Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Oct;14(10):6858–6867. doi: 10.1128/mcb.14.10.6858

PML, a growth suppressor disrupted in acute promyelocytic leukemia.

Z M Mu 1, K V Chin 1, J H Liu 1, G Lozano 1, K S Chang 1
PMCID: PMC359216  PMID: 7935403

Abstract

The nonrandom chromosomal translocation t(15;17)(q22;q21) in acute promyelocytic leukemia (APL) juxtaposes the genes for retinoic acid receptor alpha (RAR alpha) and the putative zinc finger transcription factor PML. The breakpoint site encodes fusion protein PML-RAR alpha, which is able to form a heterodimer with PML. It was hypothesized that PML-RAR alpha is a dominant negative inhibitor of PML. Inactivation of PML function in APL may play a critical role in APL pathogenesis. Our results demonstrated that PML, but not PML-RAR alpha, is a growth suppressor. This is supported by the following findings: (i) PML suppressed anchorage-independent growth of APL-derived NB4 cells on soft agar and tumorigenicity in nude mice, (ii) PML suppressed the oncogenic transformation of rat embryo fibroblasts by cooperative oncogenes, and (iii) PML suppressed transformation of NIH 3T3 cells by the activated neu oncogene. Cotransfection of PML with PML-RAR alpha resulted in a significant reduction in PML's transformation suppressor function in vivo, indicating that the fusion protein can be a dominant negative inhibitor of PML function in APL cells. This observation was further supported by the finding that cotransfection of PML and PML-RAR alpha resulted in altered normal cellular localization of PML. Our results also demonstrated that PML, but not PML-RAR alpha, is a promoter-specific transcription suppressor. Therefore, we hypothesized that disruption of the PML gene, a growth or transformation suppressor, by the t(15;17) translocation in APL is one of the critical events in leukemogenesis.

Full text

PDF
6858

Images in this article

6860Image
on p.6860
6861Image
on p.6861
6861Image
on p.6861
6862Image
on p.6862

Selected References

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

  1. Alcalay M., Zangrilli D., Pandolfi P. P., Longo L., Mencarelli A., Giacomucci A., Rocchi M., Biondi A., Rambaldi A., Lo Coco F. Translocation breakpoint of acute promyelocytic leukemia lies within the retinoic acid receptor alpha locus. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1977–1981. doi: 10.1073/pnas.88.5.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berg J. M. Zinc fingers and other metal-binding domains. Elements for interactions between macromolecules. J Biol Chem. 1990 Apr 25;265(12):6513–6516. [PubMed] [Google Scholar]
  3. Berger R., Le Coniat M., Derré J., Vecchione D., Jonveaux P. Cytogenetic studies in acute promyelocytic leukemia: a survey of secondary chromosomal abnormalities. Genes Chromosomes Cancer. 1991 Sep;3(5):332–337. doi: 10.1002/gcc.2870030503. [DOI] [PubMed] [Google Scholar]
  4. Borrow J., Goddard A. D., Sheer D., Solomon E. Molecular analysis of acute promyelocytic leukemia breakpoint cluster region on chromosome 17. Science. 1990 Sep 28;249(4976):1577–1580. doi: 10.1126/science.2218500. [DOI] [PubMed] [Google Scholar]
  5. Castaigne S., Chomienne C., Daniel M. T., Ballerini P., Berger R., Fenaux P., Degos L. All-trans retinoic acid as a differentiation therapy for acute promyelocytic leukemia. I. Clinical results. Blood. 1990 Nov 1;76(9):1704–1709. [PubMed] [Google Scholar]
  6. Chang K. S., Lu J. F., Wang G., Trujillo J. M., Estey E., Cork A., Chu D. T., Freireich E. J., Stass S. A. The t(15;17) breakpoint in acute promyelocytic leukemia cluster within two different sites of the myl gene: targets for the detection of minimal residual disease by the polymerase chain reaction. Blood. 1992 Feb 1;79(3):554–558. [PubMed] [Google Scholar]
  7. Chang K. S., Stass S. A., Chu D. T., Deaven L. L., Trujillo J. M., Freireich E. J. Characterization of a fusion cDNA (RARA/myl) transcribed from the t(15;17) translocation breakpoint in acute promyelocytic leukemia. Mol Cell Biol. 1992 Feb;12(2):800–810. doi: 10.1128/mcb.12.2.800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen S. J., Zhu Y. J., Tong J. H., Dong S., Huang W., Chen Y., Xiang W. M., Zhang L., Li X. S., Qian G. Q. Rearrangements in the second intron of the RARA gene are present in a large majority of patients with acute promyelocytic leukemia and are used as molecular marker for retinoic acid-induced leukemic cell differentiation. Blood. 1991 Nov 15;78(10):2696–2701. [PubMed] [Google Scholar]
  10. Chen T. M., Defendi V. Functional interaction of p53 with HPV18 E6, c-myc and H-ras in 3T3 cells. Oncogene. 1992 Aug;7(8):1541–1547. [PubMed] [Google Scholar]
  11. Chin K. V., Ueda K., Pastan I., Gottesman M. M. Modulation of activity of the promoter of the human MDR1 gene by Ras and p53. Science. 1992 Jan 24;255(5043):459–462. doi: 10.1126/science.1346476. [DOI] [PubMed] [Google Scholar]
  12. Chomienne C., Ballerini P., Balitrand N., Daniel M. T., Fenaux P., Castaigne S., Degos L. All-trans retinoic acid in acute promyelocytic leukemias. II. In vitro studies: structure-function relationship. Blood. 1990 Nov 1;76(9):1710–1717. [PubMed] [Google Scholar]
  13. Chomienne C., Ballerini P., Balitrand N., Huang M. E., Krawice I., Castaigne S., Fenaux P., Tiollais P., Dejean A., Degos L. The retinoic acid receptor alpha gene is rearranged in retinoic acid-sensitive promyelocytic leukemias. Leukemia. 1990 Dec;4(12):802–807. [PubMed] [Google Scholar]
  14. 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]
  15. Dermime S., Grignani F., Clerici M., Nervi C., Sozzi G., Talamo G. P., Marchesi E., Formelli F., Parmiani G., Pelicci P. G. Occurrence of resistance to retinoic acid in the acute promyelocytic leukemia cell line NB4 is associated with altered expression of the pml/RAR alpha protein. Blood. 1993 Sep 1;82(5):1573–1577. [PubMed] [Google Scholar]
  16. Doucas V., Brockes J. P., Yaniv M., de Thé H., Dejean A. The PML-retinoic acid receptor alpha translocation converts the receptor from an inhibitor to a retinoic acid-dependent activator of transcription factor AP-1. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9345–9349. doi: 10.1073/pnas.90.20.9345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Eglitis M. A., Anderson W. F. Retroviral vectors for introduction of genes into mammalian cells. Biotechniques. 1988 Jul-Aug;6(7):608–614. [PubMed] [Google Scholar]
  19. Eglitis M. A., Kantoff P., Gilboa E., Anderson W. F. Gene expression in mice after high efficiency retroviral-mediated gene transfer. Science. 1985 Dec 20;230(4732):1395–1398. doi: 10.1126/science.2999985. [DOI] [PubMed] [Google Scholar]
  20. Fagioli M., Alcalay M., Pandolfi P. P., Venturini L., Mencarelli A., Simeone A., Acampora D., Grignani F., Pelicci P. G. Alternative splicing of PML transcripts predicts coexpression of several carboxy-terminally different protein isoforms. Oncogene. 1992 Jun;7(6):1083–1091. [PubMed] [Google Scholar]
  21. Finlay C. A., Hinds P. W., Levine A. J. The p53 proto-oncogene can act as a suppressor of transformation. Cell. 1989 Jun 30;57(7):1083–1093. doi: 10.1016/0092-8674(89)90045-7. [DOI] [PubMed] [Google Scholar]
  22. Freemont P. S., Hanson I. M., Trowsdale J. A novel cysteine-rich sequence motif. Cell. 1991 Feb 8;64(3):483–484. doi: 10.1016/0092-8674(91)90229-r. [DOI] [PubMed] [Google Scholar]
  23. Giguere V., Ong E. S., Segui P., Evans R. M. Identification of a receptor for the morphogen retinoic acid. Nature. 1987 Dec 17;330(6149):624–629. doi: 10.1038/330624a0. [DOI] [PubMed] [Google Scholar]
  24. Ginsberg D., Mechta F., Yaniv M., Oren M. Wild-type p53 can down-modulate the activity of various promoters. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9979–9983. doi: 10.1073/pnas.88.22.9979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Goddard A. D., Borrow J., Freemont P. S., Solomon E. Characterization of a zinc finger gene disrupted by the t(15;17) in acute promyelocytic leukemia. Science. 1991 Nov 29;254(5036):1371–1374. doi: 10.1126/science.1720570. [DOI] [PubMed] [Google Scholar]
  26. Gorman C. M., Merlino G. T., Willingham M. C., Pastan I., Howard B. H. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. doi: 10.1073/pnas.79.22.6777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Green S., Issemann I., Sheer E. A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res. 1988 Jan 11;16(1):369–369. doi: 10.1093/nar/16.1.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Grignani F., Ferrucci P. F., Testa U., Talamo G., Fagioli M., Alcalay M., Mencarelli A., Grignani F., Peschle C., Nicoletti I. The acute promyelocytic leukemia-specific PML-RAR alpha fusion protein inhibits differentiation and promotes survival of myeloid precursor cells. Cell. 1993 Aug 13;74(3):423–431. doi: 10.1016/0092-8674(93)80044-f. [DOI] [PubMed] [Google Scholar]
  30. Hinds P. W., Finlay C. A., Frey A. B., Levine A. J. Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines. Mol Cell Biol. 1987 Aug;7(8):2863–2869. doi: 10.1128/mcb.7.8.2863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Huang M. E., Ye Y. C., Chen S. R., Chai J. R., Lu J. X., Zhoa L., Gu L. J., Wang Z. Y. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood. 1988 Aug;72(2):567–572. [PubMed] [Google Scholar]
  32. Hunter T. Cooperation between oncogenes. Cell. 1991 Jan 25;64(2):249–270. doi: 10.1016/0092-8674(91)90637-e. [DOI] [PubMed] [Google Scholar]
  33. Ishii S., Xu Y. H., Stratton R. H., Roe B. A., Merlino G. T., Pastan I. Characterization and sequence of the promoter region of the human epidermal growth factor receptor gene. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4920–4924. doi: 10.1073/pnas.82.15.4920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Kastner P., Perez A., Lutz Y., Rochette-Egly C., Gaub M. P., Durand B., Lanotte M., Berger R., Chambon P. Structure, localization and transcriptional properties of two classes of retinoic acid receptor alpha fusion proteins in acute promyelocytic leukemia (APL): structural similarities with a new family of oncoproteins. EMBO J. 1992 Feb;11(2):629–642. doi: 10.1002/j.1460-2075.1992.tb05095.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kim S. J., Lee H. D., Robbins P. D., Busam K., Sporn M. B., Roberts A. B. Regulation of transforming growth factor beta 1 gene expression by the product of the retinoblastoma-susceptibility gene. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3052–3056. doi: 10.1073/pnas.88.8.3052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Land H., Chen A. C., Morgenstern J. P., Parada L. F., Weinberg R. A. Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts. Mol Cell Biol. 1986 Jun;6(6):1917–1925. doi: 10.1128/mcb.6.6.1917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lanotte M., Martin-Thouvenin V., Najman S., Balerini P., Valensi F., Berger R. NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood. 1991 Mar 1;77(5):1080–1086. [PubMed] [Google Scholar]
  39. Larson R. A., Kondo K., Vardiman J. W., Butler A. E., Golomb H. M., Rowley J. D. Evidence for a 15;17 translocation in every patient with acute promyelocytic leukemia. Am J Med. 1984 May;76(5):827–841. doi: 10.1016/0002-9343(84)90994-x. [DOI] [PubMed] [Google Scholar]
  40. Longo L., Trecca D., Biondi A., Lo Coco F., Grignani F., Maiolo A. T., Pelicci P. G., Neri A. Frequency of RAS and p53 mutations in acute promyelocytic leukemias. Leuk Lymphoma. 1993 Nov;11(5-6):405–410. doi: 10.3109/10428199309067933. [DOI] [PubMed] [Google Scholar]
  41. Markowitz D., Goff S., Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol. 1988 Apr;62(4):1120–1124. doi: 10.1128/jvi.62.4.1120-1124.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  43. Moses H. L., Yang E. Y., Pietenpol J. A. TGF-beta stimulation and inhibition of cell proliferation: new mechanistic insights. Cell. 1990 Oct 19;63(2):245–247. doi: 10.1016/0092-8674(90)90155-8. [DOI] [PubMed] [Google Scholar]
  44. Pandolfi P. P., Alcalay M., Fagioli M., Zangrilli D., Mencarelli A., Diverio D., Biondi A., Lo Coco F., Rambaldi A., Grignani F. Genomic variability and alternative splicing generate multiple PML/RAR alpha transcripts that encode aberrant PML proteins and PML/RAR alpha isoforms in acute promyelocytic leukaemia. EMBO J. 1992 Apr;11(4):1397–1407. doi: 10.1002/j.1460-2075.1992.tb05185.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pandolfi P. P., Grignani F., Alcalay M., Mencarelli A., Biondi A., LoCoco F., Grignani F., Pelicci P. G. Structure and origin of the acute promyelocytic leukemia myl/RAR alpha cDNA and characterization of its retinoid-binding and transactivation properties. Oncogene. 1991 Jul;6(7):1285–1292. [PubMed] [Google Scholar]
  46. Perez A., Kastner P., Sethi S., Lutz Y., Reibel C., Chambon P. PMLRAR homodimers: distinct DNA binding properties and heteromeric interactions with RXR. EMBO J. 1993 Aug;12(8):3171–3182. doi: 10.1002/j.1460-2075.1993.tb05986.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Quitschke W. W., Lin Z. Y., DePonti-Zilli L., Paterson B. M. The beta actin promoter. High levels of transcription depend upon a CCAAT binding factor. J Biol Chem. 1989 Jun 5;264(16):9539–9546. [PubMed] [Google Scholar]
  48. Raycroft L., Wu H. Y., Lozano G. Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene. Science. 1990 Aug 31;249(4972):1049–1051. doi: 10.1126/science.2144364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Reichel H., Norman A. W. Systemic effects of vitamin D. Annu Rev Med. 1989;40:71–78. doi: 10.1146/annurev.me.40.020189.000443. [DOI] [PubMed] [Google Scholar]
  50. Reichlin M. Use of glutaraldehyde as a coupling agent for proteins and peptides. Methods Enzymol. 1980;70(A):159–165. doi: 10.1016/s0076-6879(80)70047-2. [DOI] [PubMed] [Google Scholar]
  51. Robbins P. D., Horowitz J. M., Mulligan R. C. Negative regulation of human c-fos expression by the retinoblastoma gene product. Nature. 1990 Aug 16;346(6285):668–671. doi: 10.1038/346668a0. [DOI] [PubMed] [Google Scholar]
  52. Robertson K. A., Emami B., Mueller L., Collins S. J. Multiple members of the retinoic acid receptor family are capable of mediating the granulocytic differentiation of HL-60 cells. Mol Cell Biol. 1992 Sep;12(9):3743–3749. doi: 10.1128/mcb.12.9.3743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sawyers C. L., Denny C. T., Witte O. N. Leukemia and the disruption of normal hematopoiesis. Cell. 1991 Jan 25;64(2):337–350. doi: 10.1016/0092-8674(91)90643-d. [DOI] [PubMed] [Google Scholar]
  54. Sleigh M. J. A nonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells. Anal Biochem. 1986 Jul;156(1):251–256. doi: 10.1016/0003-2697(86)90180-6. [DOI] [PubMed] [Google Scholar]
  55. Tanaka H., Abe E., Miyaura C., Kuribayashi T., Konno K., Nishii Y., Suda T. 1 alpha,25-Dihydroxycholecalciferol and a human myeloid leukaemia cell line (HL-60). Biochem J. 1982 Jun 15;204(3):713–719. doi: 10.1042/bj2040713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tsai S., Collins S. J. A dominant negative retinoic acid receptor blocks neutrophil differentiation at the promyelocyte stage. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7153–7157. doi: 10.1073/pnas.90.15.7153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Warrell R. P., Jr, Frankel S. R., Miller W. H., Jr, Scheinberg D. A., Itri L. M., Hittelman W. N., Vyas R., Andreeff M., Tafuri A., Jakubowski A. Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid). N Engl J Med. 1991 May 16;324(20):1385–1393. doi: 10.1056/NEJM199105163242002. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. Yamamoto K., Hirosawa S., Sakamaki H., Aoki N. Frequent rearrangements of retinoic acid receptor alpha gene and myl gene, and rare mutations of RAS and FMS genes in acute promyelocytic leukemia. Am J Hematol. 1992 Aug;40(4):252–258. doi: 10.1002/ajh.2830400403. [DOI] [PubMed] [Google Scholar]
  60. Yu D., Matin A., Hung M. C. The retinoblastoma gene product suppresses neu oncogene-induced transformation via transcriptional repression of neu. J Biol Chem. 1992 May 25;267(15):10203–10206. [PubMed] [Google Scholar]
  61. de Thé H., Chomienne C., Lanotte M., Degos L., Dejean A. The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor alpha gene to a novel transcribed locus. Nature. 1990 Oct 11;347(6293):558–561. doi: 10.1038/347558a0. [DOI] [PubMed] [Google Scholar]
  62. 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]

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

RESOURCES