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Apoptosis

Multi-sites cleavage of leukemogenic AML1-ETO fusion protein by caspase-3 and its contribution to increased apoptotic sensitivity

Leukemia volume 22, pages 378–386 (2008)Cite this article

Abstract

Leukemia-associated fusion protein AML1-ETO is a product of the chromosome translocation (8;21) frequently occurred in acute myeloid leukemia (AML). The fusion oncoprotein blocks leukemic cell differentiation, and it also induces growth arrest with increased sensitivity to apoptosis induction. Such dichotomous functions make it difficult to clarify the role of AML1-ETO in leukemogenesis. Here, we systematically showed that constitutively and overexpressed AML1-ETO protein was cleaved to four fragments of 70, 49, 40 and 25 kDa by activated caspase-3 during apoptosis induction by extrinsic mitochondrial and death receptor signaling pathways. The in vitro proteolytic system combined with MALDI-TOF/TOF mass spectrometer confirmed that AML1-ETO and wild-type ETO but not RUNX1 (AML1) proteins were direct substrates of apoptosis executioner caspase-3. Site-directed mutagenesis analyses identified two nonclassical aspartates (TMPD188 and LLLD368) as caspase-3-targeted sites in the AML1-ETO sequence. When these two aspartates were mutated into alanines, more intriguingly, the apoptosis-amplified action of AML1-ETO induction completely disappeared, while inducible expression of the caspase-3-cleaved 70 kDa fragment of AML1-ETO after tetracycline removal is sufficient to enhance apoptotic sensitivity. Further investigations on the potential in vivo effects of such a cleavage and its possible role in leukemogenesis would provide new insights for understanding the biology and treatment of AML1-ETO-associated leukemia.

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Acknowledgements

We appreciate Dr Lübbert M, Dr Tenen DG and Dr Hiebert SW for providing us U937-A/E 9/14/18, U937T cell line and some plasmids, respectively. This work was supported in part by National Key Program (973) for Basic Research of China (NO2002CB512805), National Natural Science Foundation (30500257, 30630034), Grants from Science and Technology Committee of Shanghai (05JC14032). Dr. GQ Chen is a Chang Jiang Scholar of Ministry of Education of China, and is supported by Shanghai Ling-Jun Talent Program.

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Authors and Affiliations

  1. Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education of China, Shanghai Jiao-Tong University School of Medicine (SJTU-SM, formerly Shanghai Second Medical University), Shanghai, China

    Y Lu, L Xia & G-Q Chen

  2. Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/SJTU-SM, Shanghai, China

    Z-G Peng, T-T Yuan, Q-Q Yin & G-Q Chen

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Correspondence to G-Q Chen.

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Lu, Y., Peng, ZG., Yuan, TT. et al. Multi-sites cleavage of leukemogenic AML1-ETO fusion protein by caspase-3 and its contribution to increased apoptotic sensitivity. Leukemia 22, 378–386 (2008). https://doi.org/10.1038/sj.leu.2405020

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