Regular Article

British Journal of Cancer (1999) 79, 1325–1331. doi:10.1038/sj.bjc.6690214 www.bjcancer.com
Published online 26 February 1999

Induction of apoptosis in myeloid leukaemic cells by ribozymes targeted against AML1/MTG8

H Matsushita1, M Kizaki1, H Kobayashi2, A Muto1 and Y Ikeda1

  1. 1Division of Haematology, Keio University School of Medicine, Tokyo, Japan
  2. 2Department of Laboratory Medicine, National Defense Medical College, Saitama, Japan

Received 12 November 1997; Revised 13 August 1998; Accepted 8 October 1998.

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Abstract

The translocation (8;21)(q22;q22) is a karyotypic abnormality detected in acute myeloid leukaemia (AML) M2 and results in the formation of the chimeric fusion gene AML1/MTG8. We previously reported that two hammerhead ribozymes against AML1/MTG8 cleave this fusion transcript and also inhibit the proliferation of myeloid leukaemia cell line Kasumi-1 which possesses t(8;21)(q22;q22). In this study, we investigated the mechanisms of inhibition of proliferation in myeloid leukaemic cells with t(8;21)(q22;q22) by ribozymes. These ribozymes specifically inhibited the growth of Kasumi-1 cells, but did not affect the leukaemic cells without t(8;21)(q22;q22). We observed the morphological changes including chromatin condensation, fragmentation and the formation of apoptotic bodies in Kasumi-1 cells incubated with ribozymes for 7 days. In addition, DNA ladder formation was also detected after incubation with ribozymes which suggested the induction of apoptosis in Kasumi-1 cells by the AML1/MTG8 ribozymes. However, the ribozymes did not induce the expression of CD11b and CD14 antigens in Kasumi-1 cells. The above data suggest that these ribozymes therefore inhibit the growth of myeloid leukaemic cells with t(8;21)(q22;q22) by the induction of apoptosis, but not differentiation. We conclude therefore that the ribozymes targeted against AML1/MTG8 may have therapeutic potential for patients with AML carrying t(8;21)(q22;q22) while, in addition, the product of the chimeric gene is responsible for the pathogenesis of myeloid leukaemia.

Keywords:

apoptosis, ribozyme, AML, AML1/MTG8, t(8;21)

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References

  1. Asou, H., Tashiro, S., Hamamoto, K., Otsuji, A., Kita, K. & Kamada, N. (1991). Establishment of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21 chromosome translocation. Blood 77: 2031–2036. | PubMed | ISI | ChemPort |
  2. Cameron, S., Taylor, D. S., TePas, E. C., Speck, N. A. & Mathey-Prevot, B. (1994). Identification of a critical regulatory site in the human interleukin-3 promoter by in vivo footprinting. Blood 83: 2851–2859. | PubMed | ISI | ChemPort |
  3. Erickson, P., Gao, J., Chang, K-S, Look, T., Whisenant, E., Raimondi, S., Lasher, R., Trujillo, J., Rowley, J. & Drabkin, H. (1992). 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 80: 1825–1831. | PubMed | ISI | ChemPort |
  4. Frank, R., Zhang, J., Uchida, H., Meyers, S., Hiebert, S. W. & Nimer, S. D. (1995). The AML1/ETO fusion protein blocks transactivation of the GM-CSF promoter by AML1B. Oncogene 11: 2667–2674. | PubMed | ISI | ChemPort |
  5. Haseloff, J. & Gerlach, W. L. (1988). Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 334: 585–591. | Article | PubMed | ISI | ChemPort |
  6. Homann, M., Tzortzakaki, S., Rittner, K., Sczakiel, G. & Tabler, M. (1993). Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1. Nucleic Acids Res 21: 2809–2814. | PubMed |
  7. Kizaki, M., Matsushita, H., Takayama, N., Muto, A., Ueno, H., Awaya, N., Kawai, Y., Asou, H., Kamada, N. & Ikeda, Y. (1996). Establishment and characterization of a novel acute promyelocytic leukemia cell line (UF-1) with retinoic acid-resistant features. Blood 88: 1824–1833. | PubMed | ISI | ChemPort |
  8. Klampfer, L., Zhang, J., Zelenetz, A. O., Uchida, H. & Nimer, S. D. (1996). The AML1/ETO fusion protein activates transcription of Bcl-2. Proc Natl Acad Sci USA 93: 14059–14064. | Article | PubMed | ChemPort |
  9. Koeffler, H. P. (1987). Syndromes of acute nonlymphocytic leukemia. Ann Intern Med 107: 748–758.
  10. Koizumi, M., Iwai, S. & Ohtsuka, E. (1988). Construction of a series of several self-cleaving RNA duplexes using synthetic 21-mer. FEBS Lett 228: 228–230. | Article | PubMed | ChemPort |
  11. Kozu, T., Sueoka, E., Okabe, S., Sueoka, N., Komori, A. & Fujiki, H. (1996). Designing of chimeric DNA/RNA hammerhead ribozymes to be targeted against AML1/MTG8 mRNA. J Cancer Res Clin Oncol 122: 254–256. | PubMed |
  12. Lange, W., Cantin, E. M., Finke, J. & Dolken, G. (1993). In vitro and in vivo effects of synthetic ribozymes targeted against BCR/ABL mRNA. Leukemia 7: 1786–1794.
  13. Lanotte, M., Martin-Thouvenin, V., Najman, S., Balerini, P., Valensi, F. & Berger, R. (1991). NB4, a maturation inducible cell line with t(15;17) marker isolated from human acute promyelocytic leukemia (M3). Blood 77: 1080–1086. | PubMed | ISI | ChemPort |
  14. Lenny, N., Meyers, S. & Hiebert, S. W. (1995). Functional domains of t(8;21) fusion protein, AML-1/MTG8. Oncogene 11: 1761–1769. | PubMed | ISI | ChemPort |
  15. Leopold, L. H., Shore, S. K., Newkirk, T. A., Reddy, R. M. V. & Reddy, E. P. (1995). Multi-unit ribozyme-mediated cleavage of bcr-abl mRNA in myeloid leukemia. Blood 85: 2162–2170.
  16. Matsushita, H., Kobayashi, H., Mori, S., Kizaki, M. & Ikeda, Y. (1995). Ribozymes cleave the AML1/MTG8 fusion transcript and inhibit proliferation of leukemic cells with t(8;21). Biochem Biophys Res Commun 215: 431–437. | Article | PubMed | ChemPort |
  17. Meyers, S., Downing, J. R. & Hiebert, S. W. (1993). 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 13: 6336–6345. | PubMed | ISI | ChemPort |
  18. Meyers, S., Lenny, N. & Hiebert, S. W. (1995). The t(8;21) fusion protein interferes with AML-1B-1 dependent transcriptional activation. Mol Cell Biol 15: 1974–1982. | PubMed | ISI | ChemPort |
  19. Miyoshi, H., Kozu, T., Shimizu, K., Enomoto, K., Maseki, N., Kaneko, Y., Kamada, N. & Ohki, M. (1993). The t(8;21)translocation in acute myeloid leukemia results in production of an AML1-MTG8 fusion transcript. EMBO J 12: 2715–2721. | PubMed | ISI | ChemPort |
  20. Miyoshi, H., Ohira, M., Shimizu, K., Mitani, K., Hirai, H., Imai, T., Yokoyama, K. & Ohki, M. (1995). Alternative splicing and genomic structure of the AML1 gene involved in acute myeloid leukemia. Nucleic Acid Res 23: 2762–2769. | Article | PubMed | ChemPort |
  21. Muto, A., Mori, S., Matsushita, H., Awaya, N., Ueno, H., Takayama, N., Okamoto, S., Kizaki, M. & Ikeda, Y. (1996). Serial quantification of minimal residual disease of t(8;21) acute myelogenous leukemia with RT-competitive PCR assay. Br J Haematol 95: 85–94. | PubMed |
  22. Nuchprayoon, I., Meyers, S., Scott, L. M., Suzow, J., Hiebert, S. & Friedman, A. D. (1994). PEBP2/CBF, the murine homologue of the human myeloid AML1 and PEBP2beta/CBFbeta proto-oncoproteins, regulates the murine myeloperoxidase and neutrophil elastase genes in immature myeloid cells. Mol Cell Biol 14: 5558–5568. | PubMed | ISI | ChemPort |
  23. Okuda, T., Deursen, J. V., Hiebert, S. W., Grosveld, G. & Downing, J. R. (1996). AML1, the target of multiple chromosomal translocation in human leukemia, is essential for normal fetal liver hematopoiesis. Cell 84: 321–330. | Article | PubMed | ISI | ChemPort |
  24. Okuda, T., Cai, Z., Yang, S., Lenny, N., Lyu, C. J., van Deursen, J. M., Harada, H. & Downing, J. R. (1998). Expression of a knocked-in AML1-ETO leukemia gene inhibits the establishment of normal definitive hematopoiesis and directly generates dysplastic hematopoietic progenitors. Blood 91: 3134–3143. | PubMed | ISI | ChemPort |
  25. Pace, U., Bockman, J. M., Miller, W. H. Jr, Dmitrovsky, E. & Goldberg, A. R. (1994). A ribozyme which discriminates in vitro between PML/RARalpha, the t(15;17)-associated fusion RNA of acute promyelocytic leukemia, and PML and RARalpha, the transcripts from the nonrearranged alleles. Cancer Res 54: 6365–6369.
  26. Pachuk, C. J., Yoon, K., Moelling, K. & Coney, L. R. (1994). Selective cleavage of bcr-abl chimeric RNAs by a ribozyme targeted to non-contiguous sequences. Nucleic Acids Res 22: 301–307.
  27. Rösl, F. (1992). A simple and rapid method for detection of apoptosis in human cells. Nucleic Acids Res 20: 5243 | Article | PubMed | ISI | ChemPort |
  28. Sakakura, C., Yamaguchi-Iwai, Y., Satake, M., Bae, S. C., Takahashi, A., Ogawa, E., Hagiwara, A., Takahashi, T., Murakami, A., Makino, K., Nakagawa, T., Kamada, N. & Ito, Y. (1994). Growth inhibition and induction of differentiation of t(8;21) acute myeloid leukemia cells by the DNA-binding domain of PEBP2 and the AML1/MTG8(ETO)-specific antisense oligonucleotide. Proc Natl Acad Sci USA 91: 11723–11727. | Article | PubMed | ChemPort |
  29. Schiffer, C. A., Lee, E. D., Tomiyasu, T., Wiernik, P. H. & Testa, J. R. (1989). Prognostic impact of cytogenetic abnormalities in patients with de novo acute nonlymphocytic leukemia. Blood 73: 263–270. | PubMed | ChemPort |
  30. Shore, S. K., Nabissa, P. M. & Reddy, E. P. (1993). Ribozyme-mediated cleavage of the BCRABL oncogene transcript: in vitro cleavage of RNA and in vivo loss of P210 protein-kinase activity. Oncogene 8: 3183–3188. | PubMed |
  31. Snyder, D. S., Wu, Y., Wang, J. L., Rossi, J. J., Swiderski, P., Kaplan, B. E. & Forman, S. J. (1993). Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line. Blood 82: 600–605. | PubMed | ChemPort |
  32. Takahashi, A., Satake, M., Yamaguchi-Iwai, Y., Bae, S. C., Lu, J., Maruyama, M., Zhang, Y. W., Oka, H., Arai, N., Arai, K. & Ito, Y. (1995). Positive and negative regulation of granulocyte-macrophage colony-stimulating factor promoter activity by AML1-related transcription factor, PEBP2. Blood 86: 607–616. | PubMed | ISI | ChemPort |
  33. Tanaka, T., Tanaka, K., Ogawa, S., Kurokawa, M., Mitani, K., Nishida, J., Shibata, Y., Yazaki, Y. & Hirai, H. (1995). An acute myeloid leukemia gene, AML1, regulates hematopoietic myeloid cell differentiation and transcriptional activation antagonistically by two alternative spliced forms. EMBO 14: 341–350.
  34. Tanaka, T., Kurokawa, M., Ueki, K., Tanaka, K., Imai, Y., Mitani, K., Okazaki, K., Sagata, N., Yazaki, Y., Shibata, Y., Kadowaki, T. & Hirai, H. (1996). The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability. Mol Cell Biol 16: 3967–3979. | PubMed | ISI | ChemPort |
  35. Tashiro, S., Kyo, T., Tanaka, K., Oguma, N., Hashimoto, T., Dohy, H. & Kamada, N. (1992). The prognostic value of cytogenetic analysis in patients with acute nonlymphocytic leukemia treated with the same intensive chemotherapy. Cancer 70: 2809–2815. | PubMed |
  36. Wang, Q., Stacy, T., Binder, M., Marin-Padilla, M., Sharpe, A. H. & Speck, N. A. (1996). Disruption of the cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc Natl Acad Sci USA 93: 3444–3449. | Article | PubMed | ChemPort |
  37. Yergeau, D. A., Hetherington, C. J., Wang, Q., Zhang, P., Sharpe, A. H., Binder, M., Martin-Padilla, M., Tener, D. G. & Speck, N. A. (1997). Embryonic lethality and impairment of haematopoiesis in mice heterozygous for an AML-ETO fusion gene. Nat Genet 15: 303–306. | Article | PubMed | ISI | ChemPort |
  38. Zhang, D., Fujioka, K., Hetherington, C. J., Shapiro, L. H., Chen, H., Look, T. & Tenen, D. G. (1994). Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1). Mol Cell Biol 14: 8085–8089. | PubMed | ISI | ChemPort |
  39. Zhang, D., Hetherington, C. J., Meyers, S., Rhoades, K. L., Larson, C. J., Chen, H., Hiebert, S. W. & Tenen, D. G. (1996). CCAAT enhancer-binding protein (C/EBP) and AML1 (CBFalpha2) synergistically activate the macrophage colony-stimulating factor receptor promoter. Mol Cell Biol 16: 1231–1240. | PubMed | ISI | ChemPort |