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. 1995 May;15(5):2383–2392. doi: 10.1128/mcb.15.5.2383

Dual functions of the AML1/Evi-1 chimeric protein in the mechanism of leukemogenesis in t(3;21) leukemias.

T Tanaka 1, K Mitani 1, M Kurokawa 1, S Ogawa 1, K Tanaka 1, J Nishida 1, Y Yazaki 1, Y Shibata 1, H Hirai 1
PMCID: PMC230467  PMID: 7739522

Abstract

The chromosomal translocation t(3;21)(q26;q22), which is found in blastic crisis in chronic myelogenous leukemias and myelodysplastic syndrome-derived leukemias, produces AML1/Evi-1 chimeric transcription factor and is thought to play important roles in acute leukemic transformation of hemopoietic stem cells. We report here the functional analyses of AML1/Evi-1. It was revealed that AML1/Evi-1 itself does not alter the transactivation level through mouse polyomavirus enhancer-binding protein 2 (PEBP2; PEA2) sites (binding site of AML1) but dominantly suppresses the transactivation by intact AML1, which is assumed to be a stimulator of myeloid cell differentiation. DNA-binding competition is a putative mechanism of such dominant negative effects of AML1/Evi-1 because it binds to PEBP2 sites with higher affinity than AML1 does. Furthermore, AML1/Evi-1 stimulated c-fos promoter transactivation and increased AP-1 activity, as Evi-1 (which is not normally expressed in hemopoietic cells) did. Experiments using deletion mutants of AML1/Evi-1 showed that these two functions are mutually independent because the dominant negative effects on intact AML1 and the stimulation of AP-1 activity are dependent on the runt domain (DNA-binding domain of AML1) and the zinc finger domain near the C terminus, respectively. Furthermore, we showed that AML1/Evi-1 blocks granulocytic differentiation, otherwise induced by granulocyte colony-stimulating factor, of 32Dcl3 myeloid cells. It was also suggested that both AML1-derived and Evi-1-derived portions of the fusion protein play crucial roles in this differentiation block. We conclude that the leukemic cell transformation in t(3;21) leukemias is probably caused by these dual functions of AML1/Evi-1 chimeric protein.

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Selected References

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  1. Andrews N. C., Faller D. V. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991 May 11;19(9):2499–2499. doi: 10.1093/nar/19.9.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angel P., Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991 Dec 10;1072(2-3):129–157. doi: 10.1016/0304-419x(91)90011-9. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bae S. C., Ogawa E., Maruyama M., Oka H., Satake M., Shigesada K., Jenkins N. A., Gilbert D. J., Copeland N. G., Ito Y. PEBP2 alpha B/mouse AML1 consists of multiple isoforms that possess differential transactivation potentials. Mol Cell Biol. 1994 May;14(5):3242–3252. doi: 10.1128/mcb.14.5.3242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bae S. C., Yamaguchi-Iwai Y., Ogawa E., Maruyama M., Inuzuka M., Kagoshima H., Shigesada K., Satake M., Ito Y. Isolation of PEBP2 alpha B cDNA representing the mouse homolog of human acute myeloid leukemia gene, AML1. Oncogene. 1993 Mar;8(3):809–814. [PubMed] [Google Scholar]
  6. Bartholomew C., Ihle J. N. Retroviral insertions 90 kilobases proximal to the Evi-1 myeloid transforming gene activate transcription from the normal promoter. Mol Cell Biol. 1991 Apr;11(4):1820–1828. doi: 10.1128/mcb.11.4.1820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bartholomew C., Morishita K., Askew D., Buchberg A., Jenkins N. A., Copeland N. G., Ihle J. N. Retroviral insertions in the CB-1/Fim-3 common site of integration activate expression of the Evi-1 gene. Oncogene. 1989 May;4(5):529–534. [PubMed] [Google Scholar]
  8. Cleary M. L. Oncogenic conversion of transcription factors by chromosomal translocations. Cell. 1991 Aug 23;66(4):619–622. doi: 10.1016/0092-8674(91)90105-8. [DOI] [PubMed] [Google Scholar]
  9. Colotta F., Polentarutti N., Sironi M., Mantovani A. Expression and involvement of c-fos and c-jun protooncogenes in programmed cell death induced by growth factor deprivation in lymphoid cell lines. J Biol Chem. 1992 Sep 15;267(26):18278–18283. [PubMed] [Google Scholar]
  10. Curran T., Miller A. D., Zokas L., Verma I. M. Viral and cellular fos proteins: a comparative analysis. Cell. 1984 Feb;36(2):259–268. doi: 10.1016/0092-8674(84)90219-8. [DOI] [PubMed] [Google Scholar]
  11. Daniel M. T., Koken M., Romagné O., Barbey S., Bazarbachi A., Stadler M., Guillemin M. C., Degos L., Chomienne C., de Thé H. PML protein expression in hematopoietic and acute promyelocytic leukemia cells. Blood. 1993 Sep 15;82(6):1858–1867. [PubMed] [Google Scholar]
  12. Dong F., van Buitenen C., Pouwels K., Hoefsloot L. H., Löwenberg B., Touw I. P. Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation. Mol Cell Biol. 1993 Dec;13(12):7774–7781. doi: 10.1128/mcb.13.12.7774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dyck J. A., Maul G. G., Miller W. H., Jr, Chen J. D., Kakizuka A., Evans R. M. A novel macromolecular structure is a target of the promyelocyte-retinoic acid receptor oncoprotein. Cell. 1994 Jan 28;76(2):333–343. doi: 10.1016/0092-8674(94)90340-9. [DOI] [PubMed] [Google Scholar]
  14. Erickson P., Gao J., Chang K. S., Look T., Whisenant E., Raimondi S., Lasher R., Trujillo J., Rowley J., Drabkin H. Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt. Blood. 1992 Oct 1;80(7):1825–1831. [PubMed] [Google Scholar]
  15. Fearon E. R., Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990 Jun 1;61(5):759–767. doi: 10.1016/0092-8674(90)90186-i. [DOI] [PubMed] [Google Scholar]
  16. Fichelson S., Dreyfus F., Berger R., Melle J., Bastard C., Miclea J. M., Gisselbrecht S. Evi-1 expression in leukemic patients with rearrangements of the 3q25-q28 chromosomal region. Leukemia. 1992 Feb;6(2):93–99. [PubMed] [Google Scholar]
  17. Fort P., Marty L., Piechaczyk M., el Sabrouty S., Dani C., Jeanteur P., Blanchard J. M. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res. 1985 Mar 11;13(5):1431–1442. doi: 10.1093/nar/13.5.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fukunaga R., Ishizaka-Ikeda E., Nagata S. Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stimulating factor receptor. Cell. 1993 Sep 24;74(6):1079–1087. doi: 10.1016/0092-8674(93)90729-a. [DOI] [PubMed] [Google Scholar]
  19. Funabiki T., Kreider B. L., Ihle J. N. The carboxyl domain of zinc fingers of the Evi-1 myeloid transforming gene binds a consensus sequence of GAAGATGAG. Oncogene. 1994 Jun;9(6):1575–1581. [PubMed] [Google Scholar]
  20. Furukawa K., Yamaguchi Y., Ogawa E., Shigesada K., Satake M., Ito Y. A ubiquitous repressor interacting with an F9 cell-specific silencer and its functional suppression by differentiated cell-specific positive factors. Cell Growth Differ. 1990 Mar;1(3):135–147. [PubMed] [Google Scholar]
  21. Garriga G., Guenther C., Horvitz H. R. Migrations of the Caenorhabditis elegans HSNs are regulated by egl-43, a gene encoding two zinc finger proteins. Genes Dev. 1993 Nov;7(11):2097–2109. doi: 10.1101/gad.7.11.2097. [DOI] [PubMed] [Google Scholar]
  22. Goldstone S. D., Lavin M. F. Prolonged expression of c-jun and associated activity of the transcription factor AP-1, during apoptosis in a human leukaemic cell line. Oncogene. 1994 Aug;9(8):2305–2311. [PubMed] [Google Scholar]
  23. Grigoriadis A. E., Schellander K., Wang Z. Q., Wagner E. F. Osteoblasts are target cells for transformation in c-fos transgenic mice. J Cell Biol. 1993 Aug;122(3):685–701. doi: 10.1083/jcb.122.3.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hunger S. P., Ohyashiki K., Toyama K., Cleary M. L. Hlf, a novel hepatic bZIP protein, shows altered DNA-binding properties following fusion to E2A in t(17;19) acute lymphoblastic leukemia. Genes Dev. 1992 Sep;6(9):1608–1620. doi: 10.1101/gad.6.9.1608. [DOI] [PubMed] [Google Scholar]
  25. Kakizuka A., Miller W. H., Jr, Umesono K., Warrell R. P., Jr, Frankel S. R., Murty V. V., Dmitrovsky E., Evans R. M. Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell. 1991 Aug 23;66(4):663–674. doi: 10.1016/0092-8674(91)90112-c. [DOI] [PubMed] [Google Scholar]
  26. Kamps M. P., Murre C., Sun X. H., Baltimore D. A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL. Cell. 1990 Feb 23;60(4):547–555. doi: 10.1016/0092-8674(90)90658-2. [DOI] [PubMed] [Google Scholar]
  27. Kania M. A., Bonner A. S., Duffy J. B., Gergen J. P. The Drosophila segmentation gene runt encodes a novel nuclear regulatory protein that is also expressed in the developing nervous system. Genes Dev. 1990 Oct;4(10):1701–1713. doi: 10.1101/gad.4.10.1701. [DOI] [PubMed] [Google Scholar]
  28. Koken M. H., Puvion-Dutilleul F., Guillemin M. C., Viron A., Linares-Cruz G., Stuurman N., de Jong L., Szostecki C., Calvo F., Chomienne C. The t(15;17) translocation alters a nuclear body in a retinoic acid-reversible fashion. EMBO J. 1994 Mar 1;13(5):1073–1083. doi: 10.1002/j.1460-2075.1994.tb06356.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  30. Liu P., Tarlé S. A., Hajra A., Claxton D. F., Marlton P., Freedman M., Siciliano M. J., Collins F. S. Fusion between transcription factor CBF beta/PEBP2 beta and a myosin heavy chain in acute myeloid leukemia. Science. 1993 Aug 20;261(5124):1041–1044. doi: 10.1126/science.8351518. [DOI] [PubMed] [Google Scholar]
  31. Marti A., Jehn B., Costello E., Keon N., Ke G., Martin F., Jaggi R. Protein kinase A and AP-1 (c-Fos/JunD) are induced during apoptosis of mouse mammary epithelial cells. Oncogene. 1994 Apr;9(4):1213–1223. [PubMed] [Google Scholar]
  32. Meyers S., Downing J. R., Hiebert S. W. Identification of AML-1 and the (8;21) translocation protein (AML-1/ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions. Mol Cell Biol. 1993 Oct;13(10):6336–6345. doi: 10.1128/mcb.13.10.6336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Migliaccio G., Migliaccio A. R., Kreider B. L., Rovera G., Adamson J. W. Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D. J Cell Biol. 1989 Aug;109(2):833–841. doi: 10.1083/jcb.109.2.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mitani K., Ogawa S., Tanaka T., Miyoshi H., Kurokawa M., Mano H., Yazaki Y., Ohki M., Hirai H. Generation of the AML1-EVI-1 fusion gene in the t(3;21)(q26;q22) causes blastic crisis in chronic myelocytic leukemia. EMBO J. 1994 Feb 1;13(3):504–510. doi: 10.1002/j.1460-2075.1994.tb06288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Miyoshi H., Kozu T., Shimizu K., Enomoto K., Maseki N., Kaneko Y., Kamada N., Ohki M. The t(8;21) translocation in acute myeloid leukemia results in production of an AML1-MTG8 fusion transcript. EMBO J. 1993 Jul;12(7):2715–2721. doi: 10.1002/j.1460-2075.1993.tb05933.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Miyoshi H., Shimizu K., Kozu T., Maseki N., Kaneko Y., Ohki M. t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10431–10434. doi: 10.1073/pnas.88.23.10431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Morishita K., Parganas E., Douglass E. C., Ihle J. N. Unique expression of the human Evi-1 gene in an endometrial carcinoma cell line: sequence of cDNAs and structure of alternatively spliced transcripts. Oncogene. 1990 Jul;5(7):963–971. [PubMed] [Google Scholar]
  38. Morishita K., Parganas E., Matsugi T., Ihle J. N. Expression of the Evi-1 zinc finger gene in 32Dc13 myeloid cells blocks granulocytic differentiation in response to granulocyte colony-stimulating factor. Mol Cell Biol. 1992 Jan;12(1):183–189. doi: 10.1128/mcb.12.1.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Morishita K., Parganas E., Parham D. M., Matsugi T., Ihle J. N. The Evi-1 zinc finger myeloid transforming gene is normally expressed in the kidney and in developing oocytes. Oncogene. 1990 Sep;5(9):1419–1423. [PubMed] [Google Scholar]
  40. Morishita K., Parganas E., William C. L., Whittaker M. H., Drabkin H., Oval J., Taetle R., Valentine M. B., Ihle J. N. Activation of EVI1 gene expression in human acute myelogenous leukemias by translocations spanning 300-400 kilobases on chromosome band 3q26. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3937–3941. doi: 10.1073/pnas.89.9.3937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Morishita K., Parker D. S., Mucenski M. L., Jenkins N. A., Copeland N. G., Ihle J. N. Retroviral activation of a novel gene encoding a zinc finger protein in IL-3-dependent myeloid leukemia cell lines. Cell. 1988 Sep 9;54(6):831–840. doi: 10.1016/s0092-8674(88)91175-0. [DOI] [PubMed] [Google Scholar]
  42. Mucenski M. L., Taylor B. A., Ihle J. N., Hartley J. W., Morse H. C., 3rd, Jenkins N. A., Copeland N. G. Identification of a common ecotropic viral integration site, Evi-1, in the DNA of AKXD murine myeloid tumors. Mol Cell Biol. 1988 Jan;8(1):301–308. doi: 10.1128/mcb.8.1.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nichols J., Nimer S. D. Transcription factors, translocations, and leukemia. Blood. 1992 Dec 15;80(12):2953–2963. [PubMed] [Google Scholar]
  44. Nourse J., Mellentin J. D., Galili N., Wilkinson J., Stanbridge E., Smith S. D., Cleary M. L. Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor. Cell. 1990 Feb 23;60(4):535–545. doi: 10.1016/0092-8674(90)90657-z. [DOI] [PubMed] [Google Scholar]
  45. Nucifora G., Begy C. R., Kobayashi H., Roulston D., Claxton D., Pedersen-Bjergaard J., Parganas E., Ihle J. N., Rowley J. D. Consistent intergenic splicing and production of multiple transcripts between AML1 at 21q22 and unrelated genes at 3q26 in (3;21)(q26;q22) translocations. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):4004–4008. doi: 10.1073/pnas.91.9.4004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ogawa E., Inuzuka M., Maruyama M., Satake M., Naito-Fujimoto M., Ito Y., Shigesada K. Molecular cloning and characterization of PEBP2 beta, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2 alpha. Virology. 1993 May;194(1):314–331. doi: 10.1006/viro.1993.1262. [DOI] [PubMed] [Google Scholar]
  47. Ogawa E., Maruyama M., Kagoshima H., Inuzuka M., Lu J., Satake M., Shigesada K., Ito Y. PEBP2/PEA2 represents a family of transcription factors homologous to the products of the Drosophila runt gene and the human AML1 gene. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6859–6863. doi: 10.1073/pnas.90.14.6859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Oval J., Smedsrud M., Taetle R. Expression and regulation of the evi-1 gene in the human factor-dependent leukemia cell line, UCSD/AML1. Leukemia. 1992 May;6(5):446–451. [PubMed] [Google Scholar]
  49. Piette J., Yaniv M. Two different factors bind to the alpha-domain of the polyoma virus enhancer, one of which also interacts with the SV40 and c-fos enhancers. EMBO J. 1987 May;6(5):1331–1337. doi: 10.1002/j.1460-2075.1987.tb02372.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rubin C. M., Larson R. A., Anastasi J., Winter J. N., Thangavelu M., Vardiman J. W., Rowley J. D., Le Beau M. M. t(3;21)(q26;q22): a recurring chromosomal abnormality in therapy-related myelodysplastic syndrome and acute myeloid leukemia. Blood. 1990 Dec 15;76(12):2594–2598. [PubMed] [Google Scholar]
  51. Sadowski I., Ma J., Triezenberg S., Ptashne M. GAL4-VP16 is an unusually potent transcriptional activator. Nature. 1988 Oct 6;335(6190):563–564. doi: 10.1038/335563a0. [DOI] [PubMed] [Google Scholar]
  52. Satake M., Inuzuka M., Shigesada K., Oikawa T., Ito Y. Differential expression of subspecies of polyomavirus and murine leukemia virus enhancer core binding protein, PEBP2, in various hematopoietic cells. Jpn J Cancer Res. 1992 Jul;83(7):714–722. doi: 10.1111/j.1349-7006.1992.tb01971.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Schneider N. R., Bowman W. P., Frenkel E. P. Translocation (3;21)(q26;q22) in secondary leukemia. Report of two cases and literature review. Ann Genet. 1991;34(3-4):256–263. [PubMed] [Google Scholar]
  54. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tanaka T., Nishida J., Mitani K., Ogawa S., Yazaki Y., Hirai H. Evi-1 raises AP-1 activity and stimulates c-fos promoter transactivation with dependence on the second zinc finger domain. J Biol Chem. 1994 Sep 30;269(39):24020–24026. [PubMed] [Google Scholar]
  56. Tanaka T., Shibasaki F., Ishikawa M., Hirano N., Sakai R., Nishida J., Takenawa T., Hirai H. Molecular cloning of bovine actin-like protein, actin2. Biochem Biophys Res Commun. 1992 Sep 16;187(2):1022–1028. doi: 10.1016/0006-291x(92)91299-6. [DOI] [PubMed] [Google Scholar]
  57. Tanaka T., Tanaka K., Ogawa S., Kurokawa M., Mitani K., Nishida J., Shibata Y., Yazaki Y., Hirai H. An acute myeloid leukemia gene, AML1, regulates hemopoietic myeloid cell differentiation and transcriptional activation antagonistically by two alternative spliced forms. EMBO J. 1995 Jan 16;14(2):341–350. doi: 10.1002/j.1460-2075.1995.tb07008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wang S., Wang Q., Crute B. E., Melnikova I. N., Keller S. R., Speck N. A. Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor. Mol Cell Biol. 1993 Jun;13(6):3324–3339. doi: 10.1128/mcb.13.6.3324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Weis K., Rambaud S., Lavau C., Jansen J., Carvalho T., Carmo-Fonseca M., Lamond A., Dejean A. Retinoic acid regulates aberrant nuclear localization of PML-RAR alpha in acute promyelocytic leukemia cells. Cell. 1994 Jan 28;76(2):345–356. doi: 10.1016/0092-8674(94)90341-7. [DOI] [PubMed] [Google Scholar]
  60. de Thé H., Lavau C., Marchio A., Chomienne C., Degos L., Dejean A. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell. 1991 Aug 23;66(4):675–684. doi: 10.1016/0092-8674(91)90113-d. [DOI] [PubMed] [Google Scholar]
  61. von Lindern M., Fornerod M., Soekarman N., van Baal S., Jaegle M., Hagemeijer A., Bootsma D., Grosveld G. Translocation t(6;9) in acute non-lymphocytic leukaemia results in the formation of a DEK-CAN fusion gene. Baillieres Clin Haematol. 1992 Oct;5(4):857–879. doi: 10.1016/s0950-3536(11)80049-1. [DOI] [PubMed] [Google Scholar]

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