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. 1992 Dec;11(12):4641–4652. doi: 10.1002/j.1460-2075.1992.tb05566.x

Hormone-regulated v-rel estrogen receptor fusion protein: reversible induction of cell transformation and cellular gene expression.

G Boehmelt 1, A Walker 1, N Kabrun 1, G Mellitzer 1, H Beug 1, M Zenke 1, P J Enrietto 1
PMCID: PMC557040  PMID: 1425595

Abstract

We describe the construction of a v-rel estrogen receptor fusion protein (v-relER) which allows the regulation of v-rel oncoprotein activity by hormone. In the presence of estrogen, v-relER readily transformed primary chicken fibroblasts and bone marrow cells in vitro. In both cell types, v-rel-specific transformation was critically dependent on the presence of estrogen or the estrogen agonist 4-hydroxytamoxifen (OHT). Withdrawal of estrogen or application of an estrogen antagonist, ICI164,384 (ICI) caused a reversal of the transformed phenotype. We also demonstrate that the v-relER protein binds to NF-kappa B sites in an estrogen-dependent manner, thereby showing that sequence-specific DNA binding of v-relER is critical for the activation of its transforming capacity. In transient transfection experiments, we failed to demonstrate a clear repressor or activator function of the v-rel moiety in v-relER. However, in v-relER-transformed bone marrow cells, estrogen and OHT induced elevated mRNA levels of two cellular genes whose expression is constitutive and high in v-rel-transformed cells. These results suggest that v-rel might exert part of its activity as an activator of rel-responsive genes.

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Selected References

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

  1. Baeuerle P. A. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim Biophys Acta. 1991 Apr 16;1072(1):63–80. doi: 10.1016/0304-419x(91)90007-8. [DOI] [PubMed] [Google Scholar]
  2. Baldwin A. S., Jr, Sharp P. A. Two transcription factors, NF-kappa B and H2TF1, interact with a single regulatory sequence in the class I major histocompatibility complex promoter. Proc Natl Acad Sci U S A. 1988 Feb;85(3):723–727. doi: 10.1073/pnas.85.3.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ballard D. W., Dixon E. P., Peffer N. J., Bogerd H., Doerre S., Stein B., Greene W. C. The 65-kDa subunit of human NF-kappa B functions as a potent transcriptional activator and a target for v-Rel-mediated repression. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1875–1879. doi: 10.1073/pnas.89.5.1875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ballard D. W., Walker W. H., Doerre S., Sista P., Molitor J. A., Dixon E. P., Peffer N. J., Hannink M., Greene W. C. The v-rel oncogene encodes a kappa B enhancer binding protein that inhibits NF-kappa B function. Cell. 1990 Nov 16;63(4):803–814. doi: 10.1016/0092-8674(90)90146-6. [DOI] [PubMed] [Google Scholar]
  5. Barth C. F., Ewert D. L., Olson W. C., Humphries E. H. Reticuloendotheliosis virus REV-T(REV-A)-induced neoplasia: development of tumors within the T-lymphoid and myeloid lineages. J Virol. 1990 Dec;64(12):6054–6062. doi: 10.1128/jvi.64.12.6054-6062.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berry M., Metzger D., Chambon P. Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J. 1990 Sep;9(9):2811–2818. doi: 10.1002/j.1460-2075.1990.tb07469.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Beug H., Leutz A., Kahn P., Graf T. Ts mutants of E26 leukemia virus allow transformed myeloblasts, but not erythroblasts or fibroblasts, to differentiate at the nonpermissive temperature. Cell. 1984 Dec;39(3 Pt 2):579–588. doi: 10.1016/0092-8674(84)90465-3. [DOI] [PubMed] [Google Scholar]
  8. Beug H., Müller H., Grieser S., Doederlein G., Graf T. Hematopoietic cells transformed in vitro by REVT avian reticuloendotheliosis virus express characteristics of very immature lymphoid cells. Virology. 1981 Dec;115(2):295–309. doi: 10.1016/0042-6822(81)90112-4. [DOI] [PubMed] [Google Scholar]
  9. Beug H., von Kirchbach A., Döderlein G., Conscience J. F., Graf T. Chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation. Cell. 1979 Oct;18(2):375–390. doi: 10.1016/0092-8674(79)90057-6. [DOI] [PubMed] [Google Scholar]
  10. Blank V., Kourilsky P., Israël A. NF-kappa B and related proteins: Rel/dorsal homologies meet ankyrin-like repeats. Trends Biochem Sci. 1992 Apr;17(4):135–140. doi: 10.1016/0968-0004(92)90321-y. [DOI] [PubMed] [Google Scholar]
  11. Burk O., Klempnauer K. H. Estrogen-dependent alterations in differentiation state of myeloid cells caused by a v-myb/estrogen receptor fusion protein. EMBO J. 1991 Dec;10(12):3713–3719. doi: 10.1002/j.1460-2075.1991.tb04939.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Capobianco A. J., Chang D., Mosialos G., Gilmore T. D. p105, the NF-kappa B p50 precursor protein, is one of the cellular proteins complexed with the v-Rel oncoprotein in transformed chicken spleen cells. J Virol. 1992 Jun;66(6):3758–3767. doi: 10.1128/jvi.66.6.3758-3767.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Capobianco A. J., Gilmore T. D. Repression of the chicken c-rel promoter by vRel in chicken embryo fibroblasts is not mediated through a consensus NF-kappa B binding site. Oncogene. 1991 Dec;6(12):2203–2210. [PubMed] [Google Scholar]
  14. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  15. Dauvois S., Danielian P. S., White R., Parker M. G. Antiestrogen ICI 164,384 reduces cellular estrogen receptor content by increasing its turnover. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4037–4041. doi: 10.1073/pnas.89.9.4037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Davis N., Bargmann W., Lim M. Y., Bose H., Jr Avian reticuloendotheliosis virus-transformed lymphoid cells contain multiple pp59v-rel complexes. J Virol. 1990 Feb;64(2):584–591. doi: 10.1128/jvi.64.2.584-591.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Davis N., Ghosh S., Simmons D. L., Tempst P., Liou H. C., Baltimore D., Bose H. R., Jr Rel-associated pp40: an inhibitor of the rel family of transcription factors. Science. 1991 Sep 13;253(5025):1268–1271. doi: 10.1126/science.1891714. [DOI] [PubMed] [Google Scholar]
  18. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Disela C., Glineur C., Bugge T., Sap J., Stengl G., Dodgson J., Stunnenberg H., Beug H., Zenke M. v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII. Genes Dev. 1991 Nov;5(11):2033–2047. doi: 10.1101/gad.5.11.2033. [DOI] [PubMed] [Google Scholar]
  20. Eilers M., Picard D., Yamamoto K. R., Bishop J. M. Chimaeras of myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells. Nature. 1989 Jul 6;340(6228):66–68. doi: 10.1038/340066a0. [DOI] [PubMed] [Google Scholar]
  21. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  22. Forrest D., Curran T. Crossed signals: oncogenic transcription factors. Curr Opin Genet Dev. 1992 Feb;2(1):19–27. doi: 10.1016/s0959-437x(05)80316-8. [DOI] [PubMed] [Google Scholar]
  23. Gilmore T. D. Malignant transformation by mutant Rel proteins. Trends Genet. 1991 Oct;7(10):318–322. doi: 10.1016/0168-9525(91)90421-l. [DOI] [PubMed] [Google Scholar]
  24. Gilmore T. D. NF-kappa B, KBF1, dorsal, and related matters. Cell. 1990 Sep 7;62(5):841–843. doi: 10.1016/0092-8674(90)90257-f. [DOI] [PubMed] [Google Scholar]
  25. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Graf T., Ade N., Beug H. Temperature-sensitive mutant of avian erythroblastosis virus suggests a block of differentiation as mechanism of leukaemogenesis. Nature. 1978 Oct 12;275(5680):496–501. doi: 10.1038/275496a0. [DOI] [PubMed] [Google Scholar]
  27. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  28. Gronemeyer H. Transcription activation by estrogen and progesterone receptors. Annu Rev Genet. 1991;25:89–123. doi: 10.1146/annurev.ge.25.120191.000513. [DOI] [PubMed] [Google Scholar]
  29. Hughes S. H., Greenhouse J. J., Petropoulos C. J., Sutrave P. Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors. J Virol. 1987 Oct;61(10):3004–3012. doi: 10.1128/jvi.61.10.3004-3012.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ibanez C. E., Lipsick J. S. trans activation of gene expression by v-myb. Mol Cell Biol. 1990 May;10(5):2285–2293. doi: 10.1128/mcb.10.5.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Inoue J., Kerr L. D., Ransone L. J., Bengal E., Hunter T., Verma I. M. c-rel activates but v-rel suppresses transcription from kappa B sites. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3715–3719. doi: 10.1073/pnas.88.9.3715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ip Y. T., Kraut R., Levine M., Rushlow C. A. The dorsal morphogen is a sequence-specific DNA-binding protein that interacts with a long-range repression element in Drosophila. Cell. 1991 Jan 25;64(2):439–446. doi: 10.1016/0092-8674(91)90651-e. [DOI] [PubMed] [Google Scholar]
  33. Israël A., Kimura A., Kieran M., Yano O., Kanellopoulos J., Le Bail O., Kourilsky P. A common positive trans-acting factor binds to enhancer sequences in the promoters of mouse H-2 and beta 2-microglobulin genes. Proc Natl Acad Sci U S A. 1987 May;84(9):2653–2657. doi: 10.1073/pnas.84.9.2653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Israël A., Le Bail O., Hatat D., Piette J., Kieran M., Logeat F., Wallach D., Fellous M., Kourilsky P. TNF stimulates expression of mouse MHC class I genes by inducing an NF kappa B-like enhancer binding activity which displaces constitutive factors. EMBO J. 1989 Dec 1;8(12):3793–3800. doi: 10.1002/j.1460-2075.1989.tb08556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Jordan V. C., Lieberman M. E., Cormier E., Koch R., Bagley J. R., Ruenitz P. C. Structural requirements for the pharmacological activity of nonsteroidal antiestrogens in vitro. Mol Pharmacol. 1984 Sep;26(2):272–278. [PubMed] [Google Scholar]
  36. Kabrun N., Hodgson J. W., Doemer M., Mak G., Franza B. R., Jr, Enrietto P. J. Interaction of the v-rel protein with an NF-kappa B DNA binding site. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1783–1787. doi: 10.1073/pnas.88.5.1783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kaufman J., Andersen R., Avila D., Engberg J., Lambris J., Salomonsen J., Welinder K., Skjødt K. Different features of the MHC class I heterodimer have evolved at different rates. Chicken B-F and beta 2-microglobulin sequences reveal invariant surface residues. J Immunol. 1992 Mar 1;148(5):1532–1546. [PubMed] [Google Scholar]
  38. Kerr L. D., Inoue J., Davis N., Link E., Baeuerle P. A., Bose H. R., Jr, Verma I. M. The rel-associated pp40 protein prevents DNA binding of Rel and NF-kappa B: relationship with I kappa B beta and regulation by phosphorylation. Genes Dev. 1991 Aug;5(8):1464–1476. doi: 10.1101/gad.5.8.1464. [DOI] [PubMed] [Google Scholar]
  39. Khandjian E. W. UV crosslinking of RNA to nylon membrane enhances hybridization signals. Mol Biol Rep. 1986;11(2):107–115. doi: 10.1007/BF00364822. [DOI] [PubMed] [Google Scholar]
  40. Knight J., Zenke M., Disela C., Kowenz E., Vogt P., Engel J. D., Hayman M. J., Beug H. Temperature-sensitive v-sea transformed erythroblasts: a model system to study gene expression during erythroid differentiation. Genes Dev. 1988 Feb;2(2):247–258. doi: 10.1101/gad.2.2.247. [DOI] [PubMed] [Google Scholar]
  41. Kroemer G., Zoorob R., Auffray C. Structure and expression of a chicken MHC class I gene. Immunogenetics. 1990;31(5-6):405–409. doi: 10.1007/BF02115020. [DOI] [PubMed] [Google Scholar]
  42. Kumar V., Green S., Staub A., Chambon P. Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J. 1986 Sep;5(9):2231–2236. doi: 10.1002/j.1460-2075.1986.tb04489.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  44. Leutz A., Beug H., Graf T. Purification and characterization of cMGF, a novel chicken myelomonocytic growth factor. EMBO J. 1984 Dec 20;3(13):3191–3197. doi: 10.1002/j.1460-2075.1984.tb02278.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Lewis R. B., McClure J., Rup B., Niesel D. W., Garry R. F., Hoelzer J. D., Nazerian K., Bose H. R., Jr Avian reticuloendotheliosis virus: identification of the hematopoietic target cell for transformation. Cell. 1981 Aug;25(2):421–431. doi: 10.1016/0092-8674(81)90060-x. [DOI] [PubMed] [Google Scholar]
  46. McDonnell P. C., Kumar S., Rabson A. B., Gélinas C. Transcriptional activity of rel family proteins. Oncogene. 1992 Jan;7(1):163–170. [PubMed] [Google Scholar]
  47. Morrison L. E., Boehmelt G., Beug H., Enrietto P. J. Expression of v-rel in a replication competent virus: transformation and biochemical characterization. Oncogene. 1991 Sep;6(9):1657–1666. [PubMed] [Google Scholar]
  48. Morrison L. E., Boehmelt G., Enrietto P. J. Mutations in the rel-homology domain alter the biochemical properties of v-rel and render it transformation defective in chicken embryo fibroblasts. Oncogene. 1992 Jun;7(6):1137–1147. [PubMed] [Google Scholar]
  49. Morrison L. E., Kabrun N., Mudri S., Hayman M. J., Enrietto P. J. Viral rel and cellular rel associate with cellular proteins in transformed and normal cells. Oncogene. 1989 Jun;4(6):677–683. [PubMed] [Google Scholar]
  50. Narayanan R., Klement J. F., Ruben S. M., Higgins K. A., Rosen C. A. Identification of a naturally occurring transforming variant of the p65 subunit of NF-kappa B. Science. 1992 Apr 17;256(5055):367–370. doi: 10.1126/science.256.5055.367. [DOI] [PubMed] [Google Scholar]
  51. Neri A., Chang C. C., Lombardi L., Salina M., Corradini P., Maiolo A. T., Chaganti R. S., Dalla-Favera R. B cell lymphoma-associated chromosomal translocation involves candidate oncogene lyt-10, homologous to NF-kappa B p50. Cell. 1991 Dec 20;67(6):1075–1087. doi: 10.1016/0092-8674(91)90285-7. [DOI] [PubMed] [Google Scholar]
  52. Pham T. A., Elliston J. F., Nawaz Z., McDonnell D. P., Tsai M. J., O'Malley B. W. Antiestrogen can establish nonproductive receptor complexes and alter chromatin structure at target enhancers. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3125–3129. doi: 10.1073/pnas.88.8.3125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Picard D., Salser S. J., Yamamoto K. R. A movable and regulable inactivation function within the steroid binding domain of the glucocorticoid receptor. Cell. 1988 Sep 23;54(7):1073–1080. doi: 10.1016/0092-8674(88)90122-5. [DOI] [PubMed] [Google Scholar]
  54. Puissant C., Houdebine L. M. An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Biotechniques. 1990 Feb;8(2):148–149. [PubMed] [Google Scholar]
  55. Radke K., Beug H., Kornfeld S., Graf T. Transformation of both erythroid and myeloid cells by E26, an avian leukemia virus that contains the myb gene. Cell. 1982 Dec;31(3 Pt 2):643–653. doi: 10.1016/0092-8674(82)90320-8. [DOI] [PubMed] [Google Scholar]
  56. Richardson P. M., Gilmore T. D. vRel is an inactive member of the Rel family of transcriptional activating proteins. J Virol. 1991 Jun;65(6):3122–3130. doi: 10.1128/jvi.65.6.3122-3130.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Royer-Pokora B., Beug H., Claviez M., Winkhardt H. J., Friis R. R., Graf T. Transformation parameters in chicken fibroblasts transformed by AEV and MC29 avian leukemia viruses. Cell. 1978 Apr;13(4):751–760. doi: 10.1016/0092-8674(78)90225-8. [DOI] [PubMed] [Google Scholar]
  58. Schroeder C., Gibson L., Zenke M., Beug H. Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors: a possible target for v-erbA oncogene action. Oncogene. 1992 Feb;7(2):217–227. [PubMed] [Google Scholar]
  59. Schwartz R. C., Witte O. N. A recombinant murine retrovirus expressing v-rel is cytopathic. Virology. 1988 Jul;165(1):182–190. doi: 10.1016/0042-6822(88)90671-x. [DOI] [PubMed] [Google Scholar]
  60. Simek S., Rice N. R. p59v-rel, the transforming protein of reticuloendotheliosis virus, is complexed with at least four other proteins in transformed chicken lymphoid cells. J Virol. 1988 Dec;62(12):4730–4736. doi: 10.1128/jvi.62.12.4730-4736.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Srikantha T., Landsman D., Bustin M. A single copy gene for chicken chromosomal protein HMG-14b has evolutionarily conserved features, has lost one of its introns and codes for a rapidly evolving protein. J Mol Biol. 1990 Jan 5;211(1):49–61. doi: 10.1016/0022-2836(90)90010-J. [DOI] [PubMed] [Google Scholar]
  62. Superti-Furga G., Bergers G., Picard D., Busslinger M. Hormone-dependent transcriptional regulation and cellular transformation by Fos-steroid receptor fusion proteins. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5114–5118. doi: 10.1073/pnas.88.12.5114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Thisse C., Perrin-Schmitt F., Stoetzel C., Thisse B. Sequence-specific transactivation of the Drosophila twist gene by the dorsal gene product. Cell. 1991 Jun 28;65(7):1191–1201. doi: 10.1016/0092-8674(91)90014-p. [DOI] [PubMed] [Google Scholar]
  64. Wakeling A. E., Bowler J. Biology and mode of action of pure antioestrogens. Drugs Exp Clin Res. 1988;14(12):729–734. [PubMed] [Google Scholar]
  65. Wakeling A. E., Bowler J. Novel antioestrogens without partial agonist activity. J Steroid Biochem. 1988 Oct;31(4B):645–653. doi: 10.1016/0022-4731(88)90014-3. [DOI] [PubMed] [Google Scholar]
  66. Zenke M., Kahn P., Disela C., Vennström B., Leutz A., Keegan K., Hayman M. J., Choi H. R., Yew N., Engel J. D. v-erbA specifically suppresses transcription of the avian erythrocyte anion transporter (band 3) gene. Cell. 1988 Jan 15;52(1):107–119. doi: 10.1016/0092-8674(88)90535-1. [DOI] [PubMed] [Google Scholar]
  67. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

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