google Scholar]
  34. McConkey D. J., Orrenius S., Jondal M. Agents that elevate cAMP stimulate DNA fragmentation in thymocytes. J Immunol. 1990 Aug 15;145(4):1227–1230. [PubMed] [Google Scholar]
  35. Nordeen S. K., Young D. A. Glucocorticoid action on rat thymic lymphocytes. Experiments utilizing adenosine to support cellular metabolism lead to a reassessment of catabolic hormone actions. J Biol Chem. 1976 Dec 10;251(23):7295–7303. [PubMed] [Google Scholar]
  36. Norman M. R., Thompson E. B. Characterization of a glucocorticoid-sensitive human lymphoid cell line. Cancer Res. 1977 Oct;37(10):3785–3791. [PubMed] [Google Scholar]
  37. Nuñez G., London L., Hockenbery D., Alexander M., McKearn J. P., Korsmeyer S. J. Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines. J Immunol. 1990 May 1;144(9):3602–3610. [PubMed] [Google Scholar]
  38. Owens G. P., Hahn W. E., Cohen J. J. Identification of mRNAs associated with programmed cell death in immature thymocytes. Mol Cell Biol. 1991 Aug;11(8):4177–4188. doi: 10.1128/mcb.11.8.4177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pezzella F., Tse A. G., Cordell J. L., Pulford K. A., Gatter K. C., Mason D. Y. Expression of the bcl-2 oncogene protein is not specific for the 14;18 chromosomal translocation. Am J Pathol. 1990 Aug;137(2):225–232. [PMC free article] [PubMed] [Google Scholar]
  40. Picard D., Yamamoto K. R. Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 1987 Nov;6(11):3333–3340. doi: 10.1002/j.1460-2075.1987.tb02654.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Roesler W. J., Vandenbark G. R., Hanson R. W. Cyclic AMP and the induction of eukaryotic gene transcription. J Biol Chem. 1988 Jul 5;263(19):9063–9066. [PubMed] [Google Scholar]
  42. Roskoski R., Jr Assays of protein kinase. Methods Enzymol. 1983;99:3–6. doi: 10.1016/0076-6879(83)99034-1. [DOI] [PubMed] [Google Scholar]
  43. Rundlett S. E., Wu X. P., Miesfeld R. L. Functional characterizations of the androgen receptor confirm that the molecular basis of androgen action is transcriptional regulation. Mol Endocrinol. 1990 May;4(5):708–714. doi: 10.1210/mend-4-5-708. [DOI] [PubMed] [Google Scholar]
  44. Sentman C. L., Shutter J. R., Hockenbery D., Kanagawa O., Korsmeyer S. J. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell. 1991 Nov 29;67(5):879–888. doi: 10.1016/0092-8674(91)90361-2. [DOI] [PubMed] [Google Scholar]
  45. Silver B. J., Bokar J. A., Virgin J. B., Vallen E. A., Milsted A., Nilson J. H. Cyclic AMP regulation of the human glycoprotein hormone alpha-subunit gene is mediated by an 18-base-pair element. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2198–2202. doi: 10.1073/pnas.84.8.2198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Strasser A., Harris A. W., Cory S. bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell. 1991 Nov 29;67(5):889–899. doi: 10.1016/0092-8674(91)90362-3. [DOI] [PubMed] [Google Scholar]
  47. Tada H., Shiho O., Kuroshima K., Koyama M., Tsukamoto K. An improved colorimetric assay for interleukin 2. J Immunol Methods. 1986 Nov 6;93(2):157–165. doi: 10.1016/0022-1759(86)90183-3. [DOI] [PubMed] [Google Scholar]
  48. Thompson E. B., Smith J. R., Bourgeois S., Harmon J. M. Glucocorticoid receptors in human leukemias and related diseases. Klin Wochenschr. 1985 Aug 1;63(15):689–698. doi: 10.1007/BF01733111. [DOI] [PubMed] [Google Scholar]
  49. Vanderbilt J. N., Miesfeld R., Maler B. A., Yamamoto K. R. Intracellular receptor concentration limits glucocorticoid-dependent enhancer activity. Mol Endocrinol. 1987 Jan;1(1):68–74. doi: 10.1210/mend-1-1-68. [DOI] [PubMed] [Google Scholar]
  50. Vaux D. L., Cory S., Adams J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988 Sep 29;335(6189):440–442. doi: 10.1038/335440a0. [DOI] [PubMed] [Google Scholar]
  51. Vedeckis W. V., Bradshaw H. D., Jr DNA fragmentation in S49 lymphoma cells killed with glucocorticoids and other agents. Mol Cell Endocrinol. 1983 May;30(2):215–227. doi: 10.1016/0303-7207(83)90049-7. [DOI] [PubMed] [Google Scholar]
  52. Vukmanović S., Zamoyska R. Anti-CD3-induced cell death in T cell hybridomas: mitochondrial failure and DNA fragmentation are distinct events. Eur J Immunol. 1991 Feb;21(2):419–424. doi: 10.1002/eji.1830210225. [DOI] [PubMed] [Google Scholar]
  53. Wells D., Kedes L. Structure of a human histone cDNA: evidence that basally expressed histone genes have intervening sequences and encode polyadenylylated mRNAs. Proc Natl Acad Sci U S A. 1985 May;82(9):2834–2838. doi: 10.1073/pnas.82.9.2834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wyllie A. H., Kerr J. F., Currie A. R. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251–306. doi: 10.1016/s0074-7696(08)62312-8. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES