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. 1994 Sep;14(9):6125–6134. doi: 10.1128/mcb.14.9.6125

Evidence for trans regulation of apoptosis in intertypic somatic cell hybrids.

H Gourdeau 1, P R Walker 1
PMCID: PMC359139  PMID: 8065345

Abstract

The genetic components required for glucocorticoid induction of apoptosis were studied by using somatic cell hybridization. Intertypic whole-cell hybrids were generated by crossing the glucocorticoid-resistant rat liver cell line Fado-2 with the glucocorticoid-sensitive mouse thymoma cell line BW5147.3. Morphological and biochemical criteria were used to assess sensitivity or resistance to glucocorticoid-induced cell death. Both phenotypes were observed, and all of the hybrids retained a functional glucocorticoid receptor as judged by their abilities to induce the metallothionein gene in response to dexamethasone (Dex). Sensitivity to apoptosis did not correlate with morphological phenotype in that not all suspension cells were sensitive. The effect of glucocorticoids on the expression of apoptosis-linked genes was analyzed in a subset of Dex-sensitive and Dex-resistant hybrids. p53 and c-myc mRNAs were present in parental cells as well as sensitive and resistant hybrid cells, and their levels were not affected by glucocorticoid treatment. bcl-2 expression was restricted to the thymoma cell line and was also not affected by glucocorticoids. We did not detect any bcl-2 mRNA in the hepatoma cell line and the hybrids, suggesting that, as with most tissue-specific genes, bcl-2 is regulated in trans. Furthermore, while the majority of hybrids analyzed retained a full complement of mouse chromosomes, sensitive hybrids were missing some rat chromosomes (preferentially chromosomes 16 and 19), indicating that apoptosis is subject to trans repression. Resistant cells thus appear to repress the activity or synthesis of a nuclear factor that interacts with a glucocorticoid-dependent gene(s) to activate the cell death pathway.

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Selected References

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

  1. Allan M., Harrison P. Co-expression of differentiation markers in hybrids between Friend cells and lymphoid cells and the influence of the cell shape. Cell. 1980 Feb;19(2):437–447. doi: 10.1016/0092-8674(80)90518-8. [DOI] [PubMed] [Google Scholar]
  2. Askew D. S., Ashmun R. A., Simmons B. C., Cleveland J. L. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene. 1991 Oct;6(10):1915–1922. [PubMed] [Google Scholar]
  3. Bakhshi A., Jensen J. P., Goldman P., Wright J. J., McBride O. W., Epstein A. L., Korsmeyer S. J. Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell. 1985 Jul;41(3):899–906. doi: 10.1016/s0092-8674(85)80070-2. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Beato M. Gene regulation by steroid hormones. Cell. 1989 Feb 10;56(3):335–344. doi: 10.1016/0092-8674(89)90237-7. [DOI] [PubMed] [Google Scholar]
  6. Boshart M., Weih F., Nichols M., Schütz G. The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-dependent protein kinase. Cell. 1991 Sep 6;66(5):849–859. doi: 10.1016/0092-8674(91)90432-x. [DOI] [PubMed] [Google Scholar]
  7. Bourgeois S., Newby R. F. Correlation between glucocorticoid receptor and cytolytic response of murine lymphoid cell lines. Cancer Res. 1979 Nov;39(11):4749–4751. [PubMed] [Google Scholar]
  8. 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]
  9. Bourgeois S., Newby R. F., Huet M. Glucorticoid resistance in murine lymphoma and thymoma lines. Cancer Res. 1978 Nov;38(11 Pt 2):4279–4284. [PubMed] [Google Scholar]
  10. Chen T. R. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res. 1977 Feb;104(2):255–262. doi: 10.1016/0014-4827(77)90089-1. [DOI] [PubMed] [Google Scholar]
  11. Clarke A. R., Purdie C. A., Harrison D. J., Morris R. G., Bird C. C., Hooper M. L., Wyllie A. H. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature. 1993 Apr 29;362(6423):849–852. doi: 10.1038/362849a0. [DOI] [PubMed] [Google Scholar]
  12. Cleary M. L., Sklar J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint-cluster region near a transcriptionally active locus on chromosome 18. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7439–7443. doi: 10.1073/pnas.82.21.7439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cowan N. J., Dobner P. R., Fuchs E. V., Cleveland D. W. Expression of human alpha-tubulin genes: interspecies conservation of 3' untranslated regions. Mol Cell Biol. 1983 Oct;3(10):1738–1745. doi: 10.1128/mcb.3.10.1738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Darlington G. J., Rankin J. K., Schlanger G. Expression of human hepatic genes in somatic cell hybrids. Somatic Cell Genet. 1982 May;8(3):403–412. doi: 10.1007/BF01538896. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Dipasquale B., Youle R. J. Programmed cell death in heterokaryons. A study of the transfer of apoptosis between nuclei. Am J Pathol. 1992 Dec;141(6):1471–1479. [PMC free article] [PubMed] [Google Scholar]
  17. Ellis R. E., Yuan J. Y., Horvitz H. R. Mechanisms and functions of cell death. Annu Rev Cell Biol. 1991;7:663–698. doi: 10.1146/annurev.cb.07.110191.003311. [DOI] [PubMed] [Google Scholar]
  18. Evan G. I., Wyllie A. H., Gilbert C. S., Littlewood T. D., Land H., Brooks M., Waters C. M., Penn L. Z., Hancock D. C. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 1992 Apr 3;69(1):119–128. doi: 10.1016/0092-8674(92)90123-t. [DOI] [PubMed] [Google Scholar]
  19. Favaloro J., Treisman R., Kamen R. Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol. 1980;65(1):718–749. doi: 10.1016/s0076-6879(80)65070-8. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Gourdeau H., Fournier R. E. Genetic analysis of mammalian cell differentiation. Annu Rev Cell Biol. 1990;6:69–94. doi: 10.1146/annurev.cb.06.110190.000441. [DOI] [PubMed] [Google Scholar]
  22. Gourdeau H., Peterson T. C., Fournier R. E. Differential activity of a tissue-specific extinguisher locus in hepatic and nonhepatic cells. Mol Cell Biol. 1989 May;9(5):1813–1822. doi: 10.1128/mcb.9.5.1813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Harmon J. M., Thompson E. B., Baione K. A. Analysis of glucocorticoid-resistant human leukemic cells by somatic cell hybridization. Cancer Res. 1985 Apr;45(4):1587–1593. [PubMed] [Google Scholar]
  25. Harmon J. M., Thompson E. B. Isolation and characterization of dexamethasone-resistant mutants from human lymphoid cell line CEM-C7. Mol Cell Biol. 1981 Jun;1(6):512–521. doi: 10.1128/mcb.1.6.512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Harrigan M. T., Baughman G., Campbell N. F., Bourgeois S. Isolation and characterization of glucocorticoid- and cyclic AMP-induced genes in T lymphocytes. Mol Cell Biol. 1989 Aug;9(8):3438–3446. doi: 10.1128/mcb.9.8.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hockenbery D. M., Zutter M., Hickey W., Nahm M., Korsmeyer S. J. BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6961–6965. doi: 10.1073/pnas.88.16.6961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Hyman R., Stallings V. Complementation patterns of Thy-1 variants and evidence that antigen loss variants "pre-exist" in the parental population. J Natl Cancer Inst. 1974 Feb;52(2):429–436. doi: 10.1093/jnci/52.2.429. [DOI] [PubMed] [Google Scholar]
  30. Jones K. W., Shapero M. H., Chevrette M., Fournier R. E. Subtractive hybridization cloning of a tissue-specific extinguisher: TSE1 encodes a regulatory subunit of protein kinase A. Cell. 1991 Sep 6;66(5):861–872. doi: 10.1016/0092-8674(91)90433-y. [DOI] [PubMed] [Google Scholar]
  31. Killary A. M., Fournier R. E. A genetic analysis of extinction: trans-dominant loci regulate expression of liver-specific traits in hepatoma hybrid cells. Cell. 1984 Sep;38(2):523–534. doi: 10.1016/0092-8674(84)90507-5. [DOI] [PubMed] [Google Scholar]
  32. Kozak C. A., Lawrence J. B., Ruddle F. H. A sequential staining technique for the chromosomal analysis of the interspecific mouse/hamster and mouse/human somatic cell hybrids. Exp Cell Res. 1977 Mar 1;105(1):109–117. doi: 10.1016/0014-4827(77)90156-2. [DOI] [PubMed] [Google Scholar]
  33. Kwast-Welfeld J., de Belle I., Walker P. R., Isaacs R. J., Whitfield J. F., Sikorska M. Changes in cyclic adenosine monophosphate-responsive element binding proteins in rat hepatomas. Cancer Res. 1991 Jan 15;51(2):528–535. [PubMed] [Google Scholar]
  34. Lowe S. W., Schmitt E. M., Smith S. W., Osborne B. A., Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature. 1993 Apr 29;362(6423):847–849. doi: 10.1038/362847a0. [DOI] [PubMed] [Google Scholar]
  35. Mayo K. E., Palmiter R. D. Glucocorticoid regulation of metallothionein-I mRNA synthesis in cultured mouse cells. J Biol Chem. 1981 Mar 25;256(6):2621–2624. [PubMed] [Google Scholar]
  36. Nazareth L. V., Harbour D. V., Thompson E. B. Mapping the human glucocorticoid receptor for leukemic cell death. J Biol Chem. 1991 Jul 15;266(20):12976–12980. [PubMed] [Google Scholar]
  37. Negrini M., Silini E., Kozak C., Tsujimoto Y., Croce C. M. Molecular analysis of mbcl-2: structure and expression of the murine gene homologous to the human gene involved in follicular lymphoma. Cell. 1987 May 22;49(4):455–463. doi: 10.1016/0092-8674(87)90448-x. [DOI] [PubMed] [Google Scholar]
  38. Nguyen T. T., Nabholz M. Glucocorticoid resistance is a dominant trait in hybrids between cytolytic T-lymphocyte lines and AKR thymomas. Somat Cell Mol Genet. 1985 Nov;11(6):593–604. doi: 10.1007/BF01534724. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. PITOT H. C., PERAINO C., MORSE P. A., Jr, POTTER V. R. HEPATOMAS IN TISSUE CULTURE COMPARED WITH ADAPTING LIVER IN VIVO. Natl Cancer Inst Monogr. 1964 Apr;13:229–245. [PubMed] [Google Scholar]
  43. Pearson S. J., Tetri P., George D. L., Francke U. Activation of human alpha 1-antitrypsin gene in rat hepatoma x human fetal liver cell hybrids depends on presence of human chromosome 14. Somatic Cell Genet. 1983 Sep;9(5):567–592. doi: 10.1007/BF01574259. [DOI] [PubMed] [Google Scholar]
  44. Peterson T. C., Killary A. M., Fournier R. E. Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells. Mol Cell Biol. 1985 Sep;5(9):2491–2494. doi: 10.1128/mcb.5.9.2491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pfahl M., Bourgeois S. Analysis of steroid resistance in lymphoid cell hybrids. Somatic Cell Genet. 1980 Jan;6(1):63–74. doi: 10.1007/BF01538696. [DOI] [PubMed] [Google Scholar]
  46. Pfahl M., Kelleher R. J., Jr, Bourgeois S. General features of steroid resistance on lymphoid cell lines. Mol Cell Endocrinol. 1978 Apr;10(2):193–207. doi: 10.1016/0303-7207(78)90125-9. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Tenniswood M. P., Guenette R. S., Lakins J., Mooibroek M., Wong P., Welsh J. E. Active cell death in hormone-dependent tissues. Cancer Metastasis Rev. 1992 Sep;11(2):197–220. doi: 10.1007/BF00048064. [DOI] [PubMed] [Google Scholar]
  49. Thayer M. J., Fournier R. E. Hormonal regulation of TSE1-repressed genes: evidence for multiple genetic controls in extinction. Mol Cell Biol. 1989 Jul;9(7):2837–2846. doi: 10.1128/mcb.9.7.2837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thompson E. A. Glucocorticoid insensitivity of P1798 lymphoma cells is associated with production of a factor that attenuates the lytic response. Cancer Res. 1991 Oct 15;51(20):5551–5556. [PubMed] [Google Scholar]
  51. Thompson E. A. Insensitivity to the cytolytic effects of glucocorticoids in vivo is associated with a novel "slow death" phenotype. Cancer Res. 1991 Oct 15;51(20):5544–5550. [PubMed] [Google Scholar]
  52. Thulasi R., Harbour D. V., Thompson E. B. Suppression of c-myc is a critical step in glucocorticoid-induced human leukemic cell lysis. J Biol Chem. 1993 Aug 25;268(24):18306–18312. [PubMed] [Google Scholar]
  53. Tsujimoto Y., Gorham J., Cossman J., Jaffe E., Croce C. M. The t(14;18) chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining. Science. 1985 Sep 27;229(4720):1390–1393. doi: 10.1126/science.3929382. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Vaux D. L. Toward an understanding of the molecular mechanisms of physiological cell death. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):786–789. doi: 10.1073/pnas.90.3.786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Walker P. R., Kwast-Welfeld J., Gourdeau H., Leblanc J., Neugebauer W., Sikorska M. Relationship between apoptosis and the cell cycle in lymphocytes: roles of protein kinase C, tyrosine phosphorylation, and AP1. Exp Cell Res. 1993 Jul;207(1):142–151. doi: 10.1006/excr.1993.1173. [DOI] [PubMed] [Google Scholar]
  57. Wyllie A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980 Apr 10;284(5756):555–556. doi: 10.1038/284555a0. [DOI] [PubMed] [Google Scholar]
  58. Yonish-Rouach E., Resnitzky D., Lotem J., Sachs L., Kimchi A., Oren M. Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature. 1991 Jul 25;352(6333):345–347. doi: 10.1038/352345a0. [DOI] [PubMed] [Google Scholar]
  59. Yuh Y. S., Thompson E. B. Complementation between glucocorticoid receptor and lymphocytolysis in somatic cell hybrids of two glucocorticoid-resistant human leukemic clonal cell lines. Somat Cell Mol Genet. 1987 Jan;13(1):33–45. doi: 10.1007/BF02422297. [DOI] [PubMed] [Google Scholar]
  60. Yuh Y. S., Thompson E. B. Glucocorticoid effect on oncogene/growth gene expression in human T lymphoblastic leukemic cell line CCRF-CEM. Specific c-myc mRNA suppression by dexamethasone. J Biol Chem. 1989 Jun 25;264(18):10904–10910. [PubMed] [Google Scholar]

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